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PMC3014081 | 22275798 | The Association of Medical Physicists of India (AMPI), now 30 years young, was founded in the year 1976 with the objective of promoting the application of physics in medicine and biology. It provides a forum for medical physicists in the form of annual conferences (at national and international levels) and publication of a quarterly Journal of Medical Physics for the exchange of ideas and dissemination of knowledge. The host of the AMPI Conference is decided by the general body of the association on receipt of the formal request from the head of the host institution. The association and the host institution are jointly organizing the conference.
The 27 th annual conference of the association is being organised at Bhubaneswar, Orissa during 9-12 November 2006. This is also the seventh International Conference on Medical Physics (ICMP) in India. The host of this conference, the Hemalata Hospitals and Research Centre, Bhubaneswar is equipped with all the adequate facilities for the treatment of cancer. It is pleasure to know that this hospital has come to this stage by the relentless efforts made by one of our medical physics colleagues, Dr. A.K. Rath who is also the Chairman of the organising committee of the conference.
This issue of the journal is exclusively dedicated to AMPICON 2006. About 200 abstracts were received by the organisers, which were evaluated by the scientific committee for presentation at the conference. Extended abstracts of proffered papers -both oral (63) and poster (127) - are included in this issue. The scientific programme consists of invited talks including Ramaiah Naidu Memorial oration. Ramaiah Naidu Memorial Oration (RNMO) Award was instituted by the association in 1992 in the honour of pioneer Indian medical physicist Dr. Ramaiah Naidu. The oration award consists of a citation and a silver plaque. As per the norms of AMPI, the recipient of RNMO award shall be a person of national/ international repute in the field of medical physics. The recipient of this award is selected by the Executive Committee of AMPI. There are 14 scientific sessions, including 19 invited talks, 2 guest lectures and 3 poster sessions. For the first time the scientific programme includes a debate on a topic that I am sure that all the medical physicists in radiation oncology will find it interesting. Keeping in line with the recent trends in radiation oncology three invited talks are being organised on image guided radiotherapy (IGRT) by experts from India and abroad. It is also decided to keep early morning lectures on various topics for the benefit of our young colleagues in the specialty.
While going through the abstracts, the scientific committee felt that there is scope for improvements in the methodology for abstract submission, their speedy evaluation and informing the authors on decision taken. We will certainly pass on our suggestions to the association for consideration. Couple of years down the line I hope that complete process of submission to publication of abstracts should be made on-line.
As a guest editor I take this opportunity to request all the authors of proffered papers to prepare the full manuscript of their good work and submit to the editorial office for publication in this journal.
I also thank the members of scientific and publication committees for their full cooperation and hard work in bringing out this issue in time. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2006 Jul-Sep; 31(3):97 | oa_package/11/e7/PMC3014081.tar.gz |
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PMC3014082 | 22275799 | Technology-driven delivery methods
Technological developments in computers and accelerator designs, particularly inverse treatment planning and dynamic multi-leaf collimation systems (dMLCs), have given the radiotherapy community the ability to deliver conformal and intensity-modulated radiation therapy (IMRT) treatments. With the implementation of image-guided radiation therapy (IGRT), there is a potential[ 6 ] to deliver inhomogeneous dose distributions that increase the dose inside the target by 20-30% over the minimal peripheral dose, while decreasing possible normal tissue complications by collapsing the planning target volume (PTV) onto the clinical target volume (CTV), as is often done in stereotactic radiosurgery. | Conclusion
The availability of new imaging modalities before and during radiotherapy planning and delivery has allowed us to launch new tools at every major step of the treatment process. Figure 21 shows that instead of a 3-D static patient data set, we need to incorporate dynamic 4-D patient data and corresponding beam warping to prepare a motion-compensated treatment plan. Instead of fusion of data from merely two modalities, tumors need to be characterized and localized using multimodality imaging-based information. Instead of merely delivering treatments with external patient immobilization, we need to achieve internal motion synchronized beam delivery.
Radiation therapy is unique among the cancer treatment modalities because it can be modulated in the four dimensions of time and space. Modulation in the time domain gives rise to the time-dose-fractionation problem, and this continues to be one of the most fruitful avenues for further improvement of the therapeutic ratio in radiation oncology. Spatial modulation has been greatly facilitated by recent technological advances in image guidance based treatment planning and delivery. Progress in functional and molecular imaging, together with an improved understanding of the patterns of spread of cancer, provides the tools that will allow us to prescribe 4D radiation therapy that is optimized in the individual patient. All of these developments, combined with progress in biological and chemical targeting of cancer cells, provide not only challenging and exciting research opportunities but also a real hope that more cancer patients will be cured (or left with a manageable chronic disease in place of a lethal one).
Between-patient differences can be identified and exploited with ever-greater finesse by ever-finer levels of an ever-increasing inventory of covariates, which now include an increasing number of biological and biochemical measures on continuous scales, as well as the coarser nominal and ordinal demographic measures (e.g., stage, gender, etc.) of the past.
Moreover, within-patient differences across time, e.g., the circadian variations in patient physiology and the infra-treatment organ motion that continually scans the target volume across the steep dose gradients of the IMRT radiation fields during each daily treatment can now be tracked and their effects either exploited or compensated for: Modern (‘adaptive’) radiotherapy can now routinely ‘hit a moving target.’ All of this diversity of information is very patient specific, and we are challenged to seek individual patient-specific solutions [ Figure 22 ] instead of the prior practice of mere population-based approaches.
Clinical implementation of IMRT, especially image-guided IMRT, is in a state of rapid evolution. Helical TomoTherapy, one the latest steps in this evolution, is an IMRT system whose design incorporates aspects such as infinite beam angle optimization and MVCT-based delivery verification. These features have the potential to permit the full clinical development of adaptive radiotherapy and conformal avoidance. To realize the ultimate goal of improving clinical outcomes for our patients, appropriate patient immobilization, optimized target localization, conformal avoidance of sensitive normal structures and radiobiologically-guided dose escalation are required. The clinical implementation of helical TomoTherapy and the consequent issues raised (such as radiation dose rates and dose homogeneity) present questions and opportunities that may change the current paradigm in radiation oncology. | Impressive advances in computers and materials science have fueled a broad-based confluence of basic science breakthroughs. These advances are making us reformulate our learning, teaching and credentialing methodologies and research and development frontiers. We are now in the age of molecular medicine. In the entire field of health care, a paradigm shift from population-based solutions to individual specific care is taking place. These trends are reshaping the practice of medical physics. In this short presentation, examples are given to illustrate developments in image-guided intensity-modulated and adaptive helical tomotherapy, enhanced application of intensity modulation radiotherapy (IMRT) using adaptive radiotherapy and conformal avoidance. These advances include improved normal tissue sparing and permit dose reconstruction and verification, thereby allowing significant biologically effective dose escalation and reduced radiation toxicity. The intrinsic capability of helical TomoTherapy for megavoltage CT imaging for IMRT image-guidance is also discussed. Finally developments in motion management are described. | It is a great pleasure and honor to be invited to deliver the Dr. Ramaiah Naidu oration. In the early thirties of the twentieth century, Dr. Naidu established the foundations of medical physics. He was a post-doctoral fellow of Madame Curie and was the first medical physicist at the Tata Memorial Hospital, Bombay, where he installed a Radon production facility. He implemented a similar facility at the Sloan Kettering Memorial Institute in New York. If he were here today, he would be amazed how the field has changed. Table 1 lists some of the major landmarks.
During the last decade, the complexity of detection, planning and delivery tools for medical services has increased exponentially. The computer age has been a double-edged sword. It has significantly enhanced our capabilities while also increasing the potential for greater risks in the delivery of our services. New technology not only requires a highly skilled medical physicist - it also requires continuous updating of knowledge and skills and integration of new technological developments in our practice. There are new challenges in all of the following areas: Learning, teaching, research and development and clinical services.
Challenge in learning
In the past the pace of change was slow and we used to become a qualified individual for life. In the current age of rapid change, we will need to be responsive to these changes and to be accountable to the high expectation of our clients (patients). The competencies required in the practice of clinical medical physics have grown: a professional medical physicist needs communication, managerial and scholastic competence and should also be a collaborator and advocate. To meet these obligations, training and education we receive to qualify as a certified medical physicist would require a continuous process of maintenance of certificate. It would need to be responsive to change. It would need to be an educational initiative for continuous personal development (CPD), an activity designed to enhance knowledge, skills, attitudes and competencies, required for practice. It would need to be documented and have measurable outcomes from engaging in CPD. The overall goal would be to improve practice performance, enhance the quality of patient care, safety. Moreover, nowadays all the scientific fields, similar to medical physics, are approaching a multidisciplinary paradigm. In order to keep up with the development of any field, an individual has to learn the latest changes in technology and other related skills, viz., imaging sciences, algorithm design, or use of soft computing techniques.
Challenge in teaching
Our knowledge base in the last decade has increased dramatically (The American Association of Physicists in Medicine report # 79) and continues to expand significantly. How and what we teach impacts many health sciences professionals. We teach medical physicists, radiation therapists, radiation oncologists and radiologists. Unfortunately our teaching methods have not changed and are archaic. We need to develop clinically relevant teaching tools, incorporate modern web-based technology and use interactive processes to teach clinically relevant material that reflects professional needs. The American Association of Physicists in Medicine (AAPM), American Board of Radiology (ABR) and American Society for Therapeutic Radiation Oncology (ASTRO) are all developing new syllabi and teaching tools. Some specific challenges we face are:
Retaining highly regarded faculty Declining federal funding for research support Large class sizes Realigning to the new era of medicine with focus on molecular biology and genomics Imaging to emphasize function and biological interactions, micro-imaging and pinpoint therapy Space needs for larger training programs
In the long run, with these efforts we may claim to have trained better health sciences professionals based on higher board scores or decreased mistakes; however, the real differences can be measured only in the quality of care delivered to patients.
Challenges in research and development
Significant physical and biochemical developments in the radiation oncology related fields have created potential for dose escalation in the delivery of radiation treatments. Some of these developments are based upon new compounds and models and many are from new imaging tools shown in Table 1 . These tools allow higher confidence in tumor targeting and normal tissue sparing by providing reliable mechanisms for generation and delivery of conformal dose distributions. There are significant advances in localizing the target as a function of time, particularly four-dimensional computed tomography (4D-CT). These advances allow us to shift the isodose distribution to the surface of the target volume with a rapid fall-off in the normal tissue. Several changes have been, in part, technologically mediated and to some extent due to the synergy between, and integration of, radiotherapy and other modalities. These processes are likely to continue in the next decade. Some of the major impacts will come from the following.
Radiobiological modeling
During the last 30 years, radiobiological modeling has evolved on many frontiers. The concepts of nominal standard dose[ 1 2 ] gave way to mean nominal tumor dose[ 3 ] and equivalent uniform dose (EUD)[ 4 ] and generalized equivalent uniform dose (gEUD).[ 5 ] The linear quadratic model came into prominence, and modern statistics-driven modeling tools have been outstanding in formulating new fractionation schemes. Modeling lessons have established that dose escalation improves local control, but treatment lengthening retards the gain. Tomé and Fowler[ 6 ] suggest the feasibility of increasing effects by boosting 60-80% of the target by boost-dose ratios of 1.2 to 1.3. There are few situations where a boost-dose ratio exceeding 1.3 appears to be worthwhile or necessary. Significant increases of tumor control probability, up from 50% to 75%, might be achieved for a small increase in risk of necrosis, where a substantial proportion of the target volume (60-80%) can be boosted.
Imaging advances
The need for safe delivery of inhomogeneous dose distributions without increasing risks to normal structures has provided an impetus to the development of imaging tools coupled to radiation delivery systems. Examples of some of these are given in Table 1 and Figure 1 . These range from conventional orthogonal radiographic systems to state-of-the-art TomoTherapy, cone beam computed tomography (CT), CT-PET (positron emission tomography) and magnetic resonance imaging (MRI) systems.
Molecular imaging is a rapidly evolving area, with a focus on monitoring gene expression in vivo using remote imaging devices (positron emission tomography [PET], single photon-emission computed tomography [SPECT], MRI, optical and ultrasound). Molecular imaging as now envisioned actually exploits creative synergies between these hierarchies of signal sources, resulting in fusions between these biological scales. Figure 2 shows incorporation of target definition boundaries determined using four different tissue properties in brain images. The blue contour is the volume determined from choline maps; this volume is indicative of possible microscopic extensions of the tumor. The green outline is the volume of Blood-Brain-Barrier breakdown as determined from T1-MRI; this is the standard gross target volume (GTV). The yellow outline shows the volume determined using percutaneous myocardial revascularization, indicating areas of tumor that are well perfused and hence indicates aerobic areas of the tumor. Lastly, the red contour, determined from blood oxygenation level dependent imaging combined with carbogen breathing, is indicative of chronically hypoxic tumor regions. All of these, to a degree, are important factors to be considered in the determination of the target volume, its radiation sensitivity and the prognosis of outcome.
Image-guided intensity-modulated and adaptive helical TomoTherapy
Image-guided IMRT is redefining the practice of radiation oncology. Traditional methods of implementing beam intensity modulation have included individually designed compensators, static multi-leaf collimators (MLC), dynamic MLC and sequential (serial) tomotherapy. Helical TomoTherapy[ 7 ] provides added functionality to enhance the application of IMRT. It facilitates adaptive radiotherapy and conformal avoidance. These advances improve normal tissue sparing and permit dose reconstruction and verification, thereby allowing significant biologically effective dose escalation and reduced radiation toxicity. Recent radiobiological findings can be translated into altered fractionation schemes that aim to improve the local control and long-term survival. The intrinsic capability of helical TomoTherapy for megavoltage CT (MVCT) imaging for IMRT image-guidance is an added feature aiding in further adaptation of treatments.
Helical TomoTherapy
In contrast to standard radiotherapy, helical TomoTherapy [ Figure 1 ] delivers treatment with a rotating intensity-modulated fan beam. The patient is continuously translated through a ring gantry, resulting in a helical source trajectory about the patient. The beam delivery is similar to that of helical (‘spiral’) computed tomography (CT) and requires slip rings to transmit power and data. The ring gantry provides a stable and accurate platform to perform tomographic verification of both the patient setup and delivered dose. The design of the helical TomoTherapy unit allows for continuous delivery over 360 degree beam angles.[ 8 ] The helical delivery minimizes the risk of significant high or low dose deposition in areas of overlap or junctioning.[ 9 ] Assessments of sequential units presently in use reveal that positioning errors as little as 1 mm can cause dose errors of the order of 10-20% in the abutment regions.[ 10 ] In addition to full integration of IMRT delivery, an important advance with helical TomoTherapy over the other current systems is the ability to provide accurate verification of radiation delivery via onboard tomographic imaging. Examples of conformal avoidance planning and delivery using a Linac and Tomotherpay unit are given Figure 3 .
Adaptive radiotherapy
One of the significant features of the helical TomoTherapy unit is the presence of an integrated online MVCT unit. This permits verification of patient positioning, target tumor/organ registration to assess internal motion (including geometric shift and shape/volume changes) and reconstruction of delivered dose. These capabilities offer the radiation oncology team the ability to verify and adjust the therapeutic plan as needed during the course of treatment. This concept is referred to as adaptive radiotherapy.[ 11 ] These capabilities can be viewed as a closed-circuit loop, as illustrated in Figure 4 . The integration of the MVCT and the treatment unit allows for options not possible with contemporary systems. For example, if a patient setup is found to differ from the planned position, the current approach requires moving the patient to compensate for this positioning error. With the integrated helical unit, another option is having the patient remain in the ‘incorrect’ position and modifying the treatment delivery. The success of the modification is independent of the extent and direction of the offsets, within certain limits.[ 12 ] MVCT images can be obtained at radiation doses of around 2 cGy and are comparable to that of diagnostic CT imaging[ 13 14 ] and lower than reported doses from low-dose megavoltage cone beam CT.[ 15 ] Other methods of onboard imaging have been developed recently and are available clinically.
Dose reconstruction
Newly developed dose reconstruction tools use data acquired during the delivery of a treatment to determine the actual three-dimensional dose deposited. At the time of treatment on the TomoTherapy unit, the incident energy fluence is computed from the signal detected at the exit detectors. An accurate, anatomically detailed 3D representation of the patient is also obtained. A transfer matrix then converts this signal to incident energy fluence. In other words, the matrix determines the energy fluence emitted from the MLC from the signal at the detector. The integrated CT detectors present in the TomoTherapy unit provide detailed information about the primary projection, as well as the scatter characteristics of every projection. Pathlength and detector-to-patient distance are computed from the MVCT image. Leakage and transmission plus tongue and groove penumbra are also included in the calculation. This fluence corrected for some additional MLC effects is used in the treatment dose distribution computation. Effectively, this provides an accurate daily delivered dose record, which can be compared directly with the planned dose distribution.
Dose comparison
The ability to accurately reconstruct the 3D dose distribution permits a quantitative comparison of desired and actual dose distributions delivered to the patient. An example illustrating this issue is described in Figure 5 . Desired isodose lines of a helical TomoTherapy optimization for a nasopharyngeal cancer are depicted with the parotid glands and spinal cord areas to be avoided.
The comparison of desired dose distributions to the actual delivered dose distributions is based on the methods of Van Dyk et al .[ 16 ] and Low et al .[ 17 ] The two modes of comparison are dose difference (DD) and distance-to-agreement (DTA) analyses. For regions in which both the planned and measured distributions have high dose gradients, DTA comparisons are conducted. For all other cases, DD analyses are performed. Once the mode of comparison is decided, the × index is computed by dividing the DD and DTA values by their respective tolerances.
The × value provides a measurement of quality for every voxel, indicating if they are within the desired tolerance or how far they deviate from that tolerance. Typical tolerance values in IMRT are 3% and 3 mm for DD and DTA respectively. The smaller the × value, the more accurately the compared isodose distributions are aligned. To facilitate the spatial identification of problem areas, color-wash images of × maps can be displayed [Figure 5a – d ]. Figure 7a , b illustrates the × image comparing the planned dose and the reconstructed dose for the first week of treatment in which a systematic error was made. Several error regions appear on the targets and regions at risk, mainly in the high gradient regions. Figure 7b shows a gray scale image of the re-optimized dose distribution that could be delivered during the second week of treatment in order to correct for prior misalignment. In this dose image, a pattern appears that is very similar to the × image.
Figure 8 illustrates the result of one week of treatment with a systematic error followed by a second week of either the original treatment plan given accurately ( Figure 8a ) or a re-optimized treatment plan designed to compensate for the errors of the first week ( Figure 8b ). Simply repositioning the patient and accurately delivering treatment in the subsequent week according to the originally designed radiation plan will dilute the error incurred during the first week but cannot fully compensate for the error. Radiation delivery modifications are designed to compensate for the difference between actual and desired dose distributions. In the example presented, dose reconstruction and comparison reveal that excess dose is being deposited in the spinal cord and right parotid. In Figure 9 a modified plan specifically designed to compensate for the errors of week one is instituted in the second week and the dose reconstruction is performed again. Most of the errors within the tumor region are corrected. However, a small trade-off with dose to the parotid glands and spinal cord is necessary in order to rectify the errors incurred during the first week of delivery. The specific thresholds for the trade-offs that should be accepted remain a matter of ongoing physics and clinical research.
With helical TomoTherapy, MVCT images can be registered using a full mutual information algorithm, bone extracted feature fusion (EFF) and bone and tissue EFF algorithms, with uniform down-sampling of the MVCT images along the x and z axes (to provide a time-saving by a factor of up to 4) with and without the rotational registration components. These particular algorithms take into account any changes in patient anatomy between the reference image and fusion image when the image registration is carried out.
Conformal dose-per-fraction escalation
The advances in adaptive radiotherapy, conformal avoidance and enhanced localization and immobilization have provided an impetus for novel time dose fractionation dose delivery schemes to improve local control and possibly increase survival rates. These dose-fractionation regimens are target specific (lung, prostate, etc.) and provide significant sparing of normal tissues, increase the total dose and dose per fraction, and reduce the number of fractions delivered to reduce the overall treatment time.
Motion management
Many IMRT approaches rely on increasing the number of beam directions and modulating beam intensity such that multiple tiny sub-beams are created. While this improves dose distributions, the clinical applicability of such pencil beams requires immense precision; the slightest patient/organ/tumor motion is likely to result in unintended errors. Therefore, although highly irregular dose distributions and dose volume histograms (DVHs) can be created, their clinical application is uncertain. Some of the patient immobilization and motion management systems to address this issue are described below.
Optical guidance
Noninvasive head frame systems often rely on external contours of the head and face and have immobilization errors of 2-4 mm in favorable circumstances. Such systems include the Heidelberg system, which has a reported accuracy of 2 mm;[ 18 ] the Laitinen’s stereoadapter with a measured error of approximately 2-3 mm;[ 19 ] and the Gill-Thomas-Cosman, which uses a maxillary bite block system to yield reproducible immobilization and repeat fixation[ 20 ] with a reported accuracy of 0.5-1 mm. The optically guided FSRT/IMRT approach in use at the University of Wisconsin is a noninvasive system shown in Figure 10 , where localization is separated from immobilization. This is accomplished through detection of four markers attached to a custom rigid bite plate. The location of these markers in space (tracked relative to the isocenter) is accomplished in real time using an optical position-sensor system mounted to the ceiling of the accelerator vault and interfaced with a computer. The interfraction translational error and rotational error is within 0.3 mm and 0.3 degrees respectively. Tomé et al .[ 19 – 20 ] have shown that this optically guided system, in conjunction with IMRT planning, allows the generation of highly conformal treatment plans that exhibit smaller 90, 70 and 50% prescription isodose volumes, improved PITV ratios (the ratio of the prescription isodose volume to tumor volume), comparable or improved EUD, smaller NTDmean for critical structures and an inhomogeneity index that is within generally accepted limits. In addition, optically guided treatments allow real-time monitoring of treatment delivery, providing further confidence in the patient’s actual delivered dose distribution. For helical TomoTherapy, such optical guidance will be useful in verifying that the patient has not moved between MVCT and treatment.
To demonstrate the clinical relevance of optical guidance, an early implementation of this system is illustrated in Figures 11 and 12 . This patient with an astrocytoma required radiotherapy and was planned with 3-D conformal techniques, multi-non-coplanar field FSRT and IMRT. In all three treatment planning scenarios, CT-MR fusion was utilized and the defined GTV/CTV was constant. A mathematical descriptor PITV (the ratio of the prescription isodose volume to tumor volume) is frequently employed to evaluate dose conformality. The ideal PITV ratio is 1; values up to 2 are commensurate with good stereotactic radiosurgery plans. In the example presented in Figure 11 , the PITV values are 3.16, 1.65 and 1.45 for the 3-field conventional, 3-D FSRT and helical TomoTherapy plans respectively. The DVHs in Figure 12 reveal substantial improvement in brain stem dose reduction as the technical approach becomes more sophisticated. The superior patient immobilization, day-to-day alignment and position-verification afforded by the FSRT and the IMRT systems using an optically guided system allowed for a substantial reduction in the PTV margins.[ 20 ] This margin reduction alone can have a significant impact in improving the DVH. The conformality afforded by FSRT and IMRT leads to dosimetric improvement that might be further improved using helical TomoTherapy techniques. The application of immobilization devices is practical, and the incorporation of optical guidance provides a high degree of reliability in terms of daily positional reproducibility and for monitoring intra-treatment motion, potentially maximizing the inherent benefits of helical TomoTherapy.
Ultrasound guidance
The system described above works well as long as the rigid body approximation holds, as is the case for intracranial lesions. However, outside the cranium, soft-tissue targets can move relative to rigid fixation points (e.g., bony structures) between the times of image acquisition, treatment planning and treatment delivery. Real time imaging is useful in determining target location at the time of treatment delivery. A system based on 3D-ultrasound guidance (SonArrayTM, ZMed, Inc., Ashland, MA) can be used to correct for these misalignments at the time of treatment. Ultrasound is chosen because it is a flexible and inexpensive imaging modality that can easily be adapted for use in a radiation therapy treatment room. The interpretation of two-dimensional ultrasound images can be challenging and is highly dependent on the skill of the operator in manipulating the transducer and mentally transforming the 2D images into a 3D structure. Three-dimensional ultrasound imaging overcomes this limitation. The 3D ultrasound data sets are generated through optical tracking of free-hand acquired 2D ultrasound images. The position and angulation of the ultrasound probe are determined using an array of four infrared light-emitting diodes (IRLEDs) attached to the probe. An infrared camera is used to determine the positions of the IRLEDs and this information is input to the computer workstation. The position of each image plane can therefore be determined using the IRLEDs and an ultrasound volume can be reconstructed by coupling the position information with the images. In addition to building the 3D image volume, optical guidance is used to determine the absolute position of the ultrasound image volume in the treatment room coordinate system. Because the relative positions of the 3D-image volume and the ultrasound are fixed, knowledge of the probe position in the treatment room coordinate system at the time of image acquisition is sufficient to determine the position of the image volume relative to the linac isocenter. The image-to-probe relationship is determined by a calibration step performed at the time of system installation.[ 21 ] In this way, ultrasound guidance will allow greater accuracy of treatment delivery via helical TomoTherapy to various extracranial sites.
Currently, ultrasound is being used in conjunction with MVCT. We are presently conducting a comparison of ultrasound with MVCT. Ultimately, it may be that MVCT alone will be the only image guidance necessary.
Respiratory gating
Two strategies have emerged to deal with the problem of respiratory motion. One strategy is to ‘immobilize’ the lung during one phase of respiration and to gate radiation to this phase. This requires the ability of patients to hold breath for a short period of time, which may be difficult in patients with respiratory cancers. The second strategy is to radiate at a predetermined period during respiration using dynamic aperture tracking. Online verification of the correct phase of respiration requires a respiratory monitoring device.[ 22 ] Another option provided by the helical TomoTherapy unit is to determine the phase of respiration using the MVCT. Individual patient respiratory patterns will be assessed during planning, and an MVCT can be performed at each treatment. The treatment will then be gated to the predicted respiratory phase with the appropriate dosimetric plan.
Motion management
An attempt is made here to give an overview of some of the recent developments. The concepts of motion synchronization of treatment delivery will be elaborated. During the last 5 years, there has been significant development in breathing-synchronized delivery (BSD) of 4-D optimized IMRT treatment plans.[ 23 – 35 ] Due to space limitations, a snapshot of some of the completed work is given in Figures 13 – 20 .
In BSD, patient breathing and treatment delivery are synchronized by instructing the patient to breathe following a breathing guide. We have previously developed a 4-D TomoTherapy treatment technique based on BSD. We have extended the application of BSD into conventional radiation treatment using dMLCs. Dynamic 4D-CT images are acquired for treatment planning, and the images at full exhalation phase are used as treatment planning images. A typical IMRT treatment plan is developed that yields dynamic leaf sequences for sliding window delivery of IMRT. Average target trajectory is obtained by deformable image registration of 4-D data sets and is smoothed by Fourier filtering. Assuming the patient breathes with a reproducible breathing pattern and a constant dose delivery rate, the treatment process is corre-lated with the breathing phase in BSD treatment planning. Figures 13 – 17 show the correlation of breathing phases, deformable registration, motion envelope multileaf collimation generation and delivery processes. The instantaneous target displacement is overlaid to the dMLC position at the corresponding phase.
A custom-built mobile [ Figure 18 ] phantom was used in dosimetry verification. The phantom, driven by a computer-controlled stepper motor, is able to move according to the linear pattern used in BSD treatment planning. The conventional plan is delivered on the phantom with and without motion. The BSD plan was also delivered on the phantom that moved with the prescheduled pattern and synchro-nized with the delivery of each beam. Film dosimetry results [Figures 19 and 20 ] revealed that without incorporating motion, underdose and overdose (over 20%) were noticed at the superior and inferior regions of the target respectively. BSD delivery, on the other hand, obtained dose distribution very similar to that planned. No region in the target had dose delivery error of more than 20%, as planned.
The BSD technique described for use with dMLC is similar to the method of real-time tracking. This method basically needs no hard-ware modification and avoids the difficulties of real-time tracking. Unlike cardiac motion, breathing motion, to some extent, can be voluntarily controlled by some patients. Studies have shown that many patients may be able to maintain a regular breathing pattern with some instruction. We are currently developing a patient train-ing device to allow more practice for the patient. Since BSD allows continuous air exchange, it is better tolerated than breath-hold gating.
We realize that breathing motion is an extremely challenging frontier in the delivery of precision radiotherapy and is a function of several physiological, behav-ioral and mechanical processes. Even though we may have developed an idealized 4D-CT/PET and patient-guiding cycle based on optimized IMRT treatment, its delivery is significantly susceptible to the above-mentioned processes during the delivery of the plan. The baseline filling of the lung has also been identified as a significant factor in the determination of the daily position of the target volume, which makes daily pretreatment imaging indispensable. We believe that further refinement of the BSD technique will be possible using the on-board imaging verification processes offered by TomoTherapy and kilovoltage cone-beam CT. | I wish to acknowledge the contribution of the physics staff of the departments of Medical Physics and Human Oncology. I am in particular thankful for the contribu-tions made by Dinesh Tewatia, Tiezhi Zhang, Wolfgang Tomé, Hazim Jaradat, Nigel Orton and Li Xu. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2006 Jul-Sep; 31(3):98-108 | oa_package/d9/58/PMC3014082.tar.gz |
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PMC3014084 | 21209794 | Introduction
The 2006 World Health Organization (WHO) guidelines on antiretroviral therapy (ART) established a worldwide standard of care for patients with HIV infection [1] . Since this publication, new evidence has emerged on how to treat patients infected with HIV, and this evidence formed the basis for the WHO 2010 ART guidelines update [2] . These revisions aim to better align global standards with those already adopted in well-resourced countries [3] , [4] . Specifically, revised guidelines recommend an increased number of sequential ART regimens, routinely available CD4 count monitoring, earlier ART initiation thresholds (CD4<350 cells/μl versus CD4<200 cells/μl), and replacement of stavudine with the less-toxic drug tenofovir.
As WHO expands treatment recommendations, many countries in resource-limited settings still struggle to implement 2006 guidelines [5] . In Malawi, for example, most HIV disease is monitored clinically; CD4 count monitoring is limited to pregnant women and children [6] , [7] . In South Africa, ART is available to only 22%–36% of those reported to be in need [8] . In settings confronted with numerous new recommendations, not all of which are immediately feasible, the relevant policy question is: What to do first? Should countries begin by replacing stavudine with tenofovir or by making CD4 count monitoring universally available? To assist policy makers in this prioritization process, we use a model-based analysis with data from South Africa to project the clinical and economic outcomes of alternative stepwise implementation scenarios toward the 2010 WHO ART guidelines. | Methods
Analytic Overview
The Cost Effectiveness of AIDS Complications (CEPAC)–International model is a Monte Carlo simulation model of the natural history and treatment of HIV disease (see Text S1 for model details) [9] – [11] . We populate the model with South African clinical and resource utilization data to project survival and costs under alternative guideline prioritization scenarios. We use a “no ART” scenario for comparison and assume that baseline care (designated the “reference strategy”) is a one-line stavudine-containing regimen, initiated at WHO stage III or IV disease, without CD4 count monitoring capacity. We then examine every feasible sequence of the following implementation elements: (1) widespread CD4 count monitoring capacity, allowing for ART initiation at CD4<200 cells/μl (and biannual monitoring), (2) earlier ART initiation, at CD4<350 cells/μl (assumes CD4 count availability), (3) an available second-line ART regimen upon first-line failure, and (4) replacement of stavudine with tenofovir in the first-line regimen. To refer to these strategies, we use the following nomenclature: nucleoside analog in first line/ART initiation criterion/number of regimens [e.g., stavudine/<200/μl/two-lines]). The combined implementation elements result in twelve possible strategies, in addition to no ART ( Figures 1 and S1 ). We examine the short- and long-term survival benefits and cost-effectiveness of each stepwise, incremental policy change from the reference strategy to full 2010 guideline implementation. We also evaluate the cost and survival impact of imposing an additional “equity” constraint—i.e., that all members of the cohort at any given time are provided the same treatment program. Finally, we use sensitivity analyses to examine the efficacy and cost input parameters necessary to change the conclusions.
When reporting clinical outcomes alone (per-person life expectancy), we provide undiscounted results. When clinical and economic results are used to create cost-effectiveness ratios, we adhere to established convention in discounting both at 3% per annum [12] ; cost-effectiveness ratios are reported in US dollars per year of life saved (dollars/YLS) (See Text S1 for details). We conduct an “incremental” assessment of economic costs and health benefits, as recommended by the US Panel on Cost-Effectiveness in Health and Medicine [12] . Cost and health outcomes are estimated for all 12 strategies (as well as no ART). These are then ranked in order of increasing cost. After eliminating all “dominated” strategies (i.e., strategies that both cost more and confer fewer benefits than any combination of other strategies), we compute the ratio of incremental costs to incremental benefits for each strategy, comparing it to its next-least-costly, non-dominated alternative [12] .
Costs are converted to 2008 US dollars using the South African gross domestic product deflators and the 2008 mean exchange rate between the South African rand and the US dollar (8.23 rand = US$1) [13] , [14] . Guided by the recommendations of the WHO Commission on Macroeconomics and Health, we consider interventions to be cost-effective in a given country if their cost-effectiveness ratio is less than 3 times the national per capita gross domestic product (South African 2008 gross domestic product = US$5,700) [14] .
The CEPAC-International Model
The CEPAC-International model simulates the progression of disease in a hypothetical cohort of patients infected with HIV as a sequence of monthly transitions between health states. Health states are defined to be clinically and economically representative of the disease course and are stratified by current CD4 count, current HIV RNA level, and history of opportunistic disease. A graphical representation of a patient trace in South Africa is presented in Figure S2 , illustrating CD4 cell count, HIV RNA, and clinical events, including tuberculosis, over a hypothetical patient's lifetime. We are careful to distinguish in the model “actual” CD4 cell count and HIV RNA—i.e., the underlying immunologic and virologic state, regardless of whether they are measured by a laboratory test—from “observed” CD4 cell count and HIV RNA—that which is measured by a test and upon which clinical decisions can be made. Actual CD4 cell count determines the frequency of opportunistic diseases, while ART influences actual HIV RNA levels and CD4 cell counts. Health states therefore reflect the underlying disease process, and clinical decisions (ART initiation or switch) are based on observed factors such as presentation with an opportunistic disease, or CD4 count, if monitoring is available. Reflecting standards of care in most sub-Saharan African nations, HIV RNA monitoring is assumed to be unavailable [1] . Patients are followed from entry into HIV care through death.
In strategies without available CD4 monitoring, decisions regarding ART initiation and switching are made based upon observation of any of the following severe opportunistic diseases representative of WHO stage III/IV disease: severe bacterial infection, severe fungal infection, tuberculosis, toxoplasmosis, nontuberculous mycobacteriosis, Pneumocystis jiroveci pneumonia, or other WHO stage IV defining diseases. Two mild opportunistic diseases (fungal and other) result in resource utilization but no changes in the ART decision-making process. Patients die in the model from an acute event (e.g., an opportunistic disease or a drug-related toxicity), from chronic HIV disease, or at South African age- and sex-adjusted background mortality rates [15] .
The frequency of clinical and laboratory assessments in the model is user-defined. For this analysis, we have chosen clinical assessments to occur every 3 mo; in strategies where CD4 counts are available, they are modeled as being performed biannually. ART is initiated when one of two criteria is met: falling below a defined CD4 count threshold or the development of WHO III/IV disease (i.e., severe opportunistic disease). Effective ART in the model results in actual virologic suppression (independent of gender), a CD4 count increase, and a CD4-independent reduction in risk of opportunistic diseases and chronic AIDS death [16] – [19] . Because HIV RNA monitoring is unavailable, virologic failure on an antiretroviral regimen is itself not detected. However, the impact of virologic failure is ultimately observed when it manifests with immunologic dysfunction through either a documented opportunistic disease or a CD4 decline that is revealed by laboratory testing. Six months after ART initiation, observed treatment failure is defined as meeting any one of the following three criteria: the development of a severe opportunistic disease, observation of a 50% decline from peak on-treatment CD4 count, or observation of two consecutive CD4 counts below 100 cells/μl [1] . Upon observed treatment failure, ART is switched if a subsequent regimen is available or, if not, the failed regimen is continued until death to maintain its modest decreases in the rates of opportunistic disease and death [17] , [18] . For the purposes of this analysis, we assume no treatment interruptions.
Stavudine- or tenofovir-related toxicity occurs with a one-time probability, distributed over time since drug initiation. Depending on the nature of the toxicity, toxicity results in a one-time cost and/or a duration of costs spanning the time of increased need for care. Certain types of toxicity—including lactic acidosis, lipodystropy, neuropathy, and nephrotoxicity—also result in a single drug switch to zidovudine.
Evaluating Uncertainty
To converge on stable model output, we run a simulated cohort of 1 million patients infected with HIV. Because the cohort size can be varied in the simulation—i.e., we might also simulate 2 million or 5 million patients—95% confidence intervals and standard deviations (SDs) do not adequately capture uncertainty in simulation modeling. Instead, we adhere to the guidance of the US Panel on Cost-Effectiveness in Health and Medicine for reporting uncertainty in deterministic methods [12] . We use univariate sensitivity analysis to examine the impact of variation in individual input parameters. Having identified those variables that exert the greatest influence on our conclusions, we then turn to multivariate sensitivity analyses to examine the impact of simultaneous variation in multiple parameters. This approach results in a large variety of univariate and multivariate sensitivity analyses. We report those instances in which variation of an underlying parameter value has material impact on the findings and conclusions. A more comprehensive description of relevant sensitivity analyses is provided in Texts S2 and S3 .
Input Parameters
Data sources for individual input parameters are referenced in Table 1 and in Text S1 .
Cohort characteristics
We define an ART-naïve cohort of patients with HIV in South Africa, with mean age 32.8 y [20] . We intentionally choose an initial cohort with a relatively high mean CD4 cell count of 375 cells/μl (SD, 25 cells/μl). A cohort with a lower mean CD4 cell count would not clearly demonstrate the benefits of an ART initiation threshold of CD4<350 cells/μl, as illustrated in sensitivity analyses ( Text S2 ). Over 40% of the cohort has HIV RNA>100,000 copies/ml ( Table 1 ) [21] . In the model, this ART-naïve cohort is then subject to the policies of ART initiation and drug availability as indicated by each of the 12 strategies. In the absence of ART, the model tracks the patients' natural history of disease for use in comparing the incremental clinical benefits and costs. Figure S3 illustrates the internal validation of South African data used to derive critical model input parameters such as monthly mortality and opportunistic disease incidence rates, stratified by CD4 count.
Opportunistic disease prophylaxis and ART efficacy
All patients at model entry are provided co-trimoxazole prophylaxis, conferring protection against mild and severe bacterial infections, P. jiroveci̧ and toxoplasmosis [22] , [23] . We assume a non-nucleoside reverse transcriptase inhibitor–based ART regimen that includes stavudine. This regimen results in a 24-wk virologic suppression rate of 75% with a mean 48-wk CD4 count rise of 136 cells/μl among those with suppression [19] . The monthly probability of virologic failure after 48 wk is 0.02. When stavudine is replaced with tenofovir in first-line regimens, in the absence of reliable efficacy data for a tenofovir-based regimen in resource-limited settings, we use a virologic suppression rate of 85% at 24 wk, as reported in clinical trials [24] , [25] . Despite the improved rates of virologic suppression, we want to maintain conservative assumptions with regard to CD4 benefit among those suppressed, so we use the same benefit (136 cells/μl) as that used for the stavudine-based regimen [19] . From these studies, the monthly probability of failure of tenofovir-based ART after 48 wk is 0.01 [24] , [25] .
When second-line ART is available, it is a lopinavir/ritonavir-based regimen with a 24-wk suppressive efficacy of 78%, a resultant CD4 count increase of 151 cells/μl, and a 0.03 monthly probability of virologic failure after 48 wk [16] . In sensitivity analyses, we examine the impact of improved efficacy of first-line ART associated with the use of tenofovir and the impact of alternative second-line ART efficacies ( Text S3 ).
Costs
We consider HIV-associated direct medical costs, including inpatient days, outpatient visits, medication costs, and laboratory tests, when available ( Table 1 ). Direct non-medical costs and indirect costs are excluded. Costs attributable to inpatient hospitalization resulting from an opportunistic infection are calculated as the mean cost of each inpatient day multiplied by the mean length of stay for any given opportunistic disease. Outpatient care costs include the mean cost of each visit, inclusive of standard laboratory tests and procedures. Routine care costs are stratified by CD4 cell count to account for the increased frequency of visits that may be attributable to lower CD4 cell counts ( Table 1 ). The stavudine-based first-line regimen costs US$100 per person-year (stavudine component = US$36), and the tenofovir-based regimen costs US$204 per person-year (tenofovir component = US$135) [26] ; all other first-line regimen costs are identical. Second-line ART regimens, when available, cost US$669 per person-year [26] ; CD4 count tests cost US$25 each [27] , [28] . Tenofovir, second-line ART, and CD4 monitoring costs are each varied in sensitivity analyses. | Results
Prioritization by Survival Benefits (Undiscounted)
An untreated HIV-infected South African cohort starting with a mean CD4 count of 375 cells/μl (SD, 25 cells/μl) has a mean undiscounted life expectancy of 47.9 mo. A single-line stavudine-based ART regimen, initiated on development of WHO stage III/IV disease (“reference strategy”; stavudine/WHO/one-line) increases life expectancy to 99.0 mo. Table 2 provides the projected 5-y survival and life expectancies of alternative stepwise progressions toward the 2010 WHO recommendations. Compared to stavudine/WHO/one-line (step 1), 5-y survival is largest (87% survival) with the addition of CD4 count availability and ART initiation at CD4<350 cells/μl (stavudine/<350/μl/one-line). In this initial step, tenofovir/WHO/one-line (66%), stavudine/<200/μl/one-line (80%), or stavudine/WHO/two-lines (66%) each yield lower projected short-term survival. Considering each of the guideline components, stavudine/<350/μl/one-line also produces the greatest anticipated life expectancy increase, Δ25.3 mo. With stavudine/<350/μl/one-line (step 2), adding a second-line regimen results in the next largest life expectancy increase (stavudine/<350/μl/two-lines, Δ53.3 mo). The final step replaces stavudine with tenofovir (tenofovir/<350/μl/two-lines, Δ16.0 mo, step 3), resulting in a comprehensive strategy concordant with the 2010 WHO guidelines, a 5-y survival of 91%, and a projected life expectancy of 193.6 mo ( Table 2 ).
Model-generated survival curves are provided for no ART, the reference strategy, and the three steps in Table 2 , which act stepwise to maximize life expectancy ( Figure 2 ). Marked differences in early survival are attributable to earlier ART initiation thresholds; differences in survival later in the disease course are associated with second-line ART availability.
Prioritization by Cost-Effectiveness
Incremental cost-effectiveness analysis ( Table 3 ) reveals three non-dominated strategies (i.e., strategies that attain a given survival level by the least costly means): (1) stavudine/<350/μl/one-line (US$610/YLS), (2) tenofovir/<350/μl/one-line (US$1,140/YLS), and (3) tenofovir/<350/μl/two-lines (US$2,370/YLS). All other strategies are “dominated”—i.e., they are more expensive and confer less survival benefit than some other combination of strategies. Figure 3 (upper panel) maps the 13 strategies on a discounted cost and life expectancy plane. The line connecting the non-dominated strategies designates the “efficient frontier,” which represents both the least expensive way to attain a given survival and the maximum achievable survival for any given cost [12] .
Thus, a country with a current stavudine/WHO/one-line policy ( Figure 3 , lower panel) could switch to a tenofovir/<350/μl/one-line policy (open arrow) and thereby simultaneously decrease projected per-person lifetime costs and improve survival. Similarly, a country with a stavudine/<200/μl/one-line policy could decrease per-person costs and also improve outcomes by changing to a stavudine/<350/μl/one-line policy (solid arrowhead). Countries with a stavudine/<200/μl/two-lines policy would require increased per-person expenditures to achieve the survival benefits associated with tenofovir/<350/μl/two-lines, as suggested in the revised WHO guidelines (dotted arrow).
Evaluating the Cost of Equity
Of the three efficient programs ( Table 3 ; Figure 3 ), tenofovir/<350/μl/one-line has a projected per-person lifetime cost of US$6,870, and tenofovir/<350/μl/two-lines has a projected lifetime cost of US$12,820. An HIV program budget that allows for a per-person cost between US$6,870 and US$12,820 might be achieved in several ways; two are illustrative. The first would be to proportionately divide the cohort between two of the programs along the efficient frontier, so that part of the cohort receives tenofovir/<350/μl/one-line and the rest receives tenofovir/<350/μl/two-lines. An alternative would be to provide everyone in the cohort a third program—one that lies below the efficient frontier. The opportunity cost (e.g., the anticipated net loss in discounted life expectancy associated with an alternative strategy choice) of any non-efficient strategy may be quantified by measuring its vertical distance from the efficient frontier. To illustrate this opportunity cost, we take an arbitrary affordability threshold of US$11,500 per person. In the example of a program that can afford no more than US$11,500 per person (stavudine/<200/μl/two-lines; Figure 3 , lower panel), the opportunity cost of uniformity in care (“equity”) is 14.5 mo per person of survival (shown by the bracket in Figure 3 , lower panel).
Sensitivity Analyses
Clinical parameters
In sensitivity analyses, we examine changes in clinical input data required to alter the stepwise ordering of program additions. Modest reductions in the mean CD4 count of the cohort (to 250 cells/μl) show decreased clinical benefits to earlier ART initiation but no substantial changes in cost-effectiveness. When the mean CD4 count of the cohort is less than 100 cells/μl, the benefits of a policy change to earlier ART initiation are largely irrelevant ( Text S2 ). This is because the majority of the cohort is already ART-eligible regardless of the initiation criterion (WHO stage III/IV disease, CD4<200 cells/μl, or CD4<350 cells/μl). Although CD4 monitoring still improves cohort survival compared to clinically based ART initiation, in populations with mean CD4 counts far below the policy-relevant ART initiation criteria, the addition of a second-line regimen becomes the most clinically beneficial intervention. For the anticipated life expectancy benefits of tenofovir/WHO/one-line to exceed those expected with stavudine/<350/μl/one-line, replacement of stavudine with tenofovir would have to increase the 24-wk suppressive efficacy from 85% to 95% and simultaneously decrease the monthly probability of later virologic failure by 50% (from 0.01 to 0.005) ( Text S3 ) [24] . Second-line ART maintains its position in the stepwise order (step 2) as long as its 24-wk viral suppression rate remains between 40% and 88%, even with a 3-fold increase in the rate of late failure when efficacy decreased to 40% ( Text S3 ). Increasing stavudine toxicity by 2-fold alters life expectancy estimates by less than 1 mo and does not change the recommended stepwise additions ( Text S3 ). Similarly, changes in the gender distribution of the cohort have little impact on the results ( Text S3 ).
Cost parameters
Holding efficacy constant, results are very sensitive to the price of tenofovir; a decrease in the cost of tenofovir from US$135 to US$51 per person per year would make tenofovir both more effective and less costly than stavudine. Results are less sensitive to the costs of second-line regimens (15% of base case) and CD4 monitoring (three times base case), neither of which produced meaningful changes in cost-effectiveness results ( Text S2 ). In two-way sensitivity analyses, where the cost of tenofovir is decreased and its efficacy increased, tenofovir/<350/μl/one-line dominates stavudine/<350/μl/one-line when the tenofovir regimen costs are US$153 annually (75% of the base case) and its 24-wk suppressive efficacy is 90% (5% increase from the base case).
Additional sensitivity analyses
Further sensitivity analyses are detailed in the Texts S2 and S3 . In Text S2 , we present the 1- through 5-y survival rates for all 12 strategies examined, as well as the survival curves of the stepwise strategies selected on a 5-y, rather than a 10-y, horizon ( Figure S4 ). Text S2 also provides the details of analyses under conditions of alternative mean CD4 counts for the cohort and alternative costs of both second-line regimens and CD4 monitoring. Further analyses ( Text S3 ) offer additional comprehensive analytic variations in cohort gender distributions, ART initiation criteria, first- and second-line ART efficacies, stavudine-related toxicities, and costs. Within plausible ranges, these sensitivity analyses, other than those reported above, had little impact on clinical- or policy-relevant results. | Discussion
The new 2010 WHO ART guidelines aim to promote public health interventions that “secure the greatest likelihood of survival and quality of life for the greatest number” of individuals with HIV. The reported guiding principles in the revision process include: (1) do no harm, (2) ensure access and equity, (3) promote quality and efficiency, and (4) ensure sustainability. Motivated by these tenets, the new guidelines recommend a single CD4-based ART initiation criterion for all populations, a switch from stavudine to tenofovir, and universally available second-line regimens [2] . We find that in settings where immediate implementation of all of the new WHO treatment guidelines is currently not feasible, ART initiation at CD4<350 cells/μl provides the greatest short- and long-term survival advantage and is very cost-effective. In countries that are already initiating stavudine at CD4<350 cells/μl and have access to CD4 monitoring, switching from stavudine to tenofovir increases survival and is also cost-effective. Access to second-line ART provides more clinical benefit than access to tenofovir but at substantially greater costs.
The additional outlays implied by the new guidelines stand in stark contrast to the resource-constrained reality encountered on the ground. Many countries are still striving to meet goals set by the now-superseded 2006 guidelines. The WHO estimates the current ART coverage rate across low- and middle-income countries to be 42% [5] , [29] . Meanwhile, the new guidelines recommend access to CD4 count monitoring, call for treatment of almost double the 3–5 million people already requiring treatment based on the previous guidelines [30] , and suggest replacement of the most widely used antiretroviral drug with one that costs nearly US$100 per patient-year more [26] . In most resource-limited settings, the relevant policy questions are: What is feasible now? and What to do first?
Based on projected short- and long-term survival and cost-effectiveness results, we identify three critical messages. First, countries with very limited resources and still only one line of ART available should focus first on access to CD4 count monitoring and ART initiation at CD4<350 cells/μl. These should be implemented before switching from stavudine to tenofovir and prior to providing second-line ART. Although advising to use stavudine in the first-line regimen—with its inherent toxicities—may be seen as conflicting with the primary WHO principle “first, do no harm,” the switch from stavudine to tenofovir is the recommendation that provides the least overall increase in survival, according to the results presented here. Initiating stavudine-based ART at CD4<350 cells/μl, compared with clinically based ART initiation, provides immediate and substantial short-term survival benefits, yields the greatest life expectancy compared to other guideline components, and is cost-effective by international standards. In cases where most patients present to care with CD4 counts far below the ART initiation threshold (e.g., CD4<100 cells/μl), a policy of earlier ART initiation is neutral at worst—both in terms of cost and clinical outcomes—as it serves only to increase life expectancy among patients with less advanced disease.
Second, countries with currently one line of ART available but more resources should ensure access to CD4 count monitoring with ART initiation at CD4<350 cells/μl and then switch from stavudine to tenofovir, before making second-line ART available. Indeed, some countries have already responded to the 2010 WHO guidelines and have made plans to phase out stavudine [31] . Reductions in the price of tenofovir could resolve the ongoing debate surrounding the role for stavudine in resource-limited settings. At an annual cost of US$51, tenofovir would be both less costly and more effective than stavudine.
Third, in countries with sufficient budgets to provide second-line ART, it is neither effective nor cost-effective to maintain stavudine in first-line regimens. Second-line ART may offer additional efficiencies by decreasing the prevalence of resistant virus and leaving future drug regimen options available.
Once countries have the capacity to provide early ART initiation, tenofovir, and second-line regimens, there will be additional clinical and policy questions. Policy makers will be addressing what to do upon second-line failure; optimal third-line regimens will be in question. Expanded ART regimen availability leads to clinical need for timely ART switches and forces the issue of HIV RNA laboratory availability. Finally, timely ART initiation is currently limited by late presentation to care [32] , [33] . Concurrent with scaling up to achieve the 2010 WHO ART guidelines, there should be a concerted effort to achieve the 2007 WHO HIV screening guidelines [34] ; without earlier case detection, a policy of ART initiation at CD4<350 cells/μl will never be effectively realized.
It is important to highlight that full and immediate implementation of the comprehensive set of new guidelines is cost-effective by South African standards. But, while it is helpful to critically examine the survival and economic efficiency of alternative programmatic choices, “cost-effective” does not mean “affordable.” In the setting of clear budget constraints, the question of affordability may conflict with the political imperative that all persons receive the same care package. In this case, prioritization of equity over efficiency decreases mean life expectancy—sometimes by more than 1 y per person—for the same healthcare expenditure ( Figure 3 , lower panel).
This analysis has several limitations. We report results from a cohort of HIV-infected individuals initiating ART. Although we believe the overall results would be consistent, this analysis does not specifically address ART programs with patients already in alternative stages of care, including some on first-line regimens, some on second-line regimens, and some who have previously accumulated drug-related toxicities. Such diversity within a cohort would require more individualized analyses. Additionally, a full budget impact analysis would be required to examine the number of patients in need of care, and to project the implications of each component of the WHO recommendation on program budgets over alternative time horizons.
Despite its limitations, this analysis represents the only systematic, scientific effort we are aware of that marshals the evidence base in support of implementing the WHO guidelines. The most unfortunate outcome upon release of the revised WHO guidelines would be either their complete dismissal on cost grounds alone, or the execution of more expensive—though easier to implement—interventions that offer less overall health benefit than other interventions.
In cases where the simultaneous implementation of all components of the 2010 WHO ART guidelines is beyond the reach of programs or countries, important prioritization questions emerge. This analysis suggests that CD4 count monitoring and ART initiation at CD4<350 cells/μl are the critical initial priorities. Replacing stavudine with tenofovir would further increase survival and would also be cost-effective. Adding a second-line ART regimen would provide large survival benefits, but with substantial increases in the necessary budgets. | ICMJE criteria for authorship read and met: RPW RW ALC ADP SBL XA AWS KAF. Agree with the manuscript's results and conclusions: RPW RW ALC ADP SBL XA AWS KAF. Designed the experiments/the study: RPW RW. Analyzed the data: RPW ALC SBL KAF. Collected data/did experiments for the study: RPW XA. Wrote the first draft of the paper: RPW. Contributed to the writing of the paper: RW ALC ADP SBL XA KAF. Contributed to analysis and interpretation of results: ALC ADP KAF. Developed the model: AWS.
Rochelle Walensky and colleagues use a model-based analysis to examine which of the 2010 WHO antiretroviral therapy guidelines should be implemented first in resource-limited settings by ranking them according to survival, cost-effectiveness, and equity.
Background
The new 2010 World Health Organization (WHO) HIV treatment guidelines recommend earlier antiretroviral therapy (ART) initiation (CD4<350 cells/μl instead of CD4<200 cells/μl), multiple sequential ART regimens, and replacement of first-line stavudine with tenofovir. This paper considers what to do first in resource-limited settings where immediate implementation of all of the WHO recommendations is not feasible.
Methods and Findings
We use a mathematical model and local input data to project clinical and economic outcomes in a South African HIV-infected cohort (mean age = 32.8 y, mean CD4 = 375/μl). For the reference strategy, we assume that all patients initiate stavudine-based ART with WHO stage III/IV disease and receive one line of ART (stavudine/WHO/one-line). We rank—in survival, cost-effectiveness, and equity terms—all 12 possible combinations of the following: (1) stavudine replacement with tenofovir, (2) ART initiation (by WHO stage, CD4<200 cells/μl, or CD4<350 cells/μl), and (3) one or two regimens, or lines, of available ART. Projected life expectancy for the reference strategy is 99.0 mo. Considering each of the guideline components separately, 5-y survival is maximized with ART initiation at CD4<350 cells/μl (stavudine/<350/μl/one-line, 87% survival) compared with stavudine/WHO/two-lines (66%) and tenofovir/WHO/one-line (66%). The greatest life expectancies are achieved via the following stepwise programmatic additions: stavudine/<350/μl/one-line (124.3 mo), stavudine/<350/μl/two-lines (177.6 mo), and tenofovir/<350/μl/two-lines (193.6 mo). Three program combinations are economically efficient: stavudine/<350/μl/one-line (cost-effectiveness ratio, US$610/years of life saved [YLS]), tenofovir/<350/μl/one-line (US$1,140/YLS), and tenofovir/<350/μl/two-lines (US$2,370/YLS).
Conclusions
In settings where immediate implementation of all of the new WHO treatment guidelines is not feasible, ART initiation at CD4<350 cells/μl provides the greatest short- and long-term survival advantage and is highly cost-effective.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Since 1981, acquired immunodeficiency syndrome (AIDS) has killed more than 25 million people, and about 33 million people (30 million of them in low- and middle-income countries) are now infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV destroys immune system cells (including CD4 cells, a type of lymphocyte), leaving infected individuals susceptible to other infections (so-called opportunistic infections). Early in the AIDS epidemic, most people with HIV died within 10 years of infection. Then, in 1996, highly active antiretroviral therapy (ART)—a combination of several powerful antiretroviral drugs—was developed. Now, in resource-rich countries, clinicians care for people with HIV by prescribing ART regimens tailored to each individual's needs. They also regularly measure the amount of virus in their patients' blood, test for antiretroviral-resistant viruses, and monitor the health of their patients' immune systems through regular CD4 cell counts. As a result, the life expectancy of patients with HIV in developed countries has dramatically improved.
Why Was This Study Done?
Initially, resource-limited countries could not afford to provide ART for their populations, and the life expectancy of HIV-positive people remained low. Now, through the concerted efforts of governments, the World Health Organization (WHO), and other international agencies, more than a third of the people in low- and middle-income countries who need ART are receiving it. However, many without access are still in need of ART, and ART programs in developing countries follow a public-health approach rather than an individualized approach. That is, drug regimens, clinical decision-making, and disease monitoring are all standardized and follow recommendations in the 2006 WHO ART guidelines. This year (2010), these guidelines were revised. The guidelines now recommend the following: earlier ART initiation—when the CD4 count falls below 350/μl of blood, instead of below 200/μl as in the 2006 guidelines; the provision of sequential ART regimens instead of a single regimen; and the replacement of the antiretroviral drug stavudine with tenofovir, a less toxic but more expensive drug, in first-line ART regimens. However, many resource-limited countries are still struggling to implement the 2006 guidelines, so which of these new recommendations should be prioritized? Here, the researchers use a mathematical model to address this question.
What Did the Researchers Do and Find?
The Cost Effectiveness of AIDS Complications (CEPAC)–International model simulates the natural history and treatment of HIV disease. The researchers entered South African clinical and cost data for HIV treatment into this model and then used it to project survival and costs in a hypothetical group of South African HIV-positive patients under alternative guideline prioritization scenarios. The reference strategy for the simulations (denoted as “stavudine/WHO/one-line”) assumed that patients (with a mean CD4 count of 375/μl) began a single stavudine-based ART regimen when they developed WHO stage III/IV HIV disease (i.e., when patients develop multiple opportunistic infections such as tuberculosis and pneumonia). When the new guideline recommendations were considered separately, ART initiation at CD4<350/μl (stavudine/<350/μl/one-line) maximized five-year survival. Stepwise adjustment from the reference strategy (which had a life expectancy 99.0 months) through strategies of stavudine/<350/μl/one-line (a projected life expectancy of 124.3 months), stavudine/<350/μl/two-lines (177.6 months), and tenofovir/<350/μl/two-lines (193.6 months) produced the greatest improvements in life expectancy. Finally, strategies of stavudine/<350/μl/one-line, tenofovir/<350/μl/one-line, and tenofovir/<350μl/two-lines produced incremental cost-effectiveness ratios of US$610, US$1,140, and US$2,370 per year of life saved, respectively.
What Do These Findings Mean?
As with all mathematical models, the accuracy of these findings are dependent on the assumptions included in the model and on the data populating it. Nevertheless, these findings suggest that, where resources are limited and immediate implementation of all the new WHO recommendations is impossible, ART initiation at a CD4 count of less than 350/μl would provide the greatest survival advantage and would be very cost-effective. In countries that are already initiating ART at this threshold and that have access to CD4 monitoring, a switch from stavudine to tenofovir would further increase survival and would also be cost-effective. Finally, although access to second-line ART regimen would provide more clinical benefits than access to tenofovir, the cost of this change in strategy would be substantially greater. Importantly, these findings should help to avoid the complete dismissal of the revised WHO guidelines on the basis of cost and should help policy makers adjust their ART program strategies to maximize their clinical benefits and cost effectiveness.
Additional Information
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PMC3014094 | 18939950 | Materials and Methods
Constructs
The GnTI CTS region was amplified with primers NtGnTI-1/-2 (for primer sequences see Table S1 ) from N. tabacum GnTI complementary DNA (cDNA) (47) using GoTaq DNA Polymerase (Promega) and inserted into Hin dIII/ Sal I-linearized cloning vector puc19 to produce vector puc19Nt1. The mRFP coding sequence was amplified with Turbo Pfu polymerase (Stratagene) from pRSETB-mRFP (kindly provided by Roger Tsien, UCSD, CA, USA) using primers mRFP1/2 and cloned into the Sal I/ Bam HI site of puc19Nt1 (puc19Nt1-mRFP). The GnTI-CTS-mRFP coding sequence was excised with Xba I/ Bam HI and ligated into the binary plant expression vector pPT2 (31) . In pPT2, the expression of the DNA sequences is under the control of the cauliflower mosaic virus 35 S promoter. The CT-mRFP and T-mRFP constructs were generated by ligation of overlapping primers NtGnTI-11F/-11R and NtGnTI-12F/-12R, respectively, into Xba I/ Kpn I-digested vector pPFH3, which is a derivative of pPT2 (16) . To construct the tail-mutated GnTI forms, the cytoplasmic region was excised from GnTI-CTS-mRFP with Xba I/ Kpn I, and overlapping primers NtC3F/R (GnTI-C RAA TS-mRFP), NtC4F/R (GnTI-C AAR TS-mRFP) and NtC8F/R (GnTI-C AAA TS-mRFP) were inserted.
To generate the GMII constructs, the C 10 TS region was amplified by polymerase chain reaction (PCR) from the A. thaliana GMII cDNA (14) using oligos Ath-MII-40F/-44R, the C 10AAA TS-region was amplified with Ath-MII-41F/-44R and the C 10AKA TS-region was amplified with Ath-MII-48F/-44R. The C 10 T region was amplified using primers Ath-MII-40F/-42R and C 10AAA T with Ath-MII-41F/-42R. All PCR products were Xba I/ Bam HI digested and inserted into p20F (48) to generate the GFP fusion constructs (p20F-GMII-C 10 TS, -GMII-C 10AAA TS, -GMII-C 10AKA TS and -GMII-C 10AAA T) or into p23 (48) to generate the binary vector for expression of the CFP fusion (p23-GMII-C 10 T).
The glycoprotein–GFP vectors were generated by PCR amplification of a human IgG1 heavy chain fragment from vector pTRAp-2G12-Ds (49) using primers Fc-1F/-1R. The PCR product was Bam HI/ Bgl II digested and ligated into Bam HI-linearized vector p20F to create vector p20F-Fc. GnTI-CTS was amplified using primers NtGnTI-13F/-16R and cloned into the Xba I/ Bam HI site of p20F-Fc. The cytoplasmic-domain-swap vectors were generated by Xba I/ Kpn I excision of the GnTI-C-tail and insertion of NtC8F/R (GnTI-C AAA TS) and XT-C1F/R overlapping primers (XylT-C-GnTI-TS) or the PCR product from A. thaliana genomic DNA using primers GCSI-1/-2 (GCSI-C-GnTI-TS). GCSI-CTS-GFPglyc was generated by PCR amplification from A. thaliana genomic DNA using oligos GCSI-1/-3 and cloned into the Xba I/ Bam HI site of p20F-Fc. GnTI-C-GCSI-TS-GFPglyc was generated by replacing the GnTI-TS region with a PCR fragment containing the coding sequence for the GCSI TS region obtained by using primers GCSI-4/-3.
The full-length GnTI-GFP expression vector was generated by PCR using primers NtGnTI-13F/-15R and vector pNT-GnTI-Mut as template and inserted into the Xba I/ Bam HI site of p20F. Vector pNT-GnTI-Mut was generated by site-directed mutagenesis of the Kpn I site at position 1272 of the tobacco GnTI coding sequence in vector p5/2 (47) using a quick change kit (Stratagene) and primers NtGnTI-14F/-R. Subsequently, the cytoplasmic tail was removed and the tail-mutated form inserted as described for the mRFP and glycoprotein–GFP fusion constructs to generate GnTI AAA -GFP.
To generate the mRFP-HDEL vector (pmRFP-HDEL), mRFP was amplified using primers mRFP5-F/6-R and ligated into Bam HI/ Sal I-digested vector p20ChisSP (unpublished data), which is derived from pPT2 and contains a chitinase signal peptide.
Plant material and transient expression system
Four-week-old to 5-week-old N. benthamiana plants were used for Agrobacterium tumefaciens (strain UIA143)-mediated transient expression of indicated constructs using the agroinfiltration technique described previously ( 50 , 51 ). The optical density (OD 600 ) of the bacterial suspensions used for plant transformations was 0.03 for all constructs except for ST–GFP/mRFP 0.05, ERD2-GFP 0.08, full-length GnTI-GFP and GnTI AAA -GFP 0.05. Constructs expressed in Sar1p experiments were infiltrated at the following OD 600 : ST–GFP 0.2, ERD2-GFP 0.2, Sar1-YFP 0.05, untagged Sar1[H74L]p 0.03 and GnTI-CTS-GFP 0.2.
Sampling and imaging
Sections of transformed leaves were analysed 2–4 days postinfiltration (dpi) on a Leica TCS SP2 or Zeiss LSM 510 confocal microscope both equipped with ×63 and ×100 oil immersion objectives using appropriate filters or spectral selections. Images presented in this manuscript were taken at 2 dpi, unless stated otherwise. For ease of imaging motile structures, leaf segments were incubated with n-ethylmaleimide (Sigma; stock solution, 1 m in dimethyl sulphoxide) used at a concentration of 50 m m for 10 min before confocal analysis (27) . The imaging settings were identical throughout experiments so that the images were comparable. To exclude the possibility of cross-talk between fluorophores, appropriate controls were performed. Post-acquisition image processing was performed in I mage J and A dobe P hotoshop CS.
Imaging with Zeiss microscope: GFP alone or in combination with YFP was imaged as described recently (27) . For imaging mRFP constructs, mRFP was excited with the 543-nm helium/neon laser line, and fluorescence was detected using a 488/543-nm dichroic beam splitter (DBS) and 585/615-nm band pass filter (BPF) in the single-track facility of the microscope. YFP was monitored using a 488-nm argon laser line; fluorescence was detected using a 488/543-nm DBS and a 505/550-nm BPF. For imaging coexpression of mRFP and YFP constructs, the 488-nm argon laser line for YFP and the 543-nm helium/neon laser line for mRFP were used alternately with line switching using the multi-track facility of the microscope. Fluorescence was detected using a 488/543-nm DBS and a 505/550-nm BPF for YFP and 585/615-nm BPF for mRFP. For imaging expression of GFP constructs, the 488-nm argon laser line was used to excite GFP, and fluorescence was monitored with a 488/543-nm DBS and 505/530-nm BPF in the single-track facility of the microscope. For imaging expression of GFP in combination with mRFP, the 488-nm argon laser line for GFP and the 543-nm helium/neon laser line for mRFP were used alternately with line switching using the multi-track facility of the microscope. Fluorescence was detected using a 488/543-nm DBS and a 505/530-nm BPF for GFP and 585/615-nm BPF for mRFP.
Imaging with Leica microscope: GFP alone was imaged using a 488-nm argon laser line, and emission was recorded from 500 to 535 nm. mRFP alone was excited with a 543-nm helium/neon laser line, and emission was collected at 600–630 nm. CFP alone was imaged using a 458-nm argon laser line, and emission was detected at 465–495 nm. YFP was excited with a 514-nm helium/neon laser line and detected at 520–600 nm. For imaging GFP in combination with YFP, GFP was imaged using a 458-nm argon laser line and its emission was recorded from 475 to 510 nm, while YFP was excited using a 514-nm argon laser line and its emission was collected at 560–615 nm. Images were acquired separately and superimposed in I mage J. Dual-color imaging of cells expressing both GFP and mRFP was performed simultaneously using a 488-nm argon laser line and the 543-nm helium/neon laser line. GFP emission was recorded at 500–535 nm, whereas mRFP fluorescence was detected at 600–630 nm. For imaging cells coexpressing GFP and CFP, scans optimized for each fluorophore were collected separately and superimposed afterwards. GFP was excited with a 514-nm argon laser line and emission detected at 520–540 nm. CFP was excited with a 458-nm argon laser line, and signal was collected at 465–495 nm. When CFP and mRFP were expressed together, imaging was performed using a 458-nm argon laser line and a 543-nm helium/neon laser line. Fluorescence signals were monitored separately, and CFP emission was collected at 465–495 nm, whereas mRFP was recorded at 600–630 nm.
For quantifying the number of Golgi stacks, 4-μm thick three-dimensional z-stack images (no cropping) were captured with a ×40 oil immersion objective of a Zeiss LSM 510 using identical settings throughout the experiment. Per experiment, eight z-stacks were analysed (three experiments in total). In I mage J, a maximum intensity projection of each stack was made. To separate touching Golgi stacks as different objects, projections were thresholded manually, converted into binary images and further segmented using the ‘Watershed’ segmentation tool. Finally, the number of Golgi stacks was determined using ‘Analyse Particles’ with the following parameters input: size ‘0–3.643’ (area of largest Golgi stack measured in square pixels), circularity ‘0–1.0’ default, and show ‘outline’.
Protease protection assays
Microsomal fractions were prepared from 500 mg infiltrated N. benthamiana leaves as described previously (52) . Pellets were resuspended in 100 μL 25 m m Tris/HCl (pH 7.2) and 0.5 m m dithioerythritol (DTE), and 8.5 μL was incubated in the presence or absence of 1% Triton-X-100 with 0.5 μg trypsin (Sigma) for 60 min at 37°C. To stop the reaction, 20 μg soybean trypsin inhibitor (Sigma) was added and samples were subjected to SDS–PAGE and immunoblotting using rabbit anti-RFP (US Biological) or mouse anti-GFP (Roche) antibodies for detection.
Purification of CTS-GFP fusion proteins
Purification of the CTS-GFP fusion proteins (as listed in Figure 1C ) was performed using a modified version of a previously described protocol for antibody purification from infiltrated N. benthamiana leaves (53) . Briefly, leaves of 6-week-old plants were infiltrated with agrobacteria diluted with infiltration buffer to an OD 600 of 0.2. For purification, 800 mg infiltrated leaf material was homogenized in liquid nitrogen, resuspended in 8 mL of precooled extraction buffer [1× PBS buffer, pH 7.2, 1% Triton-X-100 and protease inhibitor cocktail (Sigma)] and incubated for 15 min at 4°C. Insoluble material was removed by several centrifugation steps at 5000 × g for 10 min at 4°C. The clear supernatant was incubated with 40 μL rProtein A–Sepharose (GE Healthcare), and the fusion proteins were eluted by boiling in Laemmli sample buffer. Protein gel blot analysis of crude extracts or purified proteins was performed using anti-HRP antibodies as described previously (54) , mouse anti-GFP antibodies (Roche) or peroxidase-conjugated concanavalin A (Sigma).
LC-ESI-MS analysis of tryptic glycopeptides
To analyse the N -glycans present on the CTS-GFPglyc glycoreporters, purified protein (500 ng) was separated by SDS–PAGE (10%) under reducing conditions, and polypeptides were detected by Coomassie Brilliant Blue staining. The corresponding band was excised from the gel, destained, carbamidomethylated, in-gel trypsin digested and analysed by LC-ESI-MS as described recently (53) . | Results
Lumenal sequences of GnTI are not required for Golgi localization
We have previously shown that the 77 N-terminal amino acids (CTS region) of N. tabacum GnTI consisting of a short cytoplasmic tail (11 amino acids), a single transmembrane domain (18 amino acids) and a lumenal stem region (48 amino acids) are sufficient for targeting of a reporter protein to the Golgi apparatus in plants (12) . This finding was confirmed by others, who further truncated the putative stem region to seven residues and found that the remaining 36 N-terminal amino acids were still sufficient to target GFP to the Golgi apparatus (15) . To test the relative contribution of the domains to in vivo localization in more detail, we generated constructs, where the GnTI CTS region was fused to the N-terminal part of monomeric red fluorescent protein (mRFP) and made deletions thereof ( Figure 1A ). These constructs were used for transient expression in Nicotiana benthamiana leaf epidermal cells. GnTI-CTS-mRFP was found predominantly in small motile bodies, resembling the Golgi apparatus ( Figure 2A ). The reticulate ER network was stained to a much lesser extent. This finding was confirmed by colocalization with the well-characterized ER/Golgi marker ERD2-GFP ( 2 , 4 , 27 ) ( Figure 2A ) and with the well-known Golgi marker ST–GFP ( 2 , 27 ) (data not shown) and is consistent with previous observations ( 12 , 15 ).
To analyse the effect of complete removal of the lumenal stem region on targeting of GnTI, the cytoplasmic tail and transmembrane domain were fused to mRFP (GnTI-CT-mRFP) and transiently expressed in N. benthamiana . GnTI-CT-mRFP was localized to punctate structures ( Figure 2B ), which colocalized with ERD2-GFP and resembled the structures observed with GnTI-CTS-mRFP. Finally, we removed the cytoplasmic tail of GnTI completely and analysed the distribution of the transmembrane domain-mRFP fusion (GnTI-T-mRFP). This fusion protein marked the cytoplasm and punctate structures of mostly unknown nature while only partially colocalizing with ERD2-GFP ( Figure 2C ). These data suggest that the cytoplasmic tail of GnTI is a major determinant of proper targeting to the Golgi. In this respect, GnTI is very similar to other plant N -glycosylation enzymes like A. thaliana Golgi α-mannosidase II (GMII) (14) and β1,2-xylosyltransferase (XylT) (13) , where the transmembrane domain alone failed to provide Golgi localization of reporter constructs.
A single arginine residue proximal to the transmembrane domain of GnTI is sufficient for Golgi localization
To further investigate the contribution of the cytoplasmic tail to Golgi localization, we decided to mutate residues, which might be involved in ER-to-Golgi transport or Golgi retention of GnTI. The cytoplasmic tail of GnTI (C: M R GY K FCCDF R , basic amino acid residues are shown in bold) contains three basic amino acids, which could promote ER exit, as demonstrated for mammalian glycosylation enzymes (23) . To test this hypothesis, these basic amino acids were replaced by alanine residues and the CTS-mRFP fusion proteins with different mutated cytoplasmic tails (C RAA TS, C AAR TS and C AAA TS; Figure 1A ) were expressed transiently in N. benthamiana . The chimeric protein lacking all three basic amino acids in the cytoplasmic tail (C AAA TS) displayed a predominant ER steady-state location with little Golgi labelling ( Figure 3A ), demonstrating their importance for efficient ER exit. In contrast, the construct harbouring only the arginine proximal to the transmembrane domain (C AAR TS) was targeted to the Golgi apparatus quite similar to the wild-type form (GnTI-CTS-mRFP) ( Figure 3B ), thus revealing that the presence of this residue is sufficient for proper Golgi targeting. Interestingly, the more distal arginine was less effective in this respect. The corresponding fusion protein (C RAA TS) was detected both in the ER and in the Golgi apparatus ( Figure 3C ), with a more pronounced labelling of the ER when compared with GnTI-CTS-mRFP ( Figure 2A ). Colocalization of the tail-mutated proteins with ST–GFP, GFP-HDEL or ERD2-GFP confirmed their ER and Golgi location, respectively.
To rule out that the ER accumulation of the tail-mutated form (C AAA TS) was caused by overexpression, we compared the expression levels in extracts 1–3 days after infiltration by immunoblotting using anti-red fluorescent protein (RFP) antibodies. A band of the expected size was clearly visible after 2 and 3 days for GnTI-CTS-mRFP and GnTI-C AAA TS-mRFP ( Figure S1 ), but a much longer exposure of the blot (data not shown) was required to detect this band after day 1. However, confocal microscopy data showed the typical localization pattern for these proteins already after 1 day, demonstrating that ER accumulation of GnTI-C AAA TS-mRFP is not a consequence of overexpression.
Basic amino acids in the cytoplasmic tail of other glycosylation enzymes are important for proper Golgi localization
We have previously shown that a truncated cytoplasmic tail (C 10 ) of GMII containing only 10 amino acids fused to its transmembrane domain is sufficient for Golgi targeting and retention (14) . To analyse whether the basic amino acids present in the C 10 tail of GMII play a similar role as observed for the positively charged amino acids of GnTI, chimeric proteins consisting of the C 10 tail (C 10 : MP RKR TLVVN; basic amino acid residues are shown in bold; Figure 1B ), transmembrane and stem regions were fused to GFP (GMII-C 10 TS-GFP), the basic amino acids were replaced by alanine residues and the proteins transiently expressed in N. benthamiana leaf epidermal cells. GMII-C 10AKA TS-GFP with one remaining basic amino acid displayed a Golgi distribution like GMII-C 10 TS-GFP ( Figure 4A ). Further exchange of the lysine residue (GMII-C 10AAA TS-GFP) resulted in a predominant ER steady-state localization ( Figure 4A ), as observed for GnTI-C AAA TS-mRFP. Removal of the stem region did not have any influence on the ER distribution of the tail-mutated GMII fusion protein (GMII-C 10AAA T-GFP). Near perfect colocalization of GMII-C 10 T-cyan fluorescent protein (CFP) with ST–mRFP ( Figure 4B ) as well as of GMII-C 10AAA T-GFP with the ER marker mRFP-HDEL ( Figure 4C ) and clear differences in the localization of GMII-C 10 T-CFP with GMII-C 10AAA T-GFP ( Figure 4D ) confirmed the result. Our data demonstrate that similar to GnTI, at least one basic amino acid in the cytoplasmic tail plays a key role in ER exit or Golgi retention of GMII. In line with this observation, exchange of the four basic amino acids in the cytoplasmic tail of another Golgi-located glycosyltransferase, XylT (C: MS KR NP K IL K , basic amino acid residues are shown in bold), with alanine residues led to accumulation of a GFP fusion protein in the ER ( Figure S2 ). It has been recently shown that the presence of the proximal lysine is sufficient for Golgi targeting of a XylT fusion protein (28) . Taken together, these results demonstrate the importance of individual basic amino acids in the cytoplasmic tails of plant glycosylation enzymes for ER exit or active Golgi targeting.
Removal of basic amino acids in the cytoplasmic tail of GnTI does not alter the membrane topology of the protein
It has been shown that the orientation of eukaryotic membrane proteins correlates with the charge difference of the flanking region, with the more positive portion of the protein facing the cytosol (29) . To rule out that the observed difference in targeting of GnTI-C AAA TS is because of a change in the membrane orientation of the fusion protein caused by removal of three charged residues, we analysed the topology of the wild-type and ER-retained mRFP fusion proteins by protease protection assays. Isolated microsomal fractions from infiltrated leaves were treated with trypsin in the presence or absence of Triton-X-100 and analysed by SDS–PAGE and western blot using anti-RFP antibodies ( Figure 5A ). Trypsin treatment in the presence of detergents led to the quantitative degradation of both chimeric proteins. In the absence of Triton-X-100, the two mRFP fusion proteins were equally resistant to trypsin digestion, indicating that the exchange of the three basic amino acids in the cytoplasmic tail of GnTI with alanine did not alter the membrane orientation.
To provide further evidence for correct topology of the tail-mutated GnTI protein, we generated a glycoprotein reporter by exchanging mRFP with GFP and inserting a protein fragment with a single N -glycosylation site derived from the human immunoglobulin G1 (IgG1) heavy chain between the GnTI-CTS region and the fluorescent protein ( Figure 1C ). The resulting proteins GnTI-CTS-GFPglyc and the tail-mutated form GnTI-C AAA TS-GFPglyc displayed predominantly Golgi and ER steady-state localization, respectively, when transiently expressed in N. benthamiana leaf epidermal cells ( Figure 5B ). Coexpression with their mRFP counterparts showed perfect colocalization of the wild-type proteins (GnTI-CTS-mRFP/GnTI-CTS-GFPglyc) and mutated forms (GnTI-C AAA TS-mRFP/GnTI-C AAA TS-GFPglyc), respectively, and different localization when a wild-type form was coexpressed with a mutated one (GnTI-CTS-mRFP/GnTI-C AAA TS-GFPglyc or GnTI-C AAA TS-mRFP/GnTI-CTS-GFPglyc) regardless of the used fluorescent protein tag (data not shown).
Correct topology of the chimeric proteins, which differ only in their cytoplasmic tail, will orient the chimeric protein with the N -glycosylation site facing the ER lumen. Thus, transfer of the oligosaccharide precursor can occur, while the opposite orientation with the chimeric glycoprotein reporter in the cytosol would not result in any N -glycosylated protein. Western blot detection of proteins extracted from infiltrated leaves showed a band of expected size (60.4 kDa without accounting for the N -glycan moiety; Figure 5C ) for both GnTI-CTS-GFPglyc and GnTI-C AAA TS-GFPglyc without any detectable amounts of free GFP or other degradation products. After affinity purification, a single discrete band was detectable on Coomassie-stained SDS–PAGE gels and immunoblots using anti-GFP antibodies ( Figure 5D ). Importantly, both purified proteins reacted with antibodies recognizing complex plant N -glycans (anti-HRP) and the mannose-binding lectin concanavalin A (Con A), indicating that both proteins are N -glycosylated and thus have proper orientation. Hence, we exclude that the observed difference in localization of the tail-mutated chimeric GnTI protein is triggered by a failure to acquire a type II membrane topology.
The tail-mutated GnTI fusion protein shows a quantitative block in ER exit
The observed differences in localization of the tail-mutated form of GnTI could either be the result of an impaired ER exit or because of an increased retrograde transport from the Golgi back to the ER. To test these two possibilities, we made use of the glycoprotein reporter ( Figure 1C ), where the CTS region can be exchanged and then effects on localization monitored by changes in the N -glycan profile. The presence of oligo-mannosidic structures (like Man8) attached to the glycoprotein is a hallmark of retention in the ER, while forward movement to the Golgi will lead to processing of N -glycans and the formation of complex N -glycans carrying β1,2-xylose and core α1,3-fucose residues (main glycoform: GnGnXF). The analysis of the purified chimeric GnTI-CTS-GFPglyc proteins by western blot with anti-HRP antibodies and Con A already indicated differences in their content of complex and oligo-mannosidic N -glycans. The signal with anti-HRP antibody was stronger for the wild-type form, while Con A gave the opposite result when equal amounts of protein were analysed ( Figure 5D ). To analyse the N -glycosylation pattern in detail, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) was performed on tryptic peptides of both purified proteins. In the case of GnTI-CTS-GFPglyc, the major peak was found to be the complex N -glycan structure GnGnXF ( Figure 6A ). In contrast, GnTI-C AAA TS-GFPglyc was found to contain a large quantity of the oligo-mannosidic N -glycan Man8 and only minor amounts of GnGnXF. A quantification of the relative amounts of complex and oligo-mannosidic structures revealed significant differences between the wild type (GnTI-CTS-GFPglyc) and the mutated form (GnTI-C AAA TS-GFPglyc). The complex-type content of the wild-type form amounted to 88%, whereas the mutant form contained much less (32%) ( Figure 6B ). The distribution of the glycoforms is consistent with the reduced number (33%) of GFP-labelled Golgi stacks in cells expressing the tail-mutated form ( Figure 6C ). These data reflect the difference in subcellular targeting between the two proteins and demonstrate that most of the tail-mutated protein is retained in the ER and never reaches the Golgi. Thus, our finding suggests that the complete removal of basic amino acids in the cytoplasmic tail impairs the ER exit of GnTI.
As a control for complete retention of a glycoreporter protein in the ER, we expressed a construct where the N-terminal targeting region of A. thaliana α-glucosidase I (GCSI) (15) was fused to GFP ( Figure 1C ). The N -glycans of this ER-retained glycoreporter (GCSI-CTS-GFPglyc) were exclusively of the oligo-mannosidic type (Man7–9; Figure 6A ), showing that this reporter is restricted to the ER.
The cytoplasmic tails of glycosylation enzymes are sufficient to redirect ER- and Golgi-located proteins
Because we found that the cytoplasmic tail plays a critical role for localization of GnTI, GMII and XylT, we asked whether the complete exchange of the tail would affect their steady-state localization and redirect proteins from the Golgi to the ER. First, we tested a chimeric protein where the cytoplasmic tail of GnTI was replaced by the tail of XylT ( Figure 1C ). This chimeric protein (XylT-C-GnTI-TS-GFPglyc) behaved similarly to wild-type GnTI and was found predominantly in the Golgi apparatus ( Figure 7A ), carrying mainly complex N -glycans ( Figure 6B ). However, replacement of the cytoplasmic tail of GnTI with the cytoplasmic tail of GCSI (GCSI-C-GnTI-TS-GFPglyc; Figure 1C ) resulted in relocation of the reporter protein to the ER ( Figure 7B ). The observed reticulate labelling of GCSI-C-GnTI-TS-GFPglyc was similar to that of GCSI-CTS-GFPglyc ( Figure 7D ) and comparable to previous observations (15) . Like GCSI-CTS-GFPglyc, the GCSI-C-GnTI-TS-GFPglyc fusion protein contained more than 99% of oligo-mannosidic N -glycans ( Figure 6B ).
To test whether an ER-resident protein can be transported further down the secretory pathway to the Golgi apparatus, the cytoplasmic tail of GnTI was fused to the TS region of GCSI. The chimeric GnTI-C-GCSI-TS-GFPglyc protein was detected in the Golgi apparatus ( Figure 7C ), and consistent with the observed subcellular localization, the majority of its N -glycans was of the complex type ( Figure 6B ).
GnTI with a mutated cytoplasmic tail is only partially functional in vivo
To analyse whether mutations in the cytoplasmic tail of GnTI lead to differences in the in vivo functionality because of altered localization of the enzyme, full-length wild type and mutated GnTI (C AAA ) with GFP fused to the C-terminus ( Figure 1D ) were expressed in A. thaliana cgl1 plants, which lack an active GnTI enzyme and produce only oligo-mannosidic N -glycans ( 30 , 31 ). We reasoned that a functional GnTI protein, which is targeted to the plant Golgi, would restore the formation of complex N -glycans in the transformed mutants, while the mutated form would display less complementation because of the ER retention. Transient expression in N. benthamiana leaf epidermal cells confirmed the expression of both constructs without any obvious differences in the levels of the full-length proteins and their endogenous degradation products (free GFP; Figure 8A ). Protease protection assays were performed to obtain information about the membrane orientation of the two full-length GnTI forms. Incubation of microsomal preparations from GnTI-GFP or GnTI AAA -GFP expressing N. benthamiana leaves with trypsin demonstrated a similar resistance of the two fusion proteins to proteinase treatment, whereas the presence of Triton-X-100 resulted in either case in complete degradation ( Figure 8B ). Thus, GnTI AAA -GFP displays a type II membrane orientation like the wild-type form. Moreover, the subcellular location of the full-length fusion proteins was identical to their GnTI-CTS forms ( Figure 8C and Figure S3 ), which is consistent with the previous findings that sequence motifs present in the lumenal catalytic domain do not contribute to Golgi localization of plant GnTI. Subsequently, stable A. thaliana lines were generated by floral dipping of cgl1 plants, and for each construct, 20 lines were propagated. As expected, analysis of cgl1 plants expressing full-length wild-type GnTI-GFP clearly showed significant complementation of the mutation, as evident by the presence of a strong staining signal with the anti-HRP antibody in all tested lines ( Figure 8D ). However, analysis of transgenic cgl1 plants expressing the full-length tail-mutated form of GnTI (C AAA ) resulted in a weak staining signal in most of the lines, and some lines did not show any staining. These findings were confirmed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of total N -glycans isolated from transformed cgl1 plants, which showed the presence of complex-type N -glycans for wild-type GnTI, whereas the tail-mutated form produced only oligo-mannosidic structures ( Figure S4 ). Taking these data together, we conclude that a GnTI protein, which lacks basic amino acids in its cytoplasmic tail, is at best partially functional in vivo .
Expression of GnTI can trigger Sar1p accumulation at ERES
In mammalian cells, the ER export of glycosyltransferases depends on the interaction of basic amino acids in the cytoplasmic tail with the small GTPase Sar1p (23) , which is responsible for initiation of COPII coat assembly at ERES (22) . Having established that the tail-mutated form of GnTI is significantly retained in the ER, we next wanted to determine whether the ER export of GnTI was COPII dependent. We expressed the untagged GTP-locked mutant version of Sar1p (Sar1[H74L]p) (20) together with GnTI-CTS-GFP in N. benthamiana leaf epidermal cells. As a control, we investigated the effect of the GTP-locked Sar1p mutant on the subcellular localization of ERD2-GFP ( Figure 9A ). The expression of the GTP-locked Sar1p form resulted in concentration of GnTI-CTS-GFP in the ER ( Figure 9B ), being consistent with previous observations demonstrating that Sar1[H74L]p causes the accumulation of membrane-bound Golgi marker proteins like ERD2-GFP ( Figure 9A ) or ST–GFP in the ER ( 18 – 20 ).
To test if expression of the wild-type GnTI fluorescent fusion protein could recruit Sar1p to ERES, as was shown for ERD2-GFP and ST–GFP (21) , we coexpressed GnTI-CTS-GFP with wild-type Sar1-yellow fluorescent protein (YFP). Cytoplasmic labelling was visible in cells expressing Sar1-YFP alone ( Figure 9C ), whereas coexpression of Sar1-YFP and ERD2-GFP resulted in the appearance of bright punctate structures highlighted with Sar1-YFP ( Figure 9D ) as observed previously ( 5 , 21 ). Coexpression of GnTI-CTS-GFP together with Sar1-YFP resulted in the formation of similar punctate structures ( Figure 9E ), as observed for ERD2-GFP. In contrast, expression of the tail-mutated GnTI form did not result in any appearance of punctate structures ( Figure 9F ). These data indicate that the ER-to-Golgi transport of GnTI can be blocked by coexpression with Sar1[H74L]p and suggest the involvement of basic amino acids present in the cytoplasmic tail of GnTI in Sar1p recruitment to ERES. | Discussion
The molecular mechanisms and signals for protein trafficking between the ER and Golgi apparatus are already fairly well characterized in mammals ( 22 , 23 , 32 ), whereas these processes are less well understood in plants ( 6 , 24 , 33 ). In this study, we aimed to answer the question to what extent the cytoplasmic tail of plant N -glycan processing enzymes contributes to ER exit and Golgi localization. Most of these N -glycan processing enzymes are predicted to display a type II membrane protein topology, with a short cytoplasmic tail, a single transmembrane domain and a stem or spacer region, which orients the catalytic domain into the ER or Golgi lumen (34) . Their conserved topology and ordered arrangement in an assembly line along the early secretory pathway makes these enzymes valuable tools to investigate Golgi targeting and retention mechanisms. For mammalian glycosyltransferases, localization relies on contributions from several domains including signals in the lumenal domains ( 34 – 36 ). In plants, evidence for spatial arrangement of N -glycan processing enzymes has been provided, but the signals and underlying mechanisms are not well understood (15) .
Our data from live-cell imaging demonstrate that the 29 N-terminal amino acids of N. tabacum GnTI, comprising the cytoplasmic tail and transmembrane domain, are sufficient for Golgi targeting and retention of a reporter protein. Being consistent with our data for N. tabacum GnTI, the N-terminal 31 amino acids of rabbit GnTI corresponding to the same domains were sufficient to concentrate a reporter protein in the Golgi apparatus in mammalian cells (37) . Additional deletion of the cytoplasmic tail from N. tabacum GnTI seems to affect ER membrane insertion because we found a large portion of GnTI-T-mRFP located in the cytoplasm. Although the possible occurrence of free mRFP cannot be completely excluded, this finding is in contrast to mammalian glycosyltransferases, where the deletion of the cytoplasmic tail from different glycosyltransferases did not disturb the ER membrane insertion but impaired Golgi concentration (23) . For these mammalian type II membrane proteins, it was shown that a conserved dibasic motif [RK]X[RK] present in the cytoplasmic tail proximal to the transmembrane domain is required for ER export. This motif is conserved in a large number of mammalian glycosyltransferases, including human GnTI (38) . N. tabacum GnTI contains three basic amino acids in the 11 residues of its cytoplasmic tail. We provide evidence that a single arginine residue proximal to the transmembrane border, which is strictly conserved in plant GnTI proteins ( 39 , 40 ), is sufficient for concentration of GnTI in the Golgi like it has been proposed for mammalian glycosylation enzymes (23) .
Our finding that basic amino acid residues in the cytoplasmic tail are critical for ER exit of GnTI and GMII is consistent with the observation that mutations of basic amino acids in the cytoplasmic tail of the type II membrane protein prolyl 4-hydroxylase from tobacco BY2 cells impaired its Golgi localization without affecting the transmembrane insertion and topology (25) . However, the mutant form of prolyl 4-hydroxylase, which still contained one basic amino acid in the cytoplasmic tail proximal to the putative transmembrane domain, located to the ER. This indicates that not all type II membrane proteins have the same requirements for efficient ER export. Recently, Maruyama et al. (28) expressed a fluorescent protein fusion consisting of a mutated cytoplasmic tail and the transmembrane domain of XylT in A. thaliana seeds. Three basic amino acids were replaced with non-charged threonine residues, while the lysine residue proximal to the transmembrane domain was not altered. This fusion protein was detected mainly in the Golgi, which is in line with our observation for GnTI-C AAR TS. In contrast, our XylT-C AAAA TS protein with four alanines instead of the basic amino acids showed a reticular distribution pattern resembling ER. Other Golgi-resident plant N -glycan processing enzymes like soybean Golgi α-mannosidase I (3) and A. thaliana N-acetylglucosyminyltransferase II (41) , core α1,3-fucosyltransferases (42) , α1,4-fucosyltransferase (43) and β1,3-galactosyltransferase (16) contain at least one basic amino acid in their cytoplasmic tail.
When transiently expressed in N. benthamiana leaf epidermis, a significant fraction of GnTI-C AAA TS was still transported to the Golgi as evident from the number of GFP-positive Golgi stacks and the presence of complex N -glycans on the respective glycoreporter. The residual ER-to-Golgi transport of the tail-mutated GnTI protein could be mediated by signals other than basic amino acids, as it has been proposed for GONST1 and CASP (24) , or through bulk flow. However, the tail-mutated full-length GnTI form almost completely failed to restore the N-glycosylation defect in the A. thaliana cgl1 mutant. Thus, the removal of basic amino acids from the cytoplasmic tail of GnTI leads to a highly efficient ER export block which impairs the in vivo activity of the tail-mutated protein.
Accumulation of Golgi-resident glycosylation enzymes with altered cytoplasmic tails in the ER could be caused by exclusion from anterograde transport or increased recruitment from the Golgi by retrograde transport. Our finding of increased amounts of unprocessed oligo-mannosidic N -glycans in the tail-mutated ER-located GnTI protein (GnTI-C AAA TS-GFP) is clearly in favour of the first hypothesis. In line with this, the observed failure for ER export could be the result of a loss of interaction between the cytoplasmic tail of the cargo protein and the COPII components. For mammalian glycosyltransferases, direct in vitro interaction with the small GTPase Sar1p, which initiates COPII coat assembly, has been demonstrated (23) . This interaction was lost by removal of the dibasic amino acid motif present in the cytoplasmic tail. Moreover, in vitro binding of the cytoplasmic tails of mammalian glycosylation enzymes to another COPII protein has been reported (44) . Furthermore, for mammalian cells, as well as for plants, it was found that increased glycosyltransferase or glycosidase expression resulted in Sar1p accumulation at peri-Golgi areas, presumably representing ERES ( 21 , 26 ). We also found accumulation of Sar1p at putative ERES upon coexpression with GnTI, which indicates the involvement of COPII proteins in ER exit of plant glycosyltransferases. The expression of the tail-mutated GnTI form did not result in any association of Sar1p with ERES or peri-Golgi areas, supporting the hypothesis that the basic amino acids present in the cytoplasmic tail of GnTI are involved in recruitment of Sar1p or other COPII components to ERES. Future experiments in our laboratory will focus on the question whether there is a direct interaction between GnTI and COPII components like it was shown for other plant proteins ( 45 , 46 ) and whether this interaction involves basic amino acids present in the cytoplasmic tail, as was proposed for mammalian glycosyltransferases. | Plant N -glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgi-resident N -acetylglucosaminyltransferase I (GnTI) and α-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivo function of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N -glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails. | The plant Golgi apparatus consists of numerous separate stacks of cisternae, which are distributed throughout the cytoplasm and often associated with the endoplasmic reticulum (ER) (1) . In many plant cell types, the Golgi stacks are highly motile and move along the tubular ER network in a microfilament-dependent way ( 2 – 4 ). In contrast to animal cells, exchange of cargo between these two organelles does not involve an ER-to-Golgi intermediate compartment and is assumed to occur either by specific transport vesicles or by permanent or transient tubular connections ( 5 , 6 ). The dynamic and close association of the plant ER and Golgi is different from that of mammalian and yeast cells and might be critical for mediating protein trafficking between the ER and Golgi ( 5 , 7 , 8 ).
The plant Golgi apparatus is not only a central organelle for protein sorting within the endomembrane system but plays also a major role in the biosynthesis of cell wall polysaccharides and maturation of glycoproteins (9) . N -glycosylation is an abundant covalent protein modification in all eukaryotic cells. The core oligosaccharide, which is transferred to nascent proteins from a lipid-linked precursor, is extensively modified by removal and addition of sugar residues in the ER and subsequently in the Golgi apparatus ( 10 , 11 ). N -glycan processing is performed by a number of ER- and Golgi-resident glycosidases and glycosyltransferases, which are thought to act on cargo glycoproteins in a highly ordered fashion in a kind of assembly line. Thus, the subcellular localization of these enzymes together with their in vivo substrate specificity determines the carbohydrate structures of glycoproteins transported through the secretory pathway. How the ER and Golgi maintain the organization of these N -glycan processing enzymes is not well understood.
A number of plant N -glycan processing enzymes have been identified and characterized recently. Evidence for Golgi localization has been provided for some of them, reflecting their function in the processing pathway ( 3 , 12 – 16 ). Most of the characterized glycosidases and glycosyltransferases are typical type II membrane proteins, consisting of a short N-terminal cytoplasmic tail, a single transmembrane domain and a stem region (together the CTS region) orienting a catalytic domain into the Golgi lumen. It has been shown that important information for concentration of these type II membrane proteins in the Golgi is present in the CTS region, without any detectable contribution from the luminal catalytic domains ( 3 , 12 – 16 ). Although the importance of the CTS region for proper intracellular targeting of plant N -glycan processing enzymes is well documented, the role of the individual CTS domains for ER exit and Golgi concentration remains to be established.
In mammalian and yeast cells, protein transport between the ER and Golgi involves the vesicular coat protein complexes COPI and COPII. The COPII machinery is required for anterograde trafficking outside the ER by actively sorting secretory proteins into COPII transport vesicles, which is the assumed default route from the ER to the Golgi complex in mammals (17) . Although the existence of COPII vesicles remains to be unequivocally shown in plants, homologues of COPII proteins have been identified, and COPII-dependent ER export has been demonstrated for soluble and transmembrane proteins ( 18 – 21 ).
Different classes of targeting signals have been identified in the cytoplasmic domains of transmembrane proteins in yeast and mammals. These motifs include cytoplasmically exposed diacidic, dihydrophobic and dibasic motifs ( 22 , 23 ). In contrast, the specific signals and underlying mechanisms that promote ER exit and retention of the corresponding enzymes in the plant Golgi apparatus are still poorly understood. So far, it has been shown that diacidic motifs present in the cytoplasmic regions of the Golgi nucleotide sugar transporter GONST1 and CASP, a member of the golgin family, contribute to ER export of these proteins (24) . Furthermore, mutation of a basic motif present in the cytoplasmic tail of prolyl 4-hydroxylase was found to impair its transport to the Golgi in tobacco BY2-cells (25) . These studies suggest that the cytoplasmic portion of transmembrane proteins harbours important information for ER exit in plants.
For mammalian glycosyltransferases involved in glycolipid synthesis, it was found that the conserved dibasic amino acid motif in the N-terminal cytoplasmic tail binds directly to the small guanosine triphosphatase (GTPase) Sar1p, indicating that the export of these glycosyltransferases occurs through the formation of COPII vesicles at ER export sites (ERES) (23) . In addition, it was found that interactions between Golgi-resident glycosyltransferases and COPII components regulate COPII coat assembly (26) . For plants, it has been shown that Golgi localization of green fluorescent protein (GFP)-tagged rat α2,6-sialyltransferase (ST–GFP) (2) occurs in a COPII-dependent way (20) , and studies on coexpression of a Sar1p isoform with ST–GFP resulted in increased recruitment of Sar1p to ERES in Nicotiana tabacum leaf epidermal cells (21) . However, the detailed mechanisms that dictate ER exit and concentrate plant N -glycan processing enzymes in the Golgi have remained elusive so far.
In this work, we investigated whether the ER export of different Golgi-resident N -glycan processing enzymes is influenced by amino acid sequence motifs present in the cytoplasmic tail. Using a series of deletion mutants, we determined the protein domains required for efficient Golgi retention. Furthermore, using site-directed mutagenesis, we identified amino acid residues required for efficient ER export. For one enzyme, Golgi-resident N -acetylglucosaminyltransferase I (GnTI), the functional significance of proper Golgi targeting was tested by complementation studies in GnTI-deficient Arabidopsis thaliana plants. Coexpression of GnTI with Sar1p forms indicated the presence of COPII-dependent transport to the Golgi. Our data show that despite the unique structural characteristics of the plant ER–Golgi interface, the ER-to-Golgi transport mechanisms of glycosyltransferases seem to be similar in mammals and plants. | This study was supported by grants from the Austrian Science Fund (P18314 to H. S. and P19494 to R. S.). We thank Shoudong Zhang and Eva Turetschek for generation of GnTI-CTS-mRFP, Richard Fischl for help in cloning of mRFP-HDEL, Mirela Curin for help with some confocal experiments and Karin Polacsek (all from BOKU) for N -glycan analysis.
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1: Time–course of N. benthamiana leaf epidermal cells expressing wild-type and tail-mutated GnTI chimeras.
Figure S2: Golgi concentration of XylT depends on basic residues within the cytoplasmic tail.
Figure S3: The subcellular localization of wild-type and tail-mutated full-length GnTI fusion proteins is identical to their GnTI-CTS forms.
Figure S4: MALDI-TOF-MS analysis of transformed cgl1 plants.
Table S1: Primer sequences cited in Materials and Methods
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. | CC BY | no | 2022-01-12 15:46:43 | Traffic. 2009 Jan 3; 10(1):101-115 | oa_package/47/01/PMC3014094.tar.gz |
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PMC3014095 | 0 | The authors regret that in the production of Table 2 for this paper, information regarding data for ritonavir boosted drugs was omitted. An amended version of Table 2 is now appearing.
The authors would like to apologise for any inconvenience this may have caused to the authors of this article and the readers of the journal. | CC BY | no | 2022-01-12 20:23:21 | Antiviral Res. 2009 Nov; 84(2):203 | oa_package/6c/d9/PMC3014095.tar.gz |
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PMC3014097 | 21224922 | All scientific journals, including this one, are concerned about plagiarism. The Publications Committee of the International Organization of Medical Physics (IOMP) had prepared an editorial on plagiarism. The editorial is reproduced here (with permission from the IOMP) with slight modifications to enhance its relevance to the audience of this journal.
Plagiarism (from the Latin “ plagiare ”, meaning “to kidnap”) is defined as “the appropriation or imitation of the language, ideas and thoughts of another author and representation of them as one's original work” ( The Random House Dictionary of the English Language - unabridged). Plagiarism is a serious breach of research ethics that, if committed intentionally, is considered research misconduct. Plagiarism may result in serious sanctions, including public disclosure, loss of research funding, loss of professional stature and termination of employment. Plagiarism undermines the authenticity of research manuscripts and the journals in which they are published and compromises the integrity of the scientific process and the public regard for science. Plagiarism violates the literary rights of individuals who are plagiarized and the property rights of copyright holders. Violation of literary or property rights' may result in legal action against the individual(s) committing plagiarism. Although plagiarism has existed since the beginning of science, it seems to be increasing because the World Wide Web (Internet) facilitates finding and copying the work of others
It is possible not only to plagiarize the works of others, but also one's own work through reuse of identical or nearly identical portions of manuscripts without acknowledgement or citation. Simultaneous or subsequent submission of similar manuscripts with only minor differences and without citation between the manuscripts is, unfortunately, a rather common practice among authors hoping to acquire multiple publications from a research project. Scientific journals discourage this practice, and usually will not permit it if exposed before publication. Occasionally, the same (or a very similar) article may be published in two journals, because the journals reach different audiences and the article is of interest to both. This practice must be approved by the editors of both journals, and the duplication must be acknowledged in each article
When there is a possibility of plagiarism (often through an allegation of plagiarism by the original author, a reviewer or an interested third party), the journal's editor should act quickly. The editor should examine the original material and the publication alleged to constitute plagiarism. If the editor concludes that no plagiarism has occurred, the accuser should be notified, and no further action is necessary. If the evidence suggests that plagiarism may have occurred, the editor should contact the accused author(s), the author(s) whose work may have been plagiarized and the copyright holder of the original material if different from the author(s). The correspondence should include the alleged plagiarizing language and a copy of the original and suspected work. If all parties agree that plagiarism (whether intentional or unintentional) has occurred, a written letter of apology should be sent promptly by the offending author(s) to the editor and to the author(s) and copyright holder whose work has been plagiarized. If the offending work has been published, a notice of plagiarism, citing both the plagiarized and the offending articles and containing the exact text that has been plagiarized, should be published in the next available article of the journal in which the offending article was published. The plagiarizing authors must agree that all dissemination of the offending article will to be accompanied by the notice of plagiarism
If the accused author(s) deny that plagiarism has occurred, the editor must explore the accusation further, preferably through a mechanism already established by the journal to investigate allegations of scientific misconduct. All parties to the allegation should be encouraged to submit corroborating evidence, and the accused author(s) should be granted an opportunity (at no expense to the journal) to testify in person in defense against the allegation. The investigation should be concluded within a reasonable period of time (e.g. 3 months)
If the mechanism to investigate the allegation of plagiarism concludes in support of the allegation, then the process for the case in which plagiarism is admitted should be instituted. Further, the editor must decide whether the plagiarism should be reported to the guilty parties' supervisor, employer and/or professional organization. If the mechanism rules against the accusation of plagiarism, a letter stating this ruling should be provided to the accuser, the author(s) accused of plagiarism, the author(s) of the original work and the copyright holder if different from the author(s). In either case, these actions should constitute closure of the allegation of plagiarism
An allegation of plagiarism is a serious accusation and should never be taken lightly. On the other hand, self-policing is a major strength of the scientific community, and plagiarism should always be reported when it is suspected to have occurred | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):143-144 | oa_package/05/f3/PMC3014097.tar.gz |
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PMC3014098 | 21224923 | Materials and Methods
All PTCA procedures were performed using Philips Integris H5000 (the Netherlands) machine dedicated for cardiovascular procedures. The machine incorporated preprogrammed radiographic factors to suit the region of anatomical interest. It also had several selection modes with specific table-top dose rates such as low, normal and high for fluoroscopy screening. In the initial stages of dose auditing, the table-top dose rate for ‘low fluoroscopy mode’ was set to 43.8 mGy/min, ‘normal’ to 87.5 mGy/min and ‘high’ to 175 mGy/min. The Philips H5000 machine incorporated a minimum filtration of 1.5 mm Al equivalent; and added filtration (spectral filters) of 0.1, 0.2 and 0.4 mm copper. The spectral filters were automatically selected by the machine according to the dose rate settings selected by the operator. The machine had a Charged Couple Device (CCD) camera fixed along with the image intensifier. The advantage of using CCD camera was that it necessitated lower exposure factors, thus imparting less radiation dose to patients.
The optimization process (P2*) involved halving the dose rates to 21.9, 43.8 and 87.5 mGy/min for fluoroscopy selection modes of low, normal and high respectively. This optimization was achieved by increasing the existing filtration specified by the manufacturer and by increasing tube potentials and lowering the tube current. The dose optimization techniques using copper filters are still practiced, and more literature is required in the current scenario due to the arrival of flat panel machines. Ideally, the kVp and the filter should be preferably chosen so that for the resultant X-ray spectrum, the sensitivity of the detector or detector combination should be maximum. In addition, the energy should be optimized for better visibility of narrow blood vessels with contrast agents. For the kVp and filter used in our study, cardiologists found that the images were of optimum quality for performing the procedures.
PTCA procedure
The PTCA involves opening stenosed segments in coronary vessels using balloon angioplasty and deploying stents for long-term patency in majority of cases. After gaining femoral arterial access using the Seldinger technique, patients are taken for PTCA directly if a coronary angiogram (CA) was already available. Using dedicated guiding catheters, the coronary ostea is cannulated. After proper angiographic delineation, the optimum views are selected to show the site and length of lesion without fore shortening. The lesion is crossed with 0.014 inch sized wires and dilated with appropriate sized balloons. Adequately sized premounted stents are then deployed at the lesion site for long-term patency. Final cine runs in orthogonal views confirmed the adequacy of the procedure. The PTCA was performed by one consultant cardiologist and one resident cardiologist. The PTCA was categorized into two groups namely patients who had a single stent placement and patients who had multiple stent placement.
It is possible to use one of the three image intensifier formats (IIFs) or field sizes with diameters such as 23, 17 and 14 cm available on both the machines. A 23-cm IIF was used during fluoroscopic screening in PTCA procedure for tracing the path of the catheter from the region of femoral puncture and in the cardiac valve region. The fluoroscopic screening along the femoral region while tracing the path of the catheter from the region of femoral puncture was done only if there was a problem in the smooth insertion of the wire. A 17-cm IIF was used for the oblique, caudal, cranial and lateral projections delineating the coronary anatomy during the procedure. The 14-cm IIF was not used unless there was a necessity for finer details while performing the procedure. Selection of these IIFs was at the discretion of the personnel performing the procedure.
Dosimetry
Radiation dose imparted to patients who underwent PTCA procedure was measured using DAP meter (diamentor PTW; Freiburg, Germany) which was fitted to the collimator assembly [ Figure 1 ]. The DAP meter is constructed using transparent plastic, and it is therefore completely unobstructive to the examination. The DAP meter measured radiation dose contributed from fluoroscopy screening and cine runs. During the course of the examination, personnel involved in the data collection continuously monitored DAP values and this facilitated acquisition of DAP values pertaining to fluoroscopy screening and cine runs separately. Periodic calibration of the DAP meter was done by company engineers every 6 months in the presence of medical physicists. | Results
Out of the 44 patients who underwent PTCA procedure, 6 were female patients and 38 were male patients. Table 1 shows patient-related parameters along with the exposure factors used during fluoroscopic screening and cine runs. The exposure parameters used for single stent placement and multiple stent placement during PTCA with company preset values for the H5000 Philips Integris machine are shown as P2 in Table 1 . The P2* represents the optimized values used in the same machine. After the optimization, it is found that tube potentials during fluoroscopic screening were higher for P2* compared those used in P2; however, the tube current in P2* was less than that used in P2. The kVp and mA were only slightly increased for cine runs, and the tube potential was increased as for fluoroscopic screening in both P2 and P2*. The fluoroscopic time duration during the single stent placement ranged from 4.9 to 29.9 min; while for multiple stent placement, it ranged from 7.9 to 25.8 min. The PTCAs were performed under automatic brightness control, in which the tube potential and tube current were adjusted.
Table 2 shows the DAP values for PTCA. Though the time duration of fluoroscopic screening was higher than the time duration of cine runs, the percentage of radiation dose imparted to patients from cine runs was higher than that from fluoroscopic screening. The mean DAP value for single stent placement in P2 was 66.16 Gy cm 2 , whereas the mean DAP value after optimization (P2*) was 48.67 Gy cm 2 . Similarly for multiple stent placement, the mean DAP value in P2 was 122.68 Gy cm 2 and mean DAP value for P2* was 65.44 Gy cm 2 . The Student's ‘ t ’ test showed that the DAP value from P2 for PTCA was significantly higher than that for P2* ( P < 0.001). Table 2 also shows the dose rates during cine runs and fluoroscopic screening. After the optimization (P2*), the use of high tube filtration and low tube currents has reduced the dose rates by about 40-50% for fluoroscopic screening and cine runs respectively and hence has an effect on the overall radiation dose imparted to patients during PTCA. | Discussion
The PTCA is an interventional cardiologic procedure involving high fluoroscopic doses and replaces a sizable fraction of cardiovascular surgeries in the current scenario. Repeated PTCAs may be required for some patients in order to achieve the desired result. In spite of being a procedure of imparting high radiation doses to patients, the benefits obtained from this procedure far outweigh the risk involved from radiation. The potential deleterious consequence of this procedure involves stochastic effects as well as deterministic effects.[ 11 ]
There are no standard protocols available for PTCA; hence it is necessary to audit and optimize radiation doses during this procedure. During the optimization process in the current study, adequate copper filtration with high tube potentials and low tube currents was selected. It has been observed that with suitable thickness of copper filtration combined with optimized exposure factors, absorbed dose during fluoroscopic procedure can be reduced with little or no loss of image quality.[ 12 ] As pointed out by Fenner et al ., there is a reduction of 40% of absorbed dose with only minimal loss of image quality with the using 0.2 mm Cu filter.[ 13 ] Use of tube potentials of 120-125 kV instead of 90-100 kV and corresponding mAs could reduce entrance surface doses by 50% and effective dose by 43%.[ 14 ]
In the current study, the radiation doses after optimization (P2*) were found to be 27% lower for single stent placement and 47% lower for multiple stent placement compared to those in P2 using company preset values. It is also noteworthy in this context that the mean DAP value of 65.44 Gy cm 2 for multiple stenting in P2* was comparable to the mean DAP value of 66.16 Gy cm 2 for single stenting in P2. From Table 2 it is also noted that dose rates for cine runs and fluoroscopy screening are reduced in P2*. Since the PTCA is usually preceded by a diagnostic CA, it would be recommended to include radiation dose from CA along with that involved in PTCA also, since radiation doses have a cumulative effect. The mean DAP value for patients undergoing CA procedure before optimization was 55.86 Gy cm 2 . In a separate study conducted earlier, the mean DAP value for CA procedure was 27.71 Gy cm 2 .[ 15 ] Adding the DAP value after the optimization for corresponding patients for both CA and PTCA would therefore give 76.38 and 93.15 Gy cm 2 for single stent and multiple stent placement respectively. No ill effects of radiation, such as deterministic or stochastic, were reported for any patient during the course of the study.
The mean DAP value after the optimization in the current study for single stent placement was 48.67 Gy cm 2 , and this lies well within the values of radiation doses reported in literature. Table 3 shows a comparison of DAP values with those from studies reported in literature.[ 4 8 16 – 21 ] Kuon et al ., in their study, have optimized radiation doses up to 14.4 Gy cm 2 for PTCA by adopting techniques like training the staff in the fluoroscopy room, using low fluoroscopy modes and by limiting the number of cine runs wherever possible to one heart cycle length.[ 21 ]
High radiation doses are imparted to patients who require cranio-caudal angulations since they require maximum X-ray output and involve high tube loadings. Mottled appearance was visualized in both P2 and P2* during these extensive angulations. Short cine runs were required to visualize the vessels prominently if the images gave mottled appearance during fluoroscopic screening, and this requires increase of radiation doses. However, after the optimization, the images did not produce significant mottled appearance which could degrade the diagnostic information. In the current study, the length of the procedure and the duration of fluoroscopic screening and cine runs acquired depended upon the complexity of anatomy of the patient and the number of stents needed to be implanted. The increase in the number of cine runs and frames acquired during cine runs depended on the personnel performing the procedure and complexity of the disease. A few cardiologists required higher number of frames for each run in order to visualize the vessels more prominently. Radiation doses were high during the placement of stent when high degree of tube angulations, especially cranio-caudal positions, was selected for certain patients.
The cardiovascular machines are specifically designed for interventional cardiologic procedures and have the option of using different IIFs. Appropriate selection of IIFs based on exposure parameters, dose rates and image magnification required to obtain the necessary clinical information could reduce radiation dose imparted to the patient significantly. The cardiovascular machines dedicated for cardiac procedures are invariably imported from developed countries and the default preset exposure settings are based on the patient size of those specified locations. Since the weight and body surface area of Indian patients are lower in comparison with patients of other countries, it would be advisable to set up appropriate dose modes for the Indian population. To achieve low dose levels during cardiac procedures, the image intensifier entrance dose rates could be reduced according to the body mass index of the Indian population. However, radiation-intensive angulations do not enable substantially better image quality despite increasing image intensifier dose levels.[ 22 ]
The dose reduction achieved by these methods is likely to translate into low doses to the operators as well. Further dose reduction is possible by adopting pulsed fluoro mode operation. As pointed out by Kuon et al ., use of a 25 ps −1 would also impart low radiation dose to patients if adequate radiation safety standards are maintained.[ 22 ] The dose can be further reduced by using 12.5 ps −1 in cine mode rather than using 25 ps −1 . Maintenance and quality assurance checks at regular intervals are mandatory. Improperly calibrated machines can also inadvertently impart large doses to patients without the knowledge of the operator.[ 23 ]
The reference DAP dose levels presently available for developed countries are 45 and 75 Gy cm 2 for CA and PTCA respectively, and our center fulfils the above criteria for these interventional procedures. In accordance with the ALARA principle, interventionists should therefore vary cine image intensifier dose rates and may to a certain extent compromise optimal image quality in accordance with diagnostic and therapeutic requirements, documented structure, tube angulation and body mass index.[ 22 ] | Conclusion
Though the radiation dose imparted to patients does not present any alarming situation with regard to ill effects of radiation, it would be prudent to optimize radiation dose to patients undergoing PTCA and take efforts towards achieving reduction in radiation dose to the patients. Since there is a frequent change in the various imaging modalities, reference dose levels should be audited on a time-to-time basis so as to keep the doses as low as reasonably practical. Enhanced knowledge of ‘radiation dose’-reduction techniques significantly reduces patient radiation hazards in invasive cardiology. | The percutaneous transluminal coronary angioplasty (PTCA) is one of the interventional procedures which impart high radiation doses to patients compared to the other cardiologic procedures. This study intends to audit and optimize radiation dose imparted to patients undergoing PTCA. Forty-four patients who underwent PTCA involving single or multiple stent placement guided under cardiovascular X-ray machine were included in the study. Radiation doses were measured using dose area product (DAP) meter for patients undergoing single and multiple stent placements during PTCA. A dose reduction of 27-47% was achieved using copper filters and optimal exposure parameters. The mean DAP values before optimization were 66.16 and 122.68 Gy cm 2 for single and multiple stent placement respectively. These values were 48.67 and 65.44 Gy cm 2 respectively after optimization. In the present scenario, due to the increase in the number of PTCAs performed and the associated risk from radiation, periodical audit of radiation doses for interventional procedures are recommended. | Cardiovascular interventions are complex procedures performed with dedicated fluoroscopy machines. In recent times, these interventions are rapidly replacing sizable fraction of cardiovascular surgeries. The PTCA involves applications such as stent deployment, resulting in high radiation doses due to increasingly long exposure times[ 1 2 ] The duration of PTCA depends on the complexity of the disease. The more complex the anatomy, the longer the duration of fluoroscopic screening and the number of cine runs acquired. During PTCA, the radiation doses to patients are relatively high[ 3 – 6 ] It is therefore prudent to perform dose auditing on this procedure by implementing radiation safety protocols, ensuring good work practices and comparing risks involved with benefits obtained. Establishing reference levels for interventional examinations presents a problem since patient numbers are limited and these interventional procedures are often performed at a few specialist centers.[ 7 ]
Radiation doses imparted to patients can be measured using a dose area product (DAP) meter. The DAP is the most reliable measurement technique for dynamic examinations such as fluoroscopy in which the projection technique and the technique parameters are continually varying.[ 7 8 ] Moreover, DAP allows the estimation of patient dose in complex examinations and does not depend on the distance from the X-ray tube.[ 9 ] The DAP is particularly useful for assessing and comparing radiation doses from screening procedures, and it provides useful indication of the overall patient exposure rather than measurement of surface dose and interprets doses to particular organs.[ 10 ] Though DAP is an ancient measurement tool, it still remains as one of the best ways to measure radiation doses from diagnostic and interventional cardiologic and radiological procedures.
Dose auditing during radiological procedures is of great value in optimizing radiation dose to patients without compromising diagnostic yield. Modification of key imaging parameters such as tube voltage, mAs, field collimation, geometric magnification and radiation exposure to the image receptor has an impact on image quality and the radiation dose to the patient and these require optimization. The present study is intended to audit and optimize radiation dose imparted to patients during PTCA procedure. | The authors would like to thank all the staff members in the catheterization lab for their cooperation with the team for collecting the data. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):145-149 | oa_package/71/f4/PMC3014098.tar.gz |
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PMC3014099 | 21224924 | Materials and Methods
The first integrated PET/CT of the country, Discovery ST (GE Medical Systems, USA), was installed in the Bio-Imaging Unit of our hospital in November 2004 and has been in clinical use since January 2005. The Discovery ST (D-ST) consists of one integrated gantry containing (a) 16-slice, slip-ring design CT X-ray tube and Hi-light matrix II detector assembly, (b) ‘BGO crystal’-based PET and (c) a common imaging table with the provision of flat carbon fiber table top for radiotherapy application.
System description
The PET in D-ST consists of 24 detector rings comprising 12,096 BGO crystals. Each crystal is of size 0.63 × 0.63 × 3 cm 3 and is organized in blocks. Each block, containing 6 × 6 crystals, is coupled to a single photomultiplier tube with four anodes and constitutes a module, which is then arranged in the 24 rings. It allows 47 images to be obtained per bed position, spaced by 0.327 cm and covering an axial field of view (FOV) of 15.7 cm. The arrangement of modules in detector rings is shown in Figure 1 . The image acquisition can be carried out either in 2D or 3D mode by inserting or retracting the tungsten septa of 0.8 cm thickness and 5.4 cm length. In both acquisition modes, the system operates with an energy window of 375-650 keV and 11.7 ns coincidence time window. Mechanical design integration provides a wide 70 cm bore, 88.6 cm ring diameter, short tunnel length and compact scanner design. PET is also equipped with an auto-loading pin radioactive source of 68 Ge (55.5 MBq) for system calibration and daily quality control.
Attenuation correction is based entirely on CT numbers to PET attenuation transformation factors. Image reconstruction in 2D mode can be performed with either filtered backprojection (FBP) or ordered-subset expectation maximization (OSEM), whereas the 3D image reconstruction supports both 3D reprojection and Fourier rebinning (FORE) followed by either FBP or a weighted least squares (WLS) OSEM iterative reconstruction. Both 2D and 3D iterative reconstructions include attenuation compensation within the model to more accurately preserve the statistical nature of the input data. Scatter correction is calculated with the Bergstrom convolution in 2D and an analytic model-based technique in 3D. Random correction can be conducted with delayed-event coincidence measurements or from an estimate of randoms generated from the crystal singles rate.
NEMA 2001 (N-01) measurements
The set of tests performed under N-01 using recommended phantoms were (i) spatial resolution, (ii) sensitivity, (iii) scatter fraction (SF) and counting rate performance.
Spatial resolution : It represents the ability of the system to distinguish between two points of radioactivity in an image. Three 18 F point sources (<0.1 cm extending in any direction), each having activity concentration of 185 MBq/cc (5 mCi/cc), were prepared and contained in capillary tube with an inner diameter (ID) less than 0.1 cm. The capillary tubes were aligned parallel to the long axis of the tomography and distributed such that point sources were placed at the locations corresponding to X = 0 cm, Y = 1 cm; X = 0 cm, Y = 10 cm; and X = 10 cm, Y = 0 cm in the center of the axial FOV of the scanner as shown in Figure 2 . Data were acquired in 2D and 3D modes with each acquisition set for 60 s. The images were reconstructed using the FBP (2D) and FORE followed by FBP (3D) algorithm into a 256 × 256 matrix with a ramp filter and a 0.63-cm cutoff. The reconstruction FOV was set to 25 cm and centered at X = 5 cm, Y = −5 cm. Spatial resolution was determined from the reconstructed 2D and 3D data by measuring the full width at half maximum (FWHM) and tenth width at half maximum (TWHM) of the point spread functions in all three directions through the peak of the activity distribution in the three orthogonal directions.
Sensitivity : The sensitivity of a scanner represents its ability to detect annihilation radiation. In the N-01 standard, the absolute sensitivity of a scanner was measured as the coincidence event rate per unit radioactivity (cps/MBq) from a sufficiently low activity line source suspended within the scanner FOV in the absence of attenuating media. A uniform line source was prepared by filling with 9.6 MBq of 18 F in a 70-cm long plastic tube having 0.31 cm inner diameter (ID) and 5.2 ml volume. This line source after inserting in a 70-cm long concentric aluminum tubes having attenuation coefficient of 0.09965/cm and inner diameter (ID) of 0.39 cm was suspended at the center of the scanner FOV as shown in Figure 3 . Measurements (cps) were made both in 2D and 3D acquisition modes. Each data acquisition was set for duration of 1 min. Similar measurements (cps) were performed after successively adding aluminum tubes of bigger ID (0.7, 1.02, 1.34 and 1.66 cm) one above the other. Extrapolation of the response to zero absorber thickness gives an attenuation-free estimate of sensitivity. To evaluate the sensitivity of the scanner at another position of FOV, the whole measurement was repeated at a radial distance of 10 cm from the center.
Scatter fraction (SF) and count rate (CR) performance : The intrinsic scatter fraction is a measure of the relative system sensitivity to scatter. The scatter fraction (SF) is defined as the ratio of scattered events to total events, which are measured at a sufficiently low counting rate so that random coincidences, dead-time effects and pileup are negligible. Total events are the sum of unscattered events (trues) and scattered events. For the measurement of SF and count rate (CR) performance, a line source was prepared by filling with 2.59 GBq of 18 F in the same 70 cm long plastic tube. It was then inserted into the hole located at a radius of 4.5 cm off the central axis of the polyethylene cylindrical phantom (20 cm diameter and 70 cm length). This phantom, with the line source posterior, was positioned at the center of scanner FOV [ Figure 4 ] and imaged repeatedly over a period of 12 h. Data was acquired in 2D and 3D modes and recorded without delayed-event randoms data. The SF was then measured from the reconstructed data according to the N-01 standard and plotted for each slice across the axial FOV of the scanner. The average system SF was also calculated. The CR performance of the scanner was evaluated using all data acquisition time points. The total system counting rate; trues, randoms and scatter event rates; and noise equivalent counting rates (NECR) were calculated and plotted versus the activity concentration in the plastic tube. Peak values and corresponding activity concentration for these rates were also determined according to the NU-01 standard. | Results
The 2D transaxial (average of radial and tangential) and axial profiles for a point source at 1 cm off center are shown in Figure 5 , while Table 1 represents the 2D and 3D average transaxial and axial spatial resolution expressed as FWHM and FWTM of the point source located at 1 and 10 cm radial position. The average of the transaxial and axial spatial resolution measured as FWHM at 1 cm off axis was 0.561 cm in 2D and 0.593 cm in 3D. Both the 2D and 3D resolutions were degraded at 10 cm off axis and were 0.672 and 0.641 cm respectively. Figure 6 shows the 2D and 3D sensitivity profile across the available FOV of the scanner. The results of the sensitivity test in 2D and 3D acquisition configurations at both radial locations (R = 0 and R = 10 cm) are shown in Table 2 . The system has an average sensitivity of 2.56 and 2.63 cps/kBq in 2D for the two radial positions R = 0 and R = 10 cm respectively. Sensitivity in 3D was approximately 4.6 times higher compared to that in 2D. The results for the SF and CR tests in 3D mode are shown in Figures 7 and 8 respectively; whereas Table 3 summarizes the values of the count rate of the system and the corresponding activity concentration at the peak true rate, peak random rate, peak scatter rate and peak NECR ( k = 1) and NECR ( k = 2). The average scatter fraction across the axial FOV of the scanner was 19.79% in 2D and 46.19% in 3D. For the 2D data acquisition mode, the peak true rate was 309.29 kcps and occurred at 122.66 kBq cc, whereas the peak random rate was 1303.58 kcps at 134.83 kBq/cc. The peak noise equivalent counting rate (NECR) evaluated with single random subtraction ( k = 1 in NEC formula) was 89.41 kcps at 49 kBq/cc in 2D and 60 kcps at 12 kBq/cc in 3D, whereas the NECR with delayed random subtraction ( k = 2 in NEC formula) was 61.47 kcps at 40.67 kBq/cc in 2D and 45.57 kcps at 16.45 kBq/cc in 3D acquisition mode. | Discussion
Continuous efforts have been made to standardize the performance measurement of PET scanner. In 1991, a task group from the society of nuclear medicine (SNM) published a set of measurements.[ 4 ] Shortly the National Electrical Measurements Association (NEMA) formed a committee and refined the SNM test, resulting in the formation of NU 2-1994 (N-94) protocol.[ 2 ] N-94 protocol assumes axial FOV of all scanners to be lesser than 17 cm and supports only two-dimensional imaging for a 20 cm diameter and 20 cm long phantom. Thus N-94 protocol is more suitable to assess the performance of the PET scanners in conditions comparable to those of neurological studies. Since the publication of N-94 protocol, there have been several developments in PET scanner technology. In the last few years, three-dimensional whole-body 18 F-FDG study has become the prominent type of PET study performed by most centers. Moreover, the modern PET scanners have axial FOV as large as 25 cm. To respond to this changing technology, a new protocol NU 2-2001(N-01) was published in 2001.[ 3 ] In N-01, 70 cm long phantom is used to account for the activity contribution from outside the FOV, and tests are defined for both 2D and 3D acquisition configurations. This document specifies procedures for acquiring and analyzing test data using standard phantoms and sources. Margaret et al . described the details of the development of different protocols and advantages of following N-01 protocol over N-94.[ 5 ] An inter-laboratory comparison study conducted in Austria to assess the image quality of 85% of all their PET (dedicated; D-PET and coincidence camera CC-PET) scanners using NEMA-2001 demonstrated considerable differences not only between CC-PET and D-PET systems but also between individual D-PET systems, with possible consequences for clinical interpretation of images and measurement of quantitative indices such as the standardized uptake value.[ 6 ] The findings also strongly demonstrate the necessity for carrying out regular quality control programs for this ‘new imaging system’-following standard protocols.
The measured (FWHM) and manufacturer-specified spatial resolution in both 2D and 3D acquisition modes agree within ±3% (mean) at 1 and 10 cm radial position. Spatial resolution reported in the literatures varies depending on the type and dimension of the crystal size.[ 5 – 9 ] Our data is comparable with other's finding from the similar PET scanner with same crystal dimension.[ 8 9 ] The system sensitivity in both 2D and 3D acquisition modes was better than the manufacturer-specified value and data reported from similar PET.[ 8 9 ] The measured average scatter fraction in both acquisition modes was ≈5% more than the manufacturer-specified values. The noise equivalent counting (NEC) peak rate measured at 49 kBq/cc using k = 1R in 2D mode was 6.4% higher than the manufacturer-specified value. On the contrary, peak NEC ( k = 2R) at 12 kBq/cc in 3D was 4.8% lesser than the manufacturer-specified values. This variation could be due to the variation in the activity concentration level within the scanner FOV during the data acquisition. The 2D and 3D NEC peak rate for k = 1R reported from the similar PET scanner with same crystal dimension is 90.2 and 67.8 kcps at 52.5 and 12 kBq/cc respectively.[ 8 ] Image quality test has not been performed due to the non-availability of NEMA/IEC 2000 Torso phantom. | Conclusion
The performance of Discovery ST PET/CT evaluated using NEMA-2001 protocol and recommended phantoms satisfies the manufacturer-recommended specifications both in 2D and 3D acquisition modes. The test result of PET shows excellent system sensitivity with relatively uniform resolution throughout the FOV, making this scanner highly suitable for whole-body studies. | Integrated PET/CT has emerged as an integral component of oncology management because of its unique potential of providing both functional and morphological images in a single imaging session. In this work, performance of the ‘bismuth germinate (BGO) crystal’-based PET of a newly installed Discovery ST PET/CT was evaluated in 2D and 3D mode for whole-body scanning using National Electrical Manufacturers Association (NEMA) NU 2-2001 protocol and the recommended phantoms. During the entire measurements, the system operates with an energy window of 375-650 keV and 11.7 ns coincidence time window. The set of tests performed were spatial resolution, sensitivity, scatter fraction (SF) and counting rate performance. The average transaxial and axial spatial resolution measured as full width at half maximum (FWHM) of the point spread function at 1 cm (and 10 cm) off-axis was 0.632 (0.691) and 0.491 (0.653) cm in 2D and 0.646 (0.682) and 0.54 (0.601) cm in 3D respectively. The average sensitivity for the two radial positions ( R = 0 cm and R = 10 cm) was 2.56 (2.63) cps/kBq in 2D and 11.85 (12.14) cps/kBq in 3D. The average scatter fraction was 19.79% in 2D and 46.19% in 3D. The peak noise equivalent counting rate (NECR) evaluated with single random subtraction was 89.41 kcps at 49 kBq/cc in 2D and 60 kcps at 12 kBq/cc in 3D acquisition mode. The NECR with delayed random subtraction was 61.47 kcps at 40.67 kBq/cc in 2D and 45.57 kcps at 16.45 kBq/cc in 3D. The performance of the PET scanner was satisfactory within the manufacturer-specified limits. The test result of PET shows excellent system sensitivity with relatively uniform resolution throughout the FOV, making this scanner highly suitable for whole-body studies. | Positron emission tomography (PET) integrated to multi-slice computed tomography (CT) (PET/CT) has emerged as an integral component of oncology management in the recent past, due to its unique ability to provide both functional and morphological information in a single imaging session. In this increasingly popular dual-modality tomography, Florine-18 fluro-2-deoxyglucose (F-18 FDG) PET provides functional information due to preferential uptake and retention of glucose by tumors, and CT provides the anatomic details and attenuation-correction data to PET. The integration of PET to CT not only enhances PET image quality but also reduces total scan time by almost half and avoids the need for a posterior alignment by the use of co-registration algorithms. Besides its wide applications in the diagnosis of various solid cancers, F-18 FDG-PET/CT is increasingly used in radiation therapy planning for staging, tumor volume delineation, treatment response evaluation and recurrence detection.[ 1 ]
The PET image quality is degraded by several physical factors, including scatter, random events, attenuation, dead time and noise. While some can be corrected, others depend on the performance of scanner. Detectors are the most critical components of a PET scanner. Various models of PET, either standalone or integrated to multi-slice CT, make use of different scintillators such as Bismuth Germanate (BGO), Lutetium Oxyorthosilicate (LSO), Gadolinium Oxyorthosilicate (GSO) and different detector architecture like blocks of detectors and continuous pixilated detectors. The performance of this newly emerging dual-modality tomography needs to be evaluated at the time of installation and periodically thereafter to ensure optimum system performance in a reproducible and reliable manner according to accepted protocols. PET performance was characterized following National Electrical Manufacturers Association (NEMA) NU 2-1994 protocol.[ 2 ] However, increasing practice of whole-body scanning in oncological applications in the recent past has led to the formation of newer NEMA NU 2-2001 protocol.[ 3 ] This updated protocol accounts for the performance test of whole-body PET both in two and three dimensions, which was not addressed in the previous protocol NU 2-1994 (N-94). In this work, performance of the PET component of our newly installed Discovery ST PET/CT was evaluated following NEMA NU 2-2001 (N-01) protocol.
Acknowledgment
We would like to express our gratitude to Mr. Krishna Toraskar of Wipro GE Medical System for his technical assistance. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):150-155 | oa_package/fc/0f/PMC3014099.tar.gz |
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PMC3014100 | 21224925 | Materials and Methods
Twenty cases were studied for optimization, whose percentage deviation between TPS-calculated and ‘ion chamber’-measured dose was more than ±3%. These cases include head and neck, thorax and pelvic regions.
Linear accelerators: We have Elekta digital linear accelerators with 6 and 15 MV photon beams, fitted with a multileaf collimator having 40 pairs of leaves, each leaf having 1 cm width at isocenter, and 6 MV ‘step and shoot’ IMRT. Virtual simulation: Oncentra virtual simulation software from Nucletron. Treatment Planning System (TPS): Plato Sunrise 3D Treatment Planning System (TPS) with Inverse Treatment Planning (ITP). IMRT Patient Plan: A custom immobilization device was fabricated, and computed tomography (CT) scan for each patient with 2-mm slice thickness was obtained. The Planning Target Volume (PTV) and Organ at Risk (OR) was delineated in Oncentra virtual simulation software. For the IMRT plan, five equally spaced and non-opposing beams were found suitable for roughly ‘cylindrical’ PTV in thorax and pelvic regions. Beams were placed approximately 70 degrees apart, at gantry angles of 225, 325, 180, 105 and 35 degrees. For head and neck region, we had used seven equally spaced non-opposing beams, and they were placed approximately 50 degrees apart. In all the cases, the isocenter of the beams was at the geometrical center of PTV and which was ITP point also. The planning was performed by TPS with ITP. Dosimeter: Unidos electrometer with 0.125-cc ionization chamber of thimble type. The chamber outer diameter and length is 6.9 and 18.7 mm, respectively. IMRT Phantom: Universal IMRT verification phantom of PTW make [ Figure 1 ]. The dimension of IMRT phantom is 300 mm × 300 mm × 70 mm and is made of Polymethyl Methacrylate (PMMA) material. The measuring depth of ionization chambers is 60 mm.
At first, CT images of the Universal IMRT phantom with 0.125-cc chamber were taken with 2-mm slice thickness. The chamber was put in the central slot provided in the phantom. The CT images were exported to Oncentra virtual simulation software for contouring. The contoured images were imported into Plato 3D TPS for planning. A phantom plan was created for each patient treated with IMRT by superimposing the patient plan on to the IMRT phantom. All gantry angles were made to zero-degree orientation for the measurement without changing anything further so that isodose and profile remained the same, and it was called phantom plan. The dose was calculated at the reference point (ITP point) of the chamber. The reference point of chamber was at the central axis of the beam and at a depth of 6 cm (in the Universal IMRT phantom), i.e., at the isocenter. The isocenter is nothing but ITP of the patient plan. Beam incidence was perpendicular to the flat surface of the phantom. Ionization chambers were oriented with their longitudinal axes perpendicular to the direction of the MLC leaf motion. The dose was measured on PTW Unidose electrometer.
The results were expressed as percentage difference between calculated and measured dose as follows: % Difference = Measured dose - Calculated dose Calculated dose × 100
The results were expressed under a tolerance level of ±3%.
The plan which failed in the tolerance criteria between the measured and calculated dose proceeded in the following way:
Beam profiles were generated by the TPS in the GT and cross plane direction in the isocenter plane of the phantom. GT is the ‘Gun to Target’ direction in the LINAC. Marked nine points (including center of IMRT phantom), equidistant (distance between two points was 10 mm) to each other, around the chamber along GT and cross plane in the phantom as shown in Figure 2 . In order to find out the low-dose gradient, the Phantom Plan was generated for each of the marked points at 6-cm depth of the phantom, i.e., the superimposition of the ITP point over these points one by one. The best plan, where the low-dose gradient was found, was exported for measurement. According to the coordinates generated in the TPS, the couch was manually displaced at the required position and point dose was measured. The measured dose was compared with the dose calculated from TPS [ Table 1 ].
Taking the example of a Ca Pharynx patient plan consisting of seven fields [ Table 1 ], beam profiles were generated in the TPS at the isocenter plane both in the GT and cross plane and are shown in Figures 3a and 3b . In order to find optimized position for point dose measurement, nine different points were taken in the chamber plane as shown in Figure 2 . Optimized point dose values were generated in the TPS as shown in Table 2 . The couch was manually displaced in the GT and cross plane direction in accordance with the coordinates of the points generated in the TPS. The best point was the point where there was an acceptable dose difference between measured and calculated dose. | Results and Discussion
We have treated 132 IMRT patients so far. Among them, there were 17 prostate, 17 cervix and endometrium, 32 brain tumors; and rest of them belonged to other extremities. The total number of fields for each treatment plan was 5-7, and the dose per fraction varied between 1.8 and 2 Gy. The breakup of different cases and variation in point dose measurement are shown in Table 3 .
The mean percentage deviation value for the 20 cases studied, under 95% confidence interval, was found to be 2.10% ± 1.14.
It has been found that even after optimization, four cases showed more than ±3% deviation [ Table 1 ]. Among those four cases, one was having single solid tumor and others had only nodes to be treated.
The deviation in some fields occurs because of highly inhomogeneous fields, hence absolute dose measurement for IMRT beamlets is difficult due to the lack of lateral electron equilibrium. That is why ionization chambers should typically be placed in low-dose gradient regions.[ 7 ] Chamber type and dimension are very important, and smaller volumes are more sensitive to position and will have a higher response when positioned at an opposing leaf pair junction and between adjacent leaves.
To minimize the effect of volume averaging, the detector should be smaller than the homogenous region of dose to be measured. The ‘Tongue and Groove’ design of adjacent leaves can result in small regions of a field being blocked and therefore having reduced dose. The pre- and post-optimized percentage variation in point dose measurement for 132 IMRT treatment plans is given in Figure 4 . The necessary resolution of the detector depends on the resolution of the beamlet grid that is used for planning and sequencing fields for delivery. Partly due to the fact that the planning system calculation algorithm often cannot model transmission, leakage and scattering dose accurately in the low-dose regions; and partly in finding a uniform dose area for that region, the ionization chamber measurements in low-dose region often showed higher dose than predicted by the planning system. Measurements of profile and depth dose curves require stepping up of ion chamber across the field or up the field. This necessitates the displacement of couch, and so the chamber across the field for optimized dose measurement in this study. The generation of optimization point by taking random points is a time-consuming process and needs to be found through a computer algorithm.[ 8 ] | Results and Discussion
We have treated 132 IMRT patients so far. Among them, there were 17 prostate, 17 cervix and endometrium, 32 brain tumors; and rest of them belonged to other extremities. The total number of fields for each treatment plan was 5-7, and the dose per fraction varied between 1.8 and 2 Gy. The breakup of different cases and variation in point dose measurement are shown in Table 3 .
The mean percentage deviation value for the 20 cases studied, under 95% confidence interval, was found to be 2.10% ± 1.14.
It has been found that even after optimization, four cases showed more than ±3% deviation [ Table 1 ]. Among those four cases, one was having single solid tumor and others had only nodes to be treated.
The deviation in some fields occurs because of highly inhomogeneous fields, hence absolute dose measurement for IMRT beamlets is difficult due to the lack of lateral electron equilibrium. That is why ionization chambers should typically be placed in low-dose gradient regions.[ 7 ] Chamber type and dimension are very important, and smaller volumes are more sensitive to position and will have a higher response when positioned at an opposing leaf pair junction and between adjacent leaves.
To minimize the effect of volume averaging, the detector should be smaller than the homogenous region of dose to be measured. The ‘Tongue and Groove’ design of adjacent leaves can result in small regions of a field being blocked and therefore having reduced dose. The pre- and post-optimized percentage variation in point dose measurement for 132 IMRT treatment plans is given in Figure 4 . The necessary resolution of the detector depends on the resolution of the beamlet grid that is used for planning and sequencing fields for delivery. Partly due to the fact that the planning system calculation algorithm often cannot model transmission, leakage and scattering dose accurately in the low-dose regions; and partly in finding a uniform dose area for that region, the ionization chamber measurements in low-dose region often showed higher dose than predicted by the planning system. Measurements of profile and depth dose curves require stepping up of ion chamber across the field or up the field. This necessitates the displacement of couch, and so the chamber across the field for optimized dose measurement in this study. The generation of optimization point by taking random points is a time-consuming process and needs to be found through a computer algorithm.[ 8 ] | Conclusion
Of the 20 cases studied, 16 were found to be within acceptable criteria after necessary displacement of the couch. For those cases in which percentage variation between TPS-planned dose and measured dose was found to be within unacceptable criteria (beyond ±3% interval) even after optimization, plans were further studied with Film Dosimetry using radiochromic films. The parameters include gamma map, dose difference, distance-to-agreement, etc.; and final ‘accept/reject’ criteria depend upon outcome of these factors.[ 8 ]
Hence, point dose measurement at the reference point in the phantom can be an effective tool for patient-specific IMRT verification and QA, which should be further verified with film dosimetry or suitable dosimetry system like portal imaging and gel dosimetry, etc. | In some cases of Intensity-modulated radiotherapy (IMRT) point dose measurement, there exists significant deviation between calculated and measured dose at isocenter, sometimes greater than ±3%. This may be because IMRT fields generate complex profiles at the reference point. The deviation arises due to lack of lateral electronic equilibrium for small fields, and other factors such as leakage and scatter contribution. Measurements were done using 0.125-cc ion chamber and Universal IMRT phantom (both from PTW-Freiburg). The aim is to find a suitable point of measurement for the chamber to avoid discrepancy between calculated and measured dose. Various beam profiles were generated in the plane of the chamber for each field by implementing patient plan on the IMRT phantom. The profiles show that for the fields which are showing deviation, the ion chamber lies in the steep-gradient region. To rectify the problem, the TPS (Treatment Planning System) calculated dose is found out at various points in the measurement plane of the chamber at isocenter. The necessary displacement to the chamber, as noted from the TPS, was given to obtain the optimum result. Twenty cases were studied for optimization, whose percentage deviation was more than ±3%. The results were well within tolerance criteria of ±3% after optimization. The mean percentage deviation value for the 20 cases studied, with standard deviation of 2.33 under 95% confidence interval, was found out to be 2.10% ± 1.14. Those cases that have significant variation even after optimization are further studied with film dosimetry. | Intensity-Modulated Radiotherapy (IMRT) is an advanced form of treatment compared to Three-Dimensional Conformal Radiotherapy (3DCRT). It is the application of varying-intensity beams along various target volumes in a rather complex way. Delivery of intensity-modulated fields is based on the use of computer-controlled multileaf collimators attached to modern linear accelerators. Since the beams are modulated in a rather complex way, each IMRT field often includes many small, irregular, off-axis fields resulting in isodose distributions for each IMRT plan that are more conformal to the tumor target volume than those from conventional treatment plans. This necessitates implementation of vigorous Quality Assurance (QA) practice, which includes machine- and patient-specific QA. The latter generally involves mapping the plan fields onto a phantom that has been computed tomography (CT) scanned, creating what is known as ‘Hybrid-Plan,’ and comparing the results with measurements made on that phantom. It is assumed that the validity of the results for the phantom can be extrapolated to the patient.[ 1 ]
When prostate tumors are treated with IMRT techniques, sparing of the rectum and bladder is a priority concern, together with adequate coverage of the Planning Target Volume (PTV). The close proximity of the prostate to the bladder and rectum often requires high-dose gradients in the interface regions, which result in highly inhomogeneous field fluences in the treatment plan. Ionization chambers are the preferred dosimeters for measuring absolute absorbed dose in IMRT fields.[ 2 ] Dose measurement with ionization chambers reflects the average dose value over their volumes.[ 3 ] Points at low-dose gradients are usually preferred for measurement purposes.[ 4 ] Escude L et al. had developed an optimization algorithm to find the most favorable points to position an ionization chamber for QA dose measurements of prostate cancer patients. The dose measurement was made in a plastic phantom at 287 optimized points.[ 5 ] Although other devices like multi-detector arrays, films and electronic portal imaging devices can be used, they are more suited to relative dose measurement. Also, Sanchez-Doblado F et al. had found that the absolute dosimetry in the penumbra region of the IMRT beamlet could suffer from significant errors. They have observed that the largest dose errors correspond to the smaller contribution of the corresponding IMRT beamlets to the total dose delivered in the ionization chamber within PTV.[ 6 ] Our present work is a parallel work based on the above studies. The aim of this study is to measure dose at a reference point in the phantom for IMRT treatment and to find out an optimized point of measurement in order to overcome large variation between calculated and measured dose without the help of another computer-generated algorithm. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):156-160 | oa_package/f9/30/PMC3014100.tar.gz |
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PMC3014101 | 21224926 | Materials and Methods
Material
All the subjects voluntarily entered the study, after receiving information and giving informed consent by signing the relevant form. The Institutional ethics committee had approved and cleared the study protocol. We excluded patients with other conditions likely to interfere with bone integrity; and patients with malignancy, endocrine diseases (affecting thyroid, parathyroid and adrenal glands), Paget's disease, long-term immobilization, chronic renal failure or rheumatoid arthritis. Also, patients with diabetes, hyperthyroidism, bone cancers; fractures by severe trauma were excluded from the study.
Twenty-five pre-menopausal (mean age ± SD: 39.4 ± 3.8) and 45 postmenopausal women (mean age ± SD: 57.9 ± 7.9) participated in the study. (One woman had fracture in the spine at the thoracolumbar vertebra, and another had a hip fracture in the femur neck.)
Methods
Working principles of DXA:
When a three-dimensional absorber (such as human body) is scanned by X-ray, it produces two-dimensional flat image on the photographic film. The human body does not act as a homogeneous absorber; a single energy X-ray beam cannot differentiate among the different components such as fat mass, lean mass and bone. For this, dual energy X-ray technique was utilized. Bone mineral density measurement using dual energy X-ray absorptiometry (DXA) has great clinical significance in the early detection and diagnosis of osteoporosis. X-ray absorption is the basic mechanism for discrimination between organs in a body under X-ray observation (Aston, 1990). Exactly how much X-ray is absorbed by different tissues is determined by Lambert's law and is given by
where I is the X-ray intensity emerging from tissue, I o is the X-ray intensity incident on the tissue, x is the tissue thickness and μ is the mass attenuation coefficient.
The DXA principle is based on the fact that mass attenuation coefficient (μ m ) for different tissues decreases at different rates with increase in X-ray energy. At low X-ray energy, mass attenuation coefficient of bone (μ b ) is very high compared to soft tissue (μ s ); and at high X-ray energy, ‘μ b ’ is approximately equal to that of ‘μ s ’ as shown in Figure 1 .
BMD measurements by dual energy X-ray absorptiometry (DXA):
In the clinical setting, the hip and spine should be the site for BMD measurement; because hip fractures are associated with higher morbidity and mortality, and vertebral compression fractures in the thoracic and lumbar spine are the most frequent clinical manifestations of osteoporosis. In our study, the Bone Mineral Density of the right hip was measured by DXA (Hologic QDR – 4500 densitometer). In the proximal femur bone, mineral densities from different sites were measured, including femoral neck, intertrochanteric area, trochanter and Ward's triangle. Total femoral BMD was also calculated as an average value of bone mineral density for the femur sites.
Calculation of T-score:
Osteoporosis is diagnosed by measuring bone mineral density (BMD), thereby defining thresholds. This is possible due to the Gaussian distribution of bone density values, where bone density is expressed in relation to a reference population in terms of standard deviation (SD) units (Kanis JA, 2002). When SD units are used in relation to a young healthy population, the measurement is referred to as the T-score [ Table 1 ]. The T-score is the parameter that compares subject's BMD with average peak BMD of young normal population of the same gender.
Bone mineral density is defined as bone mineral content divided by the projected area of the scanned image.
BMD = BMC/area (g/cm 2 )
In our study group, the standard analysis procedure for the hip was performed on all the subjects as recommended by the manufacturer. The two measurements - namely, the femoral neck BMD (FN-BMD) and trochantar BMD (TR-BMD) - at the proximal femur were taken for the correlation with the texture parameters. The effective dose to the patient was 0.01 m Sev per DXA scan.
All the subjects underwent DXA tests and were classified as normal ( N = 40), osteopenia ( N = 22) and osteoporotic ( N = 8). The mean and standard deviations of age of the individuals in the three groups were 46.5 years ± 9, 55.7 years ± 11 and 63.1 years ± 9 respectively. One case in the female group (age 73) had a nontraumatic compression and wedge type fracture in the thoracolumbar spine, noted elsewhere on a radiograph 1 year ago. Another lady (age 62) had fracture of the left femoral neck, treated elsewhere 1 year ago. Both had medication with calcium supplements since the occurrence of fracture. Patients with history of fracture due to severe trauma (such as patients met with accidents) were excluded from the study.
Radiography and digitization procedure:
Radiograph of the calcaneum lateral view were taken for all participants, according to a standardized protocol. The X-ray tube, at a setting of 46 kV and 4 mAs (100 mA and 0.04 s), was focused on the calcaneum; and the film-tube distance was fixed to 90 cm. Patient X-ray exposure to radiation using standard X-ray equipment was 1 μSev per examination.[ 8 ] The radiographs were digitized with a Kodak Film Digitizer (12 bits per pixel) and were stored as Tagged Image File Format (TIFF) files for analysis. The regions of interest (ROIs) were selected at the clinically significant area as shown in the diagram [ Figure 2 ] and fed into the algorithm developed by us in Matlab software.
Acquisition of radiograph images for analysis:
The minimum intensity was subtracted from the cropped region of interest (ROI). The ROI was normalized (i.e., the gray level varies from 0 (intensity-value assigned for black pixel) to 255 (intensity value assigned for white pixel). Hence the gray level histogram of all the ROIs follows a normal or Gaussian distribution. These image-preprocessing techniques are carried out to make all the images uniform and also to ensure the reduction in the signal-to-noise ratio in the computation procedure.
Run-length matrix:
The gray level run length (GLRL) method is a way of extracting higher-order statistical texture features. The technique has been described and applied by Galloway (1975).[ 9 – 10 ] A set of consecutive pixels with the same gray level, collinear in a given direction, constitutes a gray level run. The run length is the number of pixels in run, and the run length value is the number of times such a run occurs in an image.
The gray level run length matrix (GLRLM) is a two-dimensional matrix in which each element p (i, j | θ) gives the total number of occurrences of runs of length ‘j’ at gray level ‘i’, in a given direction θ.
A number of scalar texture features may be computed from GLRLM - short runs emphasis (SRE), long runs emphasis (LRE), gray level non-uniformity (GLN), run-length non-uniformity (RLN), run percentage (RP), low gray level run emphasis (LGRE) and high gray level run emphasis (HGRE) [ Table 2 ]
Root mean square (RMS):
A fast Fourier transform was then performed, and the resulting power spectrum was analyzed to yield the root mean square (RMS) value.[ 2 ]
where RMS is the root mean square, |F(u,v)| 2 is the power spectral density derived from the fast Fourier transform, and M*N is the size of the region of interest (ROI).
The trabecular bone pattern in the calcaneal radiographs was analyzed by a computerized texture analysis method that has been developed by us in Matlab 6.1. The digitized radiographs were converted to a pixel depth of 8 bits/pixel, i.e., 255 gray levels. Regions of interest (ROIs), 256 × 256 pixels in size (pixel size 100 μm), were then manually cropped at the posterior compressive trabecular bodies [ Figure 1 ] and fed into the algorithm that computes the run-length matrix parameters and root mean square (RMS).
Reproducibility:
The precision of the selection of the region of interest from the same radiograph was estimated by repeatedly cropping the ROI of 256 × 256 pixels twenty five times at different intervals. The coefficients of variation for the texture parameters were 0.85%, 1.02%, 0.76%, 0.84%, 0.97% for five parameters (those mean values are found to be significant among pre- and postmenopausal women), which are all derived from run-length parameters; and was 1.08% for RMS.
The radiograph of the calcaneum was repeated for the same individual two times, and the region of interest from each digitized radiograph was cropped and the coefficient of variation was calculated and it was found to be 2.7%.
Data analysis:
The mean values of the FN-BMD, TR-BMD and the mean values of the run-length matrix derived parameters and the mean RMS value of the PSD were compared using Student's t -test for pre- and postmenopausal women. A visual representation of these values by error bars with 95% confident intervals is shown in Figure 3 , and the linear regressions are shown in Figure 4
The age has an influence on bone density and on trabecular bone texture analysis.[ 10 ] There is a notable decrease in the bone mineral density and an increase in RMS values in advanced age, as is evident from the graph. | Results
The values of mean and standard deviation of age and number of pre-menopausal and postmenopausal women falling under the groups ‘normal,’ ‘osteopenia’ and ‘osteoporosis’ based on the T-score are tabulated [ Table 3 ](classifications based on the local reference data[ 11 ]). The mean and SD of run-length-derived parameters and the RMS values for pre-menopausal women and postmenopausal women are tabulated in Table 4
The mean FN-BMD was 0.813 ± 0.087 g/cm 2 for pre-menopausal women and 0.700 ± 0.098 for postmenopausal women. The mean TR-BMD was 0.676 ± 0.075 g/cm 2 for pre-menopausal women and 0.575 ± 0.095 g/cm 2 for postmenopausal women [ Table 4 ]. The significant level and the confidence interval of the differences for all the texture parameters are given in the Table 5 . | Discussion
These results shows that the run-length matrix derived parameters and the RMS [derived from the power spectral density measure (PSD)] can predict significant alterations in the trabecular architecture, which may be comparable with the decrease in the FN-BMD and TR-BMD as age advances. Other groups have worked on nonfractal characterization of texture. The shape of the trabecular pattern on radius radiographs[ 12 ] showed that this shape correlated with lumbar BMD and age.
Several attributes were studied in parallel by one group on the radiographic bone images.[ 13 ] The statistical methods expressed some local relations between the gray levels of the image; the structural methods studied the distribution and shape of the radiographic patterns of the trabecular bone; and fractal analysis studied the roughness of the image texture by analyzing self-similarity variations over different scales.
In textural characterization, other attempts to evaluate the trabecular structure noninvasively have been undertaken. High-definition macro-radiography and fractal signature were used to analyze and to quantify the trabecular organization in vertebrae,[ 14 ] and they characterized architectural differences between groups of patients with low and high BMD. The fractal dimension was a better discriminator than lumbar spine BMD for distinguishing spine fracture cases.[ 15 ] The same type of analysis on proximal femur radiographs was done and showed that the fractal dimension correlated with compressive strength.[ 16 ] In a group of 10 cases of osteoporosis (bone density below normal and/or vertebral fracture) and a group of 10 controls, radius radiographs by a fractal method based on the Fourier transform were performed and showed that the mean fractal dimension of the two groups was significantly different ( P < 0.05).[ 17 ] | Conclusions
The texture parameters derived by the run-length matrix and the power spectral density (PSD) of bone texture on calcaneus radiographs constitute a new, simple, low-radiation and reproducible assessment of bone status. In this study we have shown that five out of seven texture parameters could predict changes in trabecular network. In addition to in-vivo BMD measure, the alterations in the trabecular network due to transition from pre-menopausal stage to postmenopausal stage were also quantified by the texture analysis of the radiographs. Hence both the BMD and micro-architecture undergo changes with respect to advancing age. This noninvasive analysis may provide information about the trabecular microarchitecture that is independent of bone density but depends on age. This method could be complementary to BMD measurements in assessing bone fragility. Adding information about the trabecular architecture to the bone mineral density would better predict osteoporosis where the DXA-derived bone mineral density had limitations in discriminating the osteoporotic fractured subjects from osteoporotic controls.
Out of seven texture measures derived from run-length matrix, five parameters and the RMS derived from PSD are found to be statistically significant P <0.05 among pre and postmenopausal women showing the evidence of notable alterations of the trabecular architecture in addition to bone mineral density. The methodology applied is simple, widely available (computer with a film scanner) and less expensive and more informative. | The in vivo evaluation of trabecular bone structure could be useful in the diagnosis of osteoporosis for the characterization of therapeutic response and understanding the role of parameters other than bone mineral density (BMD) in defining skeletal status. This study was made to evaluate changes taking place in the trabecular architecture of bone with age and menopausal status in women. The findings are compared with the femoral neck bone as well as the trochantar bone mineral density determined by dual energy X-ray absorptiometry (DXA), which is a standard reference test for evaluation of osteoporosis. Seventy females were recruited for the study, 25 premenopausal (mean age ± SD: 39.4 ± 3.8) and 45 postmenopausal (mean age ± SD: 57.9 ± 7.9) women. The right femoral neck bone mineral density was measured for them by dual energy X-ray absorptiometry (DXA). For the same individuals, lateral view radiographs of the right calcaneum were taken as well. The radiographs were digitized and the region of interest (ROI) of 256 × 256 pixels was selected, the run length matrix was computed for calculating seven parameters [ Table 1 ] and the two dimensional fast Fourier transform of the image was calculated. Using the FFT, the power spectral density (PSD) was derived and the root mean square (RMS) value was determined. Our results confirm that age has a significant influence on the texture of the trabecular bone and bone mineral density. | Osteoporosis has been defined as ‘a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk’ (WHO-1994). Data about prevalence of this disease worldwide shows that 25% of women over the age of 50 succumb to breakage of bone due to low bone mass, and half of them (12.5%) have risk of osteoporosis. In case of men, about 8% of them have been found to suffer from osteoporosis. In India the published data was scarce till 1990, though there were several cases of bone fracture and low bone mass. Now as per estimation, there are about 12 million cases of osteoporosis and further increments are likely due to greater longevity, poor calcium and vitamin D intake, nutritional fads and poor acceptability of hormone replacement therapy (HRT).
Bone mineral density is known to decrease as age advances. Osteoporosis causes significant morbidity and loss of quality of life. Mortality is greater in patients who have osteoporosis in middle-aged and older populations.[ 1 ] Especially, the condition is more frequent in postmenopausal women. Osteoporosis is characterized by an abnormal loss of bone mineral content, which leads to a tendency toward nontraumatic bone fractures or to structural deformations of bone.[ 2 ] Accurate estimation of the bone mineral density (BMD) has been an important diagnostic indicator for determining osteoporosis and for follow-up study of the patient under the therapy for osteoporosis. In this context, various BMD-measuring tools have been developed. Dual energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT) are typical methods of measuring BMD.
Though BMD is a useful concept, it does not give information about the trabecular structure of the bone. Noninvasive and/or nondestructive techniques can provide structural information about bone, beyond standard bone mineral density (BMD). While the latter provides important information about osteoporosis diagnosis and fracture risk assessment, considerable evidence indicates that BMD only partially explains bone strength and fracture resistance. Quantitative assessment of macro-structural characteristics such as geometry and section modulus; and micro-structural features such as relative trabecular volume and trabecular spacing, number and connectivity may improve our understanding of osteoporosis and our ability to estimate bone strength and predict fractures. The rationale for imaging bone macro-structure/microstructure, therefore, is to obtain information beyond BMD, improve fracture risk prediction, clarify the pathophysiology of skeletal disease, define the skeletal response to therapy and assess biomechanical relationships.
The most important aspect of osteoporosis is fractures in femoral neck and vertebrae. Especially, fracture in femur leads to about 20% mortality in case of older osteoporotic populations. Though BMD is a major indicator of bone strength, many studies have also shown that BMD alone cannot fully predict the possibility of osteoporotic fracture[ 3 ] and that other factors such as microstructure of trabecular bone and loading distribution have a significant effect on osteoporotic bone fracture.[ 3 ] In addition to the BMD measure, it is necessary to monitor the corresponding alterations in the trabecular microarchitecture.
Medical image processing has become the most important research topic, with development of various imaging tools and high-performance computer facilities. In particular, plain radiographic image processing has been extensively studied because radiography is widely available and relatively inexpensive. One of the well-known diseases which can be screened via plain radiographic images is osteoporosis.
Osteoporosis is established when decrease in bone mass greater than that expected for a person of a given age, sex and race is present and when it results in structural bone failure manifested by the occurrence of fractures following trivial trauma. Therefore, microstructure of trabecular bone, in addition to bone mineral density, should be considered to better predict the possibility of osteoporotic fractures. In the past decade, structural measures of trabecular bones have been studied in relation with osteoporotic fracture risk. Three-dimensional measure of trabecular bone may be the ideal measure. Recently, flat-panel-based micro CT has been extensively studied and will be used for real three-dimensional studies of trabecular bone. On the other hand, many studies have been focused on quantifying two-dimensional trabecular pattern in slice images of CT and magnetic resonance (MR). Studies on plain radiographs have been also performed to assess in vivo trabecular structures. These studies are mainly done on anatomic sites such as femur and the spine.[ 4 6 ] The calcaneum has been chosen as the site of measurement on the skeleton as it is rich in trabecular bone and is not covered by thick soft tissue. Trabecular structure of the bone is dynamic and contributes to the strength of the bone. Bone mineral density gives a value of the average distribution of bone mineral at the site of interest, i.e., the areal density. However, bone mineral in reality is distributed three-dimensionally in the trabecular architecture whose number and orientation contribute to bone strength. Therefore, study of the trabecular structure of bone is likely to be of great value in evaluating osteoporosis, in addition to bone mineral density. | The authors thank the Indian Council of Medical Research (ICMR) for the financial contributions for this study. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):161-168 | oa_package/f0/9a/PMC3014101.tar.gz |
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PMC3014102 | 21224927 | Materials and Methods
The TBI technique was based on a set of parallel-opposed, anterior-posterior fields using a 60 Co unit with the gantry rotated to project a horizontal beam. The beam collimator was set at 45° so that the field diagonal was projected onto the horizontal plane. The collimator was opened to its maximum field size of 35 × 35 at 80 cm from the source and the Source-Surface Distance (SSD) was 250 cm. The prescribed dose to the umbilicus was 13.5 Gy given in six fractions of 2.25 Gy each twice a day over three days.
Basic dosimetric parameters (PDD, SC, SP, DR)
In order to calculate the required time for TBI treatment, the following equation was used:
where the collimator scatter factor (SC) is defined as the ratio of the output in air for a given field to that for a reference field (10 × 10); the phantom scatter factor (SP) is defined as the ratio of the dose rate for a given field at a reference depth (depth of maximum dose) to the dose rate at the same depth for the reference field size (10 × 10) with the same collimator opening; percentage depth dose (PDD) is expressed as a percentage of absorbed dose at any depth to the absorbed dose at a fixed reference depth (depth of maximum dose) along the central axis of the beam and DR is defined as the output in the phantom for the reference field size (10 × 10) at the depth of the maximum dose.[ 1 5 ] SC, SP, PDD and dose rate (DR) were measured in this study.
Depth dose measurements
Depth dose measurements along the central beam axis were measured in a 30 × 30 × 30 cm 3 water phantom at a 250-cm source-surface distance using a 0.6-cc Farmer chamber (TM30010, PTW Freiburg) and an electrometer (PTW-UNIDOS). The depth dose fall-off from 2.5 to 25 cm was measured. For surface dose measurements and for relative measurements in the build-up region, a parallel plate chamber (TM23343-PTW Freiburg) was used as previously described.[ 4 ] This chamber was put on the water phantom and variable thicknesses of a water-equivalent sheet (T40006, RW3 slab phantom-PTW Freiburg) were placed over the chamber before measurements were taken and corrected for the slight SSD variation.[ 11 12 ] A comparison of this depth dose data was made with the measured depth dose at 80 cm SSD and transformed using the Mayneord formula to the TBI distance (SSD = 250 cm).[ 1 4 11 12 ]
Scatter factors
The SC measurement for TBI (with settings: SSD = 250 cm, field size = 35 × 35, collimator angle = 45) was performed using the Farmer ion chamber (0.6 cc) with a cylindrical cap having a radius equal to the electron build-up depth for 60 Co.[ 1 5 ] In order to measure the phantom scatter factor the following equation was used:
where in this equation, SC,P is the total scatter factor [defined as the dose rate at a reference depth for a given field size divided by the dose rate at the same point and depth to the reference field size (10 × 10)]. SC,P was acquired from measurements made in the build-up region. The irradiation was based on the calculated time through measurement data and prescribed dose to the umbilicus. In order to check the accuracy in dose delivery, a human phantom which had three sections (head and neck, trunk, hip) was used.[ 13 ] A 0.3-cc ion chamber (TM31013, PTW-Freiburg) was imbedded in the phantom at the prescribed point (umbilicus) and the delivered dose was measured.
Effect of phantom (patient) length
The dose absorbed at any given point depends on the scattering volume surrounding the point.[ 4 12 ] Measurements were made to determine the effect of patient length on absorbed dose. The central axis of the water phantom was monitored at several depths while the longitudinal extent of the phantom was changed. Initial measurements started with a 30 × 30 × 30 cm 3 water phantom and additional material of slab phantoms was added to one side.
Midplane dose determination Calibration technique
For obtaining the entrance and exit measurements of the human phantom, four p-type diodes (T60010L) were used as dosimeters connected to a MULTIDOS electrometer (T10004). For entrance measurements, calibration diodes were taped on the 30 × 30 × 30 cm 3 water phantom at an SSD of 250 cm with a 35 × 35 field size. The calibration of these diodes was performed against the 0.6 cc Farmer chamber at depth of 0.5 cm connected to a UNIDOS electrometer (T10001).
The calibration factor (F) was then determined as the ratio of the absorbed dose measured with the Farmer chamber (D) to the reading of the diode (M) in TBI experimental conditions. The exit calibration was the same as the entrance calibration with the exception that the water phantom was turned at 180°.
Target dose calculation
Using combined entrance and exit dose measurements (D en , D ex ), one can estimate the dose delivered to a point placed in the target volume (midplane at umbilicus). In our center, the midplane dose was estimated by three different algorithms: The first algorithm is the arithmetical mean of the entrance and the exit doses. The second algorithm is the geometric means of the entrance and the exit doses.[ 3 ] The conversion of entrance and exit doses to any other point is performed using the percentage depth dose corresponding to that point. For example, to obtain D mid from the entrance dose (D en ), we can multiply D en by PDD mid and to obtain D mid from the exit dose (D ex ), we can multiply D ex by the ratio of PDD mid /PDD ex as shown below:
To reduce the statistical error in the measurements, the average of Eqs. (5) and (6) was calculated and the third algorithm was expressed as:
where PDD corresponds to the percentage depth dose measured in TBI experimental conditions and D en and D ex are entrance and exit doses respectively. The validity of these algorithms was investigated using the human phantom.[ 13 ] Surface doses were measured by diodes and the positioning of these detectors was carefully carried out in order to avoid a shadowing effect. Midplane doses were measured with the 0.3 cc ionization chamber (TM31013).
Dose uniformity
The human phantom was used to investigate dose uniformity throughout the whole body. Diodes were taped on the anterior and posterior surfaces of the human phantom (at six points) and AP/PA radiation was given [ Figure 1 ]. Measured doses at different sites were compared to that at the umbilicus.[ 10 ] The delivered dose to the mid-lobe of the lung was also measured with the ionization chamber (0.3 cc). In order to reduce the lung dose, a proper Cerrobend shield (1.14 cm thickness) was built.
Lung shield
A mobile X-ray set placed at the position of 60 Co was used to take AP and PA films covering the thoracic region of the human phantom based on TBI experimental conditions described. The films used to design the lung shield made from Cerrobend. To simplify planning and treatment, the AP and PA films were overlaid by the physicist and a combined outline was used to define the outline shield.[ 14 15 ] Additionally, in order to maintain the lung shield, a TBI stand consisting of 11 steel pieces with dimensions of 70 × 100 × 210 cm 3 was built, which appears to be also useful for patient support [ Figure 2 ]. | Results
The central axis depth dose data for 60 Co for different field sizes (5 × 5 to 35 × 35 cm) at TBI treatment distance (250 cm SSD) are shown in Table 1 . The results of depth dose data measured at 100 cm SSD and transformed using the Mayneord formula to the TBI distance are also shown in Table 1 . The data transformed using the Mayneord formula is about 8% lower than the measured data for most of the distance at 2 to 25 cm depth. The mean differences between the calculated and measured percentage depth doses were 3.09% with a standard deviation of 2.51%, whereas in the large field used in treatments of TBI (35 × 35), this difference was 0.71% with a standard deviation of 0.96%.
The depth dose fall-off from 2.5 to 25 cm depth for a 35 × 35 cm field size is shown in Figure 3 .
The surface dose and the central axis depth dose in the build-up region for TBI treatment distance are shown in Figure 4 . The results show a plateau at 5 mm and a relative surface dose of 84%.
The measured dose at the umbilicus based on SC and SP ( Eq. 2 ) was 2.31 Gy. From this measurement, the accuracy in dose delivery at the prescribed point (umbilicus) was found to be 2.59%. Results presented in Table 2 show the effect of the doses measured at 0.5, 5 and 10 cm as a result of increasing scattering volume added in the phantom (patient) length direction. It shows that the increased scattering volume reaches a plateau at a depth of around 51 cm.
The ratios of the measured to the calculated midplane doses based on the three algorithms are summarized in Table 3 . The accuracy in the arithmetical mean algorithm was above 10%. The accuracy in the geometrical mean algorithm was within 4% and it was within 3.5% in our proposed algorithm. The mean difference between the calculated and measured doses in the first algorithm was 0.94 with a standard deviation of 0.61%; in the second algorithm, it was 2.82 with a standard deviation of 1.76% and in the third algorithm, it was 0.82 with a standard deviation of 0.54%.
Point dose measurements at anterior and posterior of human phantom are summarized in Table 4 . These results show that dose uniformity at the anterior surface was within 6% and it was within 5% at the posterior surface. The delivered dose to the mid-lobe of the lung with and without the shield is presented in Table 5 . It has been seen that the delivered lung dose without the shield was 14.14 Gy but by applying a proper shield (1.14 cm thickness), it has been reduced to 8.16 Gy. | Discussion
The results in Table 1 illustrate a poor coincidence between the calculated and measured percent depth doses for small and intermediate fields. But as the collimator opening was comparable with the mean range of scattered photons, the scatter contribution to the dose reached its maximum. Hence, the calculated percent depth dose using the Mayneord formula which does not account for a change in the scatter component showed increasing agreement with the measured data. However, for the largest field, deviation is minimum and there is no significant difference between them. It can be suggested that, for the simplicity, the calculated percentage depth dose be used instead of the measured percentage depth dose. The result in Figure 3 demonstrates that the depth dose fall-off from 2.5 to 25 cm is nearly linear. Hence, a combined parallel opposed irradiation produces dose uniformity across a homogeneous transverse section of the patient.
The results of the build-up dose are shown in Figure 4 . Skin sparing is undesirable for some treatment situations. For example, an adequate skin dose must be maintained for patients who have a generalized disease such as leukemia, where leukemia blast cells can be presumed to be circulating in the capillary bed immediately beneath the skin surface. Another example is neuroblastoma. This tumor is distributed widely throughout the body and in superficial bones lying less than 1 cm beneath the surface.[ 2 ] Therefore, the skin dose should be high in neuroblastomas as well. As shown in Figure 4 , the skin dose is 84%, which is one of the advantages of using 60 Co to an accelerator option in the TBI treatment, as the skin dose may be smaller with high energy accelerators. As mentioned earlier, the results based on the measurements of basic dosimetric parameters confirmed that the measured dose to the umbilicus was within 3% of the prescribed dose to the umbilicus. The accuracy and variation of the dose delivered to the umbilicus is considered to be clinically acceptable.[ 1 ] The implication of the results in Table 2 is that adding scatter material around the phantom does not changes depth doses significantly and The effect of phantom (patient) length on absorbed dose effect would be canceled. It can be seen from Table 3 that using both calculation methods, proposed and arithmetical mean algorithms, the dose agrees with the measured dose within 4%. But the geometric mean algorithm has a larger error and is not acceptable. Among the three algorithms discussed earlier, the proposed algorithm appears to be more appropriate for the determination of the midplane dose from entrance and exit measurements in TBI treatment. The results in Table 4 show that the difference between doses in the whole body relative to the umbilicus is within ±7%, which is in agreement with the results obtained by Harden.[ 15 ] The delivered dose in some areas such as the neck is higher because of the combination of reduced beam intensity and a relative lack of scattered material in this area. The lung region shows an increased dose relative to the prescribed dose to the umbilicus due to its density. However, by applying a shield, it has been reduced to 8.16 Gy which is below the tolerance level.[ 14 ] The TBI stand made in this project has the capability of being extended for shields for organs other than the lungs, for example, the eyes of patients undergoing TBI treatment. | Conclusion
Basic dosimetric parameters which are necessary in TBI treatment were measured in this study. The proposed algorithm by this work appears to be useful with an accuracy within 2.5%. Accuracy in dose delivery was within 3% and dose uniformity through the whole body was within ±7%. | Total Body Irradiation (TBI) is a form of radiotherapy used for patients prior to bone marrow or stem cell transplant to destroy any undetectable cancer cells. The dosimetry characteristics of a 60 Co unit for TBI were studied and a simple method for the calculation of the prescribed dose for TBI is presented. Dose homogeneity was verified in a human phantom. Dose measurements were made in water phantom (30 × 30 × 30 cm 3 ), using farmer ionization chamber (0.6 cc, TM30010, PTW) and a parallel plate ionization chamber (TM23343, PTW). Point dose measurements for AP/PA irradiation were measured in a human phantom using silicon diodes (T60010L, PTW). The lung dose was measured with an ionization chamber (0.3 cc, TM31013). The validity of the proposed algorithm was checked at TBI distance using the human phantom. The accuracy of the proposed algorithm was within 3.5%. The dose delivered to the mid-lobe of the lung was 14.14 Gy and it has been reduced to 8.16 Gy by applying the proper shield. Dose homogeneity was within ±7% for all measured points. The results indicate that a good agreement between the total prescribed and calculated midplane doses can be achieved using this method. Therefore, it could be possible to use calculated data for TBI treatments. | Total body irradiation (TBI) is a part of a complex treatment program for aplastic anemia, leukemia, lymphoma and certain other cancers that require chemotherapy and bone marrow transplantation. TBI also helps to cause immunosuppression that is necessary to keep the recipient's immune system from rejecting the bone marrow transplant.[ 1 2 ] Hematologists, immunologists, radiotherapists and medical physicists have been trying to increase the success rate of TBI treatments. Improvement requires an understanding of all the clinical, biological and physical aspects.[ 3 ] There is no standard TBI technique as hospital radiotherapy departments adopt their own treatment plans that are influenced by the type of treatment facility available.[ 4 ] Therefore, different treatment distances, patient set-ups, radiation beams and radiation fields are used in different radiotherapy centers for TBI. Due to the variability of technique, it is not possible to use published data as a reference for another institute. Therefore, if a center wants to implement TBI, it has to measure the basic dosimetric parameters.[ 3 5 ]
In TBI set-ups, the patient represents a very irregular and extended field. When such treatment is used as part of the preparation for a bone marrow transplant, it is important to know the dose delivered throughout the body.[ 6 7 ] The determination of the dose delivered to the body during TBI is not easy, as direct measurements are impossible.[ 3 ] In this study, we present a simple method for absorbed dose determination in 60 Co TBI using entrance and exit dose readings generated by the semiconductor detectors. The calibration of diodes is also presented here based on a previously described study.[ 8 ] The estimated accuracy in dose delivery in TBI as well as standard radiotherapy should be better than ±5%.[ 3 ]
Avoiding dose inhomogeneity is very important for TBI because it can lead to failure of TBI through either insufficient dose being delivered to the marrow stem cells or an excessive dose to the critical organs. Therefore, most techniques aim to achieve dose uniformity throughout the whole body while minimizing dose outside the volume especially to critical structures such as the lung.[ 9 10 ]
The purpose of this study were (i) to measure some basic dosimetric parameters in the TBI condition in order to check the accuracy in dose delivery. (ii) to develop an algorithm for dose calculation in TBI techniques, and (iii) to investigate dose uniformity in TBI treatment using a human phantom and semiconductor diodes. | This research has been supported by a Tehran University of Medical Science and health service grant. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):169-174 | oa_package/9a/67/PMC3014102.tar.gz |
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PMC3014103 | 21224928 | Methods
Simulations of beams of the same/close shape and size, contrast, contrast-to-noise ratio, dose and figure-of-merit
In order to simulate X-ray beams of the same/close shape and size ( spectrally equivalent beams), calculations were carried out for the matching of hardening and attenuation from aluminum and copper filters (reference filters) and other filter materials that are different from aluminum and copper. The matching exercises involved the use of the numerical algorithms earlier reported by Jennings.[ 8 ] The thicknesses of aluminum filter used were between 1 and 6 mm at 1 mm increments while those of copper were between 0.05 and 0.3 mm at 0.05 mm increments. The incident X-ray spectrals on the filter materials were determined by using polynomial functions earlier reported by Boone and Seibert.[ 12 ] Unfiltered tungsten anode spectra ɸ 0 (E i ) in units of photons/mm 2 at energy E i in keV were calculated for values of kVp ranging between 50 and 140 in 10 kVp steps.
The transmission, contrast, contrast-to-noise ratio, figure-of-merit and dose were calculated using equations presented in the theory section above. A total of 10 values of kVp, 8 alternative conventional filter materials (other than aluminum/copper) and 6 reference filter thicknesses of aluminum/copper were considered. Simulations were carried out for the evaluation of T, C, CNR, FOM and DOSE for water phantom of thicknesses ranging between 5 and 30 cm at 5 cm increments. Also considered in these simulations are three contrast media - namely, calcium, barium and iodine; and three intensifying screens, which include CaWO 4 , CsI and Gd 2 O 2 S. By similar simulation exercise, the k-edge filter materials investigated in comparison with aluminum/copper filters include lanthanium, gadolinium, holmium, thulium and tungsten. The interaction data of photons for water (patient phantom) and those for elemental filters that were considered were taken from McMaster et al .,[ 13 ] while those for the contrast media and intensifying screens were from Hubbell and Seltzer.[ 14 ] All the models reported in this work were fitted using the curve-fit least-square routines implemented in the computer package GRAF4WIN.[ 15 ] Figure 1 shows the schematic diagram for the implementation of the computer simulations.
Computer code EQSPECT.FOR
A FORTRAN source codes EQSPECT.FOR was developed for the execution of the simulations of same/close shape and size of beams, C, CNR, FOM and DOSE. In these computer codes, the algorithms reported[ 12 ] for the simulations of energy distribution of X-ray beams permit the specification of values of kVp other than those of multiples of 10 kV (say 86 kV). The interpolations for values of λ m at values of kVp other than multiples of 10 kVp were carried out using cubic spline computer routines (named DEFSAL and DEFSCU) that were embedded in EQSPECT.FOR. A brief description of the main steps in the execution of EQSPECT.FOR is presented in Table 4 . | Results
Figure 2 shows the results of comparison of T, CNR, [CNR] 2 , C, DOSE and FOM for beams transmitted through aluminum/copper and selected alternative filter materials at ‘spectrally’ equivalent thicknesses. Specifically, for peak voltage of 86 kV, Figure 3 shows the results of comparison of the spectra transmitted through 3.7 mm Al and 9.3 mm Al and those through the respective spectrally equivalent thicknesses of copper, 0.1 mm Cu and 0.25 mm Cu, at equal tube loading. Table 5 shows the results of comparison of performance characteristics of other filter materials relative to copper filter. For the purpose of comparison with the work of Nagel,[ 9 ] these results are for 0.2 mm Cu filter, imaging of patient phantom of thickness 20 cm and contrast simulated with iodine (10 mg/cm 2 ) and Gd 2 O 2 S intensifying screen (80 mg/cm 2 ). Figure 4 shows the comparison of spectral shape and size obtained by forcing the same shape and size for aluminum/copper and gadolinium filters using the algorithms reported by Jennings.[ 8 ] The results of the comparison of transmission, contrast, CNR, [CNR 2 ] DOSE and FOM obtained by the matching of spectral shape and size of beams transmitted by aluminum/copper and lanthanium are shown in Figure 5 . To further provide verification of the validity of the formulations (Eqs. 8 – 15 ) presented in this work, the results of comparison carried out for 0.088 mm Gd filter and 0.097 mm Cu filter using EQSPECT.FOR are presented in Table 6 . | Discussion
The value of CNR for the beam filtered by 0.1 mm Cu exceeds that of the beam filtered by 3.7 mm Al by 7.0% for the imaging of a 20-cm patient phantom with the contrast simulated with iodine (12 mg/cm 2 ) and CsI intensifying screen (60 mg/cm 2 ). For this same imaging, the value of CNR for beam filtered by 0.25 mm Cu exceeds that for beam filtered by 9.3 mm Al by 19.0%. For the case of 3.7 mm Al and 0.10 mm Cu filtrations, the values of T Cu , DOSE Cu and [CNR Cu ] 2 exceed those of T Al , DOSE Al and [CNR Al ] 2 by 14.5% respectively while C Cu ≅ C Al and FOM Cu ≅ FOM Al . This is in good agreement with the result from earlier work,[ 8 ] which reported a surplus of 14.8% for T Cu over T Al . At spectrally equivalent thicknesses, the difference in exposure time obtained experimentally for these aluminum and copper filters to produce beam of exactly the same shape and size was 15.6%, with the former filter requiring more exposure time.[ 8 ] This is in reasonable agreement with the theoretical formulations [Eqs. (8) and (9) of this text], which yield 14.5% as the value of ∆ × 100% (where ∆ = λ m t ref ). With the beams from these filters (copper and aluminum at ‘spectrally’ equivalent thicknesses) numerically made exactly equal in shape and size {ɸ Al (E i ) = αɸ Cu (E i )}, the results obtained by simulations were such that T Cu ≅ T Al , DOSE Cu ≅ DOSE Al , CNR Cu ≅ CNR Al , [CNR Cu ] 2 ≅ [CNR Al ] 2 , C Cu ≅ C Al and FOM Cu ≅ FOM Al .
In the case of beam filtered by 9.3 mm Al in comparison with that filtered by 0.25 mm Cu and for spectral without scaling (same mAs and heat capacity), the values of T Cu , DOSE Cu and [CNR Cu ] 2 exceed those of T Al , DOSE Al and [CNR Al ] 2 by 41.9% respectively while C Cu ≅ C Al and FOM Cu ≅ FOM Al . Theoretical result presented[ 8 ] shows this surplus value to be 41.6%. With the transmitted spectrum through the copper filter theoretically scaled {ɸ Al (E i = αɸ Cu (E i )} in comparison with aluminum filter, the results obtained by simulations were such that T Cu ≅ T Al , DOSE Cu ≅ DOSE Al , [CNR Cu ] 2 ≅ [CNR Al ] 2 , CNR Cu ≅ CNR Al , C Cu ≅ C Al and FOM Cu ≅ FOM Al .
Results of spectrometric measurements carried out by Jennings[ 8 ] show that 4.08 mmAl, 0.11 mmCu and 0.10 mm Y transmit spectrally equivalent beams. For 4.08 mm Al in comparison with 0.11 mm Cu and without the scaling of spectral (same mAs and heat capacity), using Eqs. ( 8 – 11 ), the values of T Cu , DOSE Cu and [CNR Cu ] 2 exceed those of T Al , DOSE Al and [CNR Al ] 2 by 16.2% respectively while C Cu ≅ C Al and FOM Cu ≅ FOM Al . The same surplus was found for yttrium in comparison with aluminum at the respective ‘spectrally’ equivalent thicknesses of 0.10 mm Y and 4.08 mm Al while C Y ≅ C Al and FOM Y ≅ FOM Al . This is in good agreement with the results from the work of Jennings,[ 8 ] which reported a difference of 15.3% (experimental) and 16.7% (theoretical) for T Cu and T Al with copper transmitting surplus. Also, reported by the same author[ 8 ] was a difference of 15.4% (experimental) and 16.4% (theoretical) for T Y and T Al with yttrium filter transmitting surplus. With the transmitted spectrum through the copper filter scaled (mAs and heat capacity scaled by the factor α), the results obtained from the simulations carried out in this work were such that T Al ≅ T Cu ≅ T Y , DOSE Al ≅ DOSE Cu ≅ DOSE Y , [CNR Al ] 2 ≅ [CNR Cu ] 2 ≅ [CNR Y ] 2 , C Al ≅ C Cu ≅ C Y and FOM Al ≅ FOM Cu ≅ FOM Y .
Using scaled spectrum {α Φ Cu (E i )}, operating the tube with copper filter at reduced tube loading/heat capacity (α × mAs Al or 1.35α × mAs Al × kVp) results in same value of T, DOSE, C, CNR and FOM when compared with operating the tube with aluminum filter at higher tube loading/heat capacity (mAs Al or 1.35 × mAs Al × kVp). Note that whether the beams are scaled or not scaled, once the spectral shape is the same, the values of contrast and FOM will be the same. However, beams filtered by materials with higher values of atomic numbers are more intense (bigger in shape) and produce higher values of CNR and dose to patient than those filtered by material of lower atomic numbers at ‘spectrally’ equivalent thicknesses when the tube is operated at the same values of kVp, tube loading and heat capacity [Figures 2 and 3 ]. For a given filter material when compared with aluminum/copper at ‘spectrally’ equivalent thicknesses, only the shapes of transmitted spectra are the same. Depending on the Z -value, the sizes are not the same when the tube is operated at same values of kVp, tube loading and heat capacity [ Figure 3 ]. At respective ‘spectrally’ equivalent thicknesses, the benefits derivable from the use of higher Z -value filter material in comparison with those from the use of lower Z -value include higher intensity, lower filter-generated scattered radiation and shorter tube current or exposure time. This can translate into an increase in the lifespan of tube (more filament hours by operating at reduced mAs) and reduction in the cost of dealing with the detrimental effects of heat and scattered radiation. Also, extra images could be obtained using the gain in exposure time and tube loading/heat capacity.
The spectrum obtained by numerically scaling the values of αɸ Cu (E i ) using the factor α ( Eq. 14 ) is in agreement with that transmitted by 3.7 mm Al [ Figure 3 ]. The scaled spectrum obtained from copper filter {αɸ Cu (E i )} and that directly obtained from aluminum {ɸ Al (E i )} were found to produce the same contrast, CNR, DOSE and FOM. Though not carried out in this work, experimentally at the same kVp, the spectra obtained from filter material with higher Z -value (say copper) in comparison with those with lower Z -value (say aluminum) could be obtained at the scaled intensity and dose (scaled to that obtainable from aluminum filter) by operating the tube at scaled tube loading and heat capacity. This scaling factor provides for the means of obtaining strictly equivalent (same shape and size) spectra from two different elemental filter materials (conventional filters) at the same value of kVp [ Figure 3 ].
The validity of Eq. (15) had been reported in Okunade[ 6 ] using the case of peak voltage of 86 kV and comparison of 0.10 mm Cu with the corresponding ‘spectrally’ equivalent aluminum thickness, 3.7 mm Al (reference filter). Also, Figure 3 shows that the formulation presented as T Al = α × T Cu is valid. Hence all the formulations in Eq. (15) are valid. Table 5 shows that the values of ‘spectrally’ equivalent thicknesses obtained using Eqs.( 16 – 18 ) are in good agreement with those earlier reported in literature.[ 8 – 9 ] Apart from providing comparison for aluminum/copper and any given filter material irrespective of atomic number, EQSPECT.FOR can be used to obtain relative performance for any arbitrary pair of filter materials, say silicon and iron. This is accomplishable by specifying the reference filter material as silicon and the alternative filter material as iron.
Generally, it is well known that it is not possible to have a complete spectral matching for conventional and k-edge filter materials. The k-edge discontinuity interferes substantially in the range of useful energies, resulting in beams with significant difference in shape and size. Unlike the case of the comparison of conventional filter materials (aluminum/copper) with other conventional filter materials, large values of the root-mean-square errors[ 8 ] were obtained when conventional filter materials were compared with k-edge filter materials. This implies a large difference between the actual transmission of aluminum/copper and the scaled transmission of the k-edge filters. It is not possible to derive simple mathematical formulations of the form with factors α, δ and ∆ for the comparison of performance of beams from conventional filter materials with those from k-edge filter materials since spectrals transmitted by these pair of filter materials differ in quality and quantity [ Figure 4 ]. Theoretically, the transmission below the k-edge in terms of quality and quantity could not be matched in these comparisons. However, using Eq. (9) , the results presented in Table 6 for the comparison of the overall quantity of photons in terms of the ratios of T and DOSE from beams transmitted by 0.097 mm Cu and 0.088 mm Gd are in reasonable agreement with experimentally measured ratio of exposure time reported by Nagel.[ 9 ] | Conclusions
Models and software program have been presented for the comparative evaluation of the performance characteristics (contrast, contrast-to-noise ratio and FOM) of beams transmitted at spectral equivalent thicknesses by elemental filter materials (conventional and k-edge filters). Under this condition of spectral equivalence, the mathematical formulations presented show that when compared with one of lower Z -value, conventional filter material with higher Z -value offers opportunity for extended exposure time. This extended exposure time can translate into acquiring extra images using the gain in exposure time, tube loading and heat capacity. The computer software EQSPECT.FOR developed in this work can be used as a tool for the comparative investigation of filter materials with a view to determine alternative beams that could yield optimum performance in clinical radiological practice. The results from this comparative investigation can aid in the design of X-ray machines in such a way as to produce a reference shape and size of X-ray beam with which others can be compared. This could assist in the achievement of some degree of uniformity in clinical X-ray diagnostic practice. | In order to achieve uniformity in radiological imaging, it is recommended that the concept of equivalence in shape (quality) and size (quantity) of clinical Xray beams should be used for carrying out the comparative evaluation of image and patient dose. When used under the same irradiation geometry, X-ray beams that are strictly or relatively equivalent in terms of shape and size will produce identical or relatively identical image quality and patient dose. Simple mathematical models and software program EQSPECT.FOR were developed for the comparative evaluation of the performance characteristics in terms of contrast (C), contrast to noise ratio (CNR) and figure-of-merit (FOM = CNR 2 /DOSE) for spectrally equivalent beams transmitted through filter materials referred to as conventional and k-edged. At the same value of operating potential (kVp), results show that spectrally equivalent beam transmitted through conventional filter with higher atomic number ( Z -value) in comparison with that transmitted through conventional filter with lower Z -value resulted in the same value of C and FOM. However, in comparison with the spectrally equivalent beam transmitted through filter of lower Z -value, the beam through filter of higher Z -value produced higher value of CNR and DOSE at equal tube loading (mAs) and kVp. Under the condition of equivalence of spectrum, at scaled (or reduced) tube loading and same kVp, filter materials of higher Z -value can produce the same values of C, CNR, DOSE and FOM as filter materials of lower Z -value. Unlike the case of comparison of spectrally equivalent beam transmitted through one conventional filter and that through another conventional filter, it is not possible to derive simple mathematical formulations for the relative performance of spectrally equivalent beam transmitted through a given conventional filter material and that through kedge filter material. | The use of tube voltage (peak kilovoltage, kVp), effective energy, homogeneity coefficient and half-value layer (HVL) as measures of beam quality and quantity in medical diagnostic radiology continues to produce diversities in results obtained for image quality and patient dose. As a result of this, the use of equivalent spectral as a measure of beam quality and as a tool for the standardization of medical X-ray imaging has been reported.[ 1 – 2 ] Among other factors, the quality and quantity of X-ray beams for medical diagnostic purpose depend on filter material and filter thickness. The standardization of beam quality can be achieved by providing a ranking for filter materials and filter thicknesses when the technology of X-ray tube is the same. By careful choice of thickness, two different elemental filters can be made to transmit beams that are simultaneously of the same/close shape (quantity) and size (quality).[ 3 – 6 ] When used under the same irradiation condition, beams that are strictly equivalent or relatively equivalent in terms of shape and size will produce identical or relatively identical image quality and patient dose. The results of theoretical and experimental approaches to the comparative ranking of the transmissions of filter materials on the basis of closeness of hardening (size) and attenuation (quantity) properties have been reported earlier in literature.[ 7 – 9 ] Due to the differences in attenuation and absorption properties, two different elemental filters that can independently produce beams of exactly equal shape and size do not exist.
In this paper, computer simulations were carried out to obtain spectrally equivalent X-ray beams using elemental filters - which include those referred to as conventional, such as aluminum and copper; and k-edge, such as gadolinium and tungsten. Analytical models similar to those earlier reported[ 6 ] for quantitative parameters were derived for the evaluation of differences in parameters for image quality, such as contrast-to-noise ratio (CNR) and figure-of-merit (FOM) for filter materials that transmit spectrally equivalent beams. For the purpose of clinical assessment of the performance characteristics of spectrally equivalent beams, iterative computer software EQSPECT.FOR was developed for the evaluation of these models. Also, by least square methods, parameters which can be used for the evaluation of thicknesses of other filter materials that are ‘spectrally’ equivalent to a specified thickness of copper are presented. The definitions of some of the terms used in the computer simulations and analytical formulations that were derived are presented in Table 1 .
Theoretical formulations
In this work, T (t m ) of filter material m at ‘spectrally’ equivalent thickness of t m is given by
C is defined as
E a and E b were calculated respectively using equations of the forms[ 10 ]
and
CNR and FOM were respectively calculated as
and
The absorbed dose in patient (water phantom of thickness t w centimeters) was determined using equation of the form[ 11 ]
where ε(t w ) could be obtained using an equation of the form
Details of the derivation of Eq. (7) can be found in the work reported by Okunade.[ 11 ]
From the earlier work reported,[ 6 ] for a given filter material m in comparison with aluminum filter at spectrally equivalent thickness,
and
where ∆ = λ m t Al .
The values of λ m resulting in best fit to Eq. (9) are reported in Table 1 of Okunade[ 6 ] for filter materials m with atomic numbers ranging from 12 to 39 and kVp ranging between 50 and 140 when reference filter is assumed to be aluminum.
From the simulated data and curve-fitting exercises carried out in this work,
Calculations carried out using copper as reference filter in comparison with filter material m resulted in equation of the form
where ∆= λ m t cu
The values of λ m resulting in best fit to Eq. (11) are reported in Table 2 of this text. Thus from Eqs. (5) , (9) and (10) , for spectrally equivalent beams emanating from filter material m and aluminum filter at ‘spectrally’ equivalent thicknesses respectively, FOM is of the form
and the CNR for beam transmitted through filter material m in comparison with that through a reference filter (aluminum) is of the form
In order to obtain spectral that is exactly equal in shape and size (or that will produce exactly the same values of transmittance, T, contrast, C, dose, DOSE, contrast-to-noise ratio, CNR, and figure-of-merit, FOM), the spectral data generated from filter material, say copper, in comparison with those from aluminum at ‘spectrally’ equivalent thickness have to be scaled across the entire energy spectrum by using the factor α. Mathematically, for this, we can write,
where α = 1 1 + ∆
Note that the values of number of photons (per energy bin E i ) transmitted by a filter with higher Z -value are greater than those transmitted by filter of lower Z -values when the tube is operated at the same kVp and tube loading. The use of this scaled spectral results in models of the form
The thickness of a given material, t eq , that is ‘spectrally’ equivalent to a given aluminum thickness t Al is of the form[ 6 ]
The values of ω n resulting in best fits to Eq. (16) when Z -value ranges between 12 and 39 are obtainable from Table 5 of Okunade.[ 6 ] When Z -values are 12, 13 and 14, the value of the thickness (t eq ) of other filter material that is ‘spectrally’ equivalent to copper thickness t cu can be obtained from equation of the form
where κ n is given by
When Z -values are 23, 26, 28, 30, 32 and 39, the value of the thickness (t eq ) of other filter material that is ‘spectrally’ equivalent to copper thickness t cu can be obtained from equation of the form
The values of ω n and α n resulting in best fits to Eqs. (17) and (18) are shown in Table 3 of this text. | CC BY | no | 2022-01-12 15:33:33 | J Med Phys. 2007 Oct-Dec; 32(4):175-184 | oa_package/cb/2e/PMC3014103.tar.gz |
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PMC3014110 | 20840335 | INTRODUCTION
Pathological gambling (PG) is a fairly common disorder with an estimated point prevalence of approximately 1% ( Welte et al . 2001 ). PG often results in severe psychosocial problems ( Petry & Kiluk 2002 ; Potenza et al . 2002 ). Currently, PG is classified as an impulse control disorder, but the diagnostic criteria closely resemble those of substance dependence. In addition, recent studies have shown neurobiological similarities between PG and substance dependence ( Petry & Kiluk 2002 ; Potenza et al . 2002 ; Goudriaan et al . 2004 ). As a consequence, some authors have proposed to reclassify PG as a behavioural addiction in DSM-V ( Petry 2006 ; Potenza 2006 ).
Increased cue reactivity coupled with heightened attention for addiction-related cues represents an important mechanism in the development of addictive behaviors ( Goldstein & Volkow 2002 ) and may promote relapse in substance dependence ( Cooney et al . 1997 ; Kosten et al . 2006 ; Marissen et al . 2006 ). Functional imaging studies using cue-exposure paradigms in nicotine, alcohol and cocaine dependence have reported increased ventral prefrontal, insular, amygdala, striatal, and thalamic activity, brain regions associated with emotion processing and motivational behavior. In addition, attentional and cognitive control circuitry has been implicated in neuroimaging cue reactivity studies, indicated by increased dorsolateral prefrontal, anterior cingulate cortex and parietal activation ( Kilts et al . 2001 ; Tapert et al . 2004 ; David et al . 2005 ; Kosten et al . 2006 ; McBride et al . 2006 ; Franklin et al . 2007 ).
About 50% of pathological gamblers who try to quit experience a relapse with seriously negative consequences ( Hodgins & el Guebaly 2004 ), and other studies indicate frequent relapses in treatment-seeking pathological gamblers ( Ledgerwood & Petry 2006 ). Because cue reactivity is a key mechanism in the development of addictive disorders, and because it has been associated with a higher risk of relapse in substance dependence ( Cooney et al . 1997 ; Kosten et al . 2006 ; Marissen et al . 2006 ), investigating the neurobiological mechanisms of cue reactivity in this population is highly relevant. So far, only two functional magnetic resonance imaging (fMRI) studies on exposure to gambling-related cues in pathological gamblers have been published ( Potenza et al . 2003 ; Crockford et al . 2005 ). Both studies employed video fragments of gambling-related and various control scenes, but yielded inconsistent results. In the first study among 10 pathological gamblers and 11 normal controls, PG subjects revealed decreased, rather than increased activation in the ventral anterior cingulate cortex, orbitofrontal cortex, basal ganglia and thalamus during gambling-associated versus control epochs. Increased activation during viewing of gambling-related material was found in the occipital lobe only ( Potenza et al . 2003 ). In the second study in 10 pathological gamblers and 10 healthy controls (HC) ( Crockford et al . 2005 ), PG subjects showed higher brain activation in response to gambling stimuli in the left occipital cortex, left fusiform gyrus, right parahippocampal gyrus and right prefrontal areas, compared with HC.
Thus, whereas these PG studies indicate increased activation of brain regions involved in attention, memory and visual processing, no evidence for abnormally increased activity in limbic structures during processing of gambling cues was found (e.g. increased activation in amygdala), unlike neuroimaging studies on cue reactivity in substance dependence ( Kilts et al . 2004 ; Tapert et al . 2004 ; Kosten et al . 2006 ; McBride et al . 2006 ; Franklin et al . 2007 ). Possible reasons for this discrepancy are the use of videos instead of pictures and lack of power because of small sample sizes. Furthermore, both studies enrolled gamblers recruited through advertisements, and neither study investigated whether treatment-seeking problem gamblers (PRGs) would differ in cue reactivity to gambling cues from normal controls. In an fMRI study focusing on the processing of rewards in pathological gamblers ( Reuter et al . 2005 ), a blunted response to wins versus losses was found in the limbic reward areas in pathological gamblers versus HC. When presenting pathological gamblers with gambling videos, the limbic system may thus be relatively underactivated because of a diminished response to gambling situations in which money is gained. Given this blunted response to monetary gains, the investigation of limbic activation to gambling cues versus neutral cues that do not include monetary gain may provide insight in cue reactivity to general gambling cues.
In the present study, we wanted to address these issues by investigating brain activation patterns to gambling or smoking cues in chronic PRGs seeking treatment, heavy smokers (HSM) and non-smoking non-gambling healthy controls (HC). We employed an event-related picture paradigm ( George et al . 2001 ; Myrick et al . 2004 ; Smolka et al . 2006 ) because this provides optimal flexibility with regard to stimulus timing and avoids modeling problems which may arise when analyzing video paradigm fMRI data. In order to compare cue reactivity in PRG to cue reactivity of a substance-dependent group, a comparison group of HSM was included as well. An HSM control group was chosen because the neurotoxic effects of nicotine are limited compared with those of other drugs of abuse, such as alcohol ( Sullivan 2003 ; Mudo, Belluardo & Fuxe 2007 ). Based on previous cue-reactivity studies in substance dependence, we hypothesized that gambling cues in PRG and smoking cues in HSM would elicit higher brain reactivity compared with brain reactivity in healthy non-smoking controls in brain regions associated with emotion processing and motivational behavior such as the amygdala, ventral striatum and ventral prefrontal cortex, and in attention and cognitive control-related brain areas such as the dorsal prefrontal cortex and anterior cingulate cortex (ACC). In addition, the relation between cue-related brain activity and subjective craving in PRG and HSM was studied. We hypothesized that subjective craving would be associated with increased activation in emotion and motivation-related brain areas in PRG and HSM. | MATERIALS AND METHODS
Subjects
Nineteen treatment-seeking PRG (four left-handed), 19 HSM (three left-handed) and 19 non-smoking HC (one left-handed), all males, participated in this study. For two PRG, one HSM and two HC, magnetic resonance imagine (MRI) data could not be (completely) acquired because of scanner failures. Therefore, 17 PRG, 18 HSM and 17 HC constituted the three groups used for statistical analysis. The PRG were recruited from two Dutch addiction treatment centers. The HSM and the HC group were recruited through advertisements in newspapers.
The main inclusion criterion for PRG was current treatment for gambling problems. PRG were interviewed with section T of the Diagnostic Interview Schedule ( Robins et al . 1998 ) to assess the diagnostic criteria for a DSM-IV-TR diagnosis of PG. In addition, the South Oaks Gambling Screen (SOGS; Lesieur & Blume 1987 ) was administered as a measure of problem gambling severity. Two PRG failed to meet the criteria of a current DSM-IV-TR PG diagnosis. However, because they did meet two PG criteria currently, met PG criteria in the past and their SOGS scores (7 and 8, respectively) were similar to the PRG that did fulfill diagnostic criteria for PG (see Table 1 ; mean SOGS score = 9.6 ± 2.6), these PRG were included in the analyses. All PRG were abstinent from gambling for at least 1 week. HSM were included if they smoked at least 15 cigarettes per day, and did not engage in gambling activities more than twice a year. HSM were current smokers who engaged in an experimental smoking cessation as part of this study. The Fagerström Test for Nicotine Dependence (FTND) served as an indicator of nicotine dependence severity ( Heatherton et al . 1991 ). No minimum score on the FTND was required for HSM. HSM had to be overnight smoking abstinent, filled out questionnaires in the morning and were scanned in the afternoon (16–18 hours abstinent). Abstinence was confirmed with a breath carbon monoxide measurement in the morning, using a micro+ Smokerlyzer (Bedfont Scientific, Ltd., Rochester, UK). HC never smoked, did not have a history of problem gambling and did not engage in gambling activities more than twice in the last year.
Exclusion criteria for all groups were: age under 18 years; difficulty reading Dutch; use of psychotropic medication; a lifetime diagnosis of schizophrenia or psychotic episodes; a 12-month diagnosis of manic disorder, assessed with the respective sections of the Composite International Diagnostic Interview (CIDI; Heatherton et al . 1991 ; World Health Organization 1997 ); current treatment for mental disorders other than those under study; physical conditions known to influence cognition or motor performance (e.g. multiple sclerosis, rheumatic disease); positive urine screen for alcohol, amphetamines, benzodiazepines, opioids or cocaine; consumption of more than 21 units of alcohol per week. Groups were mutually exclusive with regard to the psychiatric disorder under study. For instance, PRG and HC did not smoke (with the exception of one PRG who smoked less than five cigarettes a day). Additional exclusion criteria for HC and HSM, but not for PRG, were presence of anxiety disorders (CIDI-section D), depression (CIDI-section E), obsessive–compulsive disorder (CIDI-section K), post-traumatic stress disorder (CIDI-section K) and attention-deficit/hyperactivity disorder (Conners ADHD Rating Scales; Conners & Sparrow 1999 ). PRG with these comorbid disorders were not excluded, because problem gambling is highly comorbid with these disorders. The severity of depression symptoms was assessed with the Beck Depression Inventory (BDI-II; Beck et al . 1996 ). Problematic alcohol use was screened with the Alcohol Use Disorders Identification Test-Consumption ( Bush et al . 1998 ).
In addition to the Cue Reactivity Task, a probabilistic reversal learning task, a planning task and a stop signal task were administered. Results from the reversal learning task and the planning task are reported elsewhere ( de Ruiter et al . 2009 ). The ethical review board of the Academic Medical Center approved the study and written informed consent was obtained. Participants were reimbursed with €50 transferred to their bank account following participation.
fMRI paradigm: Cue Reactivity Task
A picture two-choice response task was used (for examples of pictures, see Fig. 1 ). Pictures were matched for complexity as follows: an equal number of overview pictures and detail pictures was selected for each condition (e.g. several persons gambling, smoking or talking, versus detailed pictures of a hand at a slot machine, a hand with a cigarette, a hand with a magazine). Second, to match for picture complexity and comparability, all pictures were taken in a similar naturalistic setting (e.g. all pictures with multiple persons were taken with multiple objects in the background), only males were included on pictures, and care was taken to match for emotional expressions between the different pictures, by including only photos with neutral face expressions. Thirty gambling pictures, 30 smoking-related pictures, 30 neutral pictures and 30 low-level baseline pictures were presented randomly, with the restriction that a stimulus of the same stimulus category was not presented more than three times in a row. Low-level baseline pictures with arrows pointing to the left or right were presented, and a left or right response had to be given, in order to be able to compare complex picture processing compared with low-level visual processing. In the gambling, smoking-related and neutral pictures, participants had to press a response button with their left index finger when a face was present in the picture and had to press a response button with their right index finger when no face was present. Fifty percent of all pictures within each category contained a face. Each picture was presented for a fixed period of 5 seconds, and participants were requested to respond within this time period. When no response was made after 5 seconds, the task proceeded. A 2.5-second blank screen was presented between each picture. No feedback was given about right or wrong responses. The scanning session lasted 15 minutes; each of the gambling, smoking-related and neutral pictures was presented once. Subjects were not encouraged to respond as quickly as possible. The task was explained and practiced outside the scanner using other pictures. The performance parameter for the task was mean reaction time to the pictures in each stimulus category.
Urge questionnaires
An 8-item gambling urge questionnaire, range 1–7 (MN Potenza & SS O'Malley, unpublished data) and a 10-item smoking urge questionnaire, range 1–7 ( Tiffany & Drobes 1991 ), were included to assess levels of gambling and nicotine craving, respectively. Participants filled out the urge questionnaires before and immediately after fMRI scanning.
Imaging acquisition and preprocessing
Imaging data was obtained using a 3.0 Tesla Philips Intera full-body fMRI scanner equipped with a standard SENSE RF head coil (Quasar gradient system, Philips Medical Systems BV, Eindhoven, the Netherlands) located at the Academic Medical Center, Amsterdam. While participants performed the task, T2*-weighted echo planar images, sensitive to blood oxygenation level-dependent (BOLD) contrast were acquired (35 axial slices, voxel size 3 × 3 × 3 mm, interslice gap 0.3 mm, matrix size 64 × 64 mm, bandwidth 90 kHz, TE 35 ms, repetition time 2.28 seconds), covering the entire brain except for the inferior regions of the cerebellum. A sagittal T1-weighed structural scan (voxel size 1 × 1 × 1 mm, 170 slices) was made in order to co-register it with the fMRI data. Imaging analysis was done using SPM2 (Statistical Parametric Mapping; Wellcome Department of Cognitive Neurology, London, UK). Images were slice-timed, reoriented and realigned to the first volume. Next, T1-coregistered volumes were normalized to an SPM T1- template (using 12 linear parameters and a set of non-linear cosine basis functions), and spatial smoothing was performed using an 8 mm FWHM Gaussian kernel.
Statistical analysis
Group differences in demographic and clinical data were analyzed using univariate analysis of variance (ANOVA) and Tukey's post hoc tests. Group differences in educational level were analyzed using Pearson's chi-square test. ANOVAs were used to analyze performance data (mean reaction time) with group as between-subject factor (PRG, HSM and HC), and stimulus category (gambling versus neutral, smoking-related versus neutral, or low-level baseline versus neutral) as within-subject factor, using group contrasts. ANOVA was used to analyze urge ratings (mean gambling urge, mean smoking urge), with time (before and after task completion) as within-subject factor. All analyses were performed two-tailed.
The mean FTND score in the HSM group was low (M = 4.0; SD = 1.5) compared with FTND scores in smokers reported in other fMRI cue reactivity studies ( Franklin et al . 2007 , FTND = 4.8; McClernon et al . 2007 , FTND = 6.4; McClernon, Kozink & Rose 2008 , FTND = 6.5), and no nicotine dependence diagnoses were available for the HSM, as in other studies ( Brody et al . 2002 ). Therefore, exploratory analyses were done, comparing HSM with high FTND scores ( n = 10, FTND-high group M = 5.4, SD = 0.5) to HSM with low FTND scores ( n = 8, FTND-low group: M = 2.9, SD = 1.0), after a median split was made. In the PRG group, no split was made between high or low severity PRG, because severity of gambling problems in our sample, as assessed with the SOGS, was comparable with severity reported in other studies in treatment-seeking pathological gamblers.
The fMRI data were analyzed in the context of the general linear model, using delta functions convolved with a synthetic hemodynamic response function to model responses to each stimulus type. For each comparison of interest, single-subject contrast images were entered into second-level (random effects) analyses. To investigate differential processing of addiction relevant stimuli between groups, one-way ANOVAs were performed and interaction effects were computed for gambling versus neutral pictures in the PRG versus the HC or HSM, and for smoking-related versus neutral pictures in the HSM (HSM total group; FTND-high group; FTND-low group) versus the PRG or the HC. Main effects and interaction effects were analyzed with one-way ANOVA implemented in SPM2 and are reported with a cluster size restriction of 10 voxels at P < 0.05 corrected for multiple comparisons according to the Family Wise Error method ( Tiffany & Drobes 1991 ; Nichols & Hayasaka 2003 ). Group interactions are reported with a cluster size restriction of 5 voxels at P < 0.001, masked with the appropriate main effect.
Gambling or smoking-related pictures versus neutral pictures were chosen for our main group-interaction contrast, because this contrast is most specific for the cue-reactivity effect: reactivity to addiction specific cues versus cues not related to addiction. The comparison of addiction-related pictures versus baseline would include various non-specific visual processes (such as stimulus processing, object recognition) that are activated when watching visually complex stimuli compared with very simple visual stimuli (an arrow pointing to the left or right). An interaction between addiction-related pictures and baseline would therefore be less specific, because visual processing would then interact with cue reactivity effects. However, in addicted populations, it is important to establish that baseline visual interpretation is similar in both addicted persons and non-addicted groups. In another study from our group, it was found that addicted persons had a larger brain response to neutral pictures compared with baseline ( Zijlstra et al . 2009 ). Therefore, we also present the contrast neutral versus baseline, to demonstrate that neutral pictures generated similar activation patterns across groups.
In addition, the potential influence of left-handedness on brain activity patterns was investigated by performing all analyses with and without left-handed participants. The activity patterns found after excluding left-handed participants were very similar to those obtained when including both left- and right-handed participants. Therefore, in the Results section, we only present data based on the whole sample.
Regression analyses were performed for the PRG and HSM separately, to investigate whether brain activation in response to addiction-related stimuli (gambling and smoking stimuli, respectively) versus the neutral pictures correlated with self-reported craving after scanning. Regression analyses were also conducted to investigate whether co-morbid ADHD [Conners Adult ADHD Rating Scales (CAARS) scores] and depressive symptoms (BDI-II scores) correlated with cue-reactivity-related brain activation (addiction-related pictures versus neutral pictures). Because the PRG scored somewhat higher on the CAARS, and much higher on the BDI-II than the other two groups (see Table 1 ), these analyses were done separately for each group. Four PRG had co-morbid psychiatric disorders (anxiety and/or depression). Therefore, group interactions including PRG were analyzed both with and without these co-morbid participants. | RESULTS
Demographic and clinical results
Table 1 summarizes demographic and clinical characteristics for the three groups. PRG had an average of almost €60 000 in gambling-related debts. Breath carbon monoxide levels were higher for the HSM, compared with PRG and HC. PRG obtained higher scores on the CAARS and BDI-II than both HSM and HC.
Results for performance data and craving ratings
Mean reaction times to gambling pictures (M: 1143 ms, SD: 340) were longer than mean reaction times to neutral pictures (M: 1006 ms, SD: 311), F (1,49) = 50.1, P < 0.0001; mean reaction times to smoking pictures (M: 929 ms, SD: 235) were shorter than mean reaction times to neutral stimuli ( F (1,49) = 12.9, P < 0.0001; and mean reaction times to the low level baseline condition (M: 717 ms, SD: 169) were shorter than to the neutral stimuli, F (1,49) = 80.3, P < 0.0001, but no stimulus type by group interactions were present (all group by stimulus contrasts F values < 1, NS). Accuracy was high; mean number of errors summed across all conditions was 1.2, and no differences in number of errors between groups or conditions were found ( F < 1, NS). ANOVA indicated that craving for smoking before scanning was higher in the HSM compared with HC, F (1,34) = 87.4, P < 0.0001, and compared with PRG F (1,34) = 57.8, P < 0.0001. Craving did not differ between the FTND-high group and the FTND-low group, F (1,17) < 1, NS. No difference between smoking craving before and after the cue reactivity task in the total group of HSM F (1,17) = 1.42, P = 0.25, nor in the FTND-high group versus the FTND-low group, F (1,16) = .29, P = 0.60 was present. Craving for gambling was higher in PRG compared with HSM and HC, F (2,51) = 6.92, P < 0.002, and a trend for increased gambling craving after the cue reactivity task was observed in PRG, F (1,16) = 3.18, P = 0.09, partial η 2 = 0.17 (defined as a large effect size, Stevens 1996 ).
fMRI cue reactivity
Main effects (pictures versus baseline)
The main effects of viewing neutral pictures versus low-level baseline pictures were observed in all three groups mainly in the ventral visual stream (occipital lobe: middle, inferior and lingual gyrus), as well as in areas related to reward/motivation, and attention and cognitive control; medial temporal lobe including the amygdala, bilateral dorsolateral prefrontal cortex (DLPFC), as well as bilateral posterior thalamus, see Fig. 2 , left panel. For gambling versus baseline pictures and smoking-related versus baseline pictures, similar regions were identified. In addition, we found bilateral activation of the ventrolateral prefrontal cortex (VLPFC) for gambling and smoking-related pictures versus baseline pictures, as well as dorsomedial prefrontal cortex activation for gambling pictures versus baseline pictures ( Fig. 2 , middle and right panels, respectively).
Group interactions
For neutral pictures versus low-level baseline pictures, no significant group interaction effects were observed. For gambling pictures versus neutral pictures, we found greater activation in left occipital cortex, bilateral parahippocampal gyrus, right amygdala and right DLPFC in PRG relative to HC. Relative to the HSM, PRG showed higher bilateral occipital cortex, bilateral parahippocampal gyrus, bilateral amygdala, bilateral DLPFC and left VLPFC activation when viewing gambling pictures versus neutral pictures ( Table 2 and Fig. 3 ). Similar group differences were observed when PRG with co-morbid psychopathology were excluded, although differences in DLPFC activation in PRG compared with HC, and differences in activation in right amygdala and left DLPFC in PRG compared with HSM ceased to be statistically significant.
No significant group by condition interactions were observed for smoking pictures in HSM compared with PRG or HC. Greater activation was present in the ventromedial prefrontal cortex (VMPFC) bilaterally, in the rostral ACC bilaterally and in the left VLPFC in the FTND-high group compared with HC and in the FTND-high group compared with the FTND-low group. Similar effects were observed when comparing the FTND-high group with PRG (see Table 3 and Fig. 4 ). In addition, in the FTND-high group, activation in the left precuneus, right insula and left middle and superior temporal gyri was greater than in the FTND-low group. No significant group by condition interactions were observed in the FTND-low group compared with either HC or PRG.
Correlations between BOLD activation, subjective craving, BDI-II and CAARS
Regression analyses indicated a positive relation between subjective craving for gambling after scanning in PRG and BOLD activation in the VLPFC, left anterior insula and left caudate head when viewing gambling pictures versus neutral pictures (see Table 2 ). A positive relation between subjective craving for nicotine after scanning in HSM and BOLD activation in the VLPFC and left amygdala region during viewing of smoking-related pictures versus neutral pictures was present ( Table 4 ).
No significant relations between BDI-II or CAARS scores and regional cerebral blood flow changes during viewing gambling or smoking-related pictures versus neutral pictures were present in PRG, HSM or HC. | DISCUSSION
This is the first study investigating cue reactivity to gambling stimuli in treatment-seeking PRG compared with HSM and HC, using an fMRI event-related picture paradigm. PRG showed higher brain activation compared with HC and HSM when viewing gambling pictures (compared with neutral pictures) in brain areas related to visual information processing and memory (bilateral occipital cortex, parahippocampal gyrus), and emotion and motivation (amygdala region, VLPFC). Specifically, upregulation of visual information processing areas has been related to altered dopaminergic transmission in neural systems implicated in substance dependence: (1) an emotion/motivational and memory/learning circuit, including orbitofrontal, subcallosal cortex, amygdala and hippocampus; and (2) an attention/control circuit, including dorsal prefrontal and ACC ( Breiter & Rosen 1999 ; Goldstein & Volkow 2002 ; Kalivas & Volkow 2005 ). Higher activation in PG in these visual information processing areas may thus be related to a higher saliency of gambling stimuli, through innervations of dopamine pathways from the nucleus accumbens, ventral tegmental area and limbic areas to this visual system. Similar brain areas were found to be activated in fMRI cue reactivity studies of smokers and alcohol-dependent persons ( George et al . 2001 ; Due et al . 2002 ; Myrick et al . 2004 ). Higher activation of the amygdala region and parahippocampal gyrus indicates that gambling pictures activated the emotion/motivation and memory-related circuitry more in PRG than in HSM and HC. The parahippocampal gyrus is involved in processing complex visual information, receives input from the nucleus accumbens and amygdala, and is an important afferent pathway to the hippocampus. Cue reactivity studies of problem gambling, alcohol dependence and nicotine dependence have also reported brain activation in the parahippocampal gyrus ( Crockford et al . 2005 ; Smolka et al . 2006 ; Park et al . 2007 ). This study is the first to show involvement of the amygdala region in a cue-reactivity study in PRG, and to observe that activation in brain areas such as insular cortex and caudate nucleus is associated with self-reported gambling craving. These findings point to the persistent emotional relevance of gambling stimuli in patients currently in treatment for gambling problems.
All PRG were being treated for PG when they participated in the study, and reported an average duration of gambling problems of 13 years (data not shown). The two fMRI cue reactivity studies in PG present in the literature ( Potenza et al . 2003 ; Crockford et al . 2005 ) focused on community-recruited PRG, and did not report amygdala, insular cortex or caudate nucleus activation. The findings of this study suggest that cue reactivity in chronic PRGs seeking treatment may be related more strongly to brain reactivity in emotional and motivational circuitries than cue reactivity in (non-chronic) PRGs who are not in treatment.
Differences in brain activation patterns to smoking pictures between the FTND-high smokers and HC or PRG were most consistently present in VLPFC, VMPFC and rostral ACC, conforming with previous fMRI cue reactivity studies in smokers ( David et al . 2005 ; Lee et al . 2005 ; McClernon et al . 2005 , 2008 ). The lack of an effect of cue-reactivity in the FTND-low HSM group compared with the PRG or the HC group is likely related to the lower level of nicotine dependence in this subgroup. It has been reported that FTND scores positively correlate with regional brain reactivity to smoking cues ( Smolka et al . 2006 ; McClernon et al . 2008 ). Therefore, in future studies, the selection of a more homogeneous group of smokers, with a minimum score on the FTND or a formal DSM-IV ND diagnosis would be advisable.
In addition to our findings of higher brain activation in VMPFC and rostral ACC in the FTND-high smokers compared with the other groups, we observed that smoking urge in HSM correlated positively with activity in brain areas related to emotion and reward/motivational processing (amygdala and VLPFC), areas previously implicated in craving for smoking ( David et al . 2005 ; McClernon et al . 2008 ).
Limitations
Although we observed increased brain activation in response to gambling pictures in PRG and to smoking cues in the FTND-high HSM group, viewing these pictures elicited only a trend for higher self-reported craving in PRG, whereas in HSM no effects of the cue reactivity task on smoking urges were present. Changes in subjective craving before and after the task may have been limited in our study because of the timing of the measurement: a paper and pencil craving questionnaire was filled out after leaving the scanner, when immediate effects of the task on craving may have subsided. In future research, computerized craving measures administered in the scanner, halfway or immediately after the cue reactivity task, are therefore preferable.
After recruiting the HSM group, it became clear that FTND scores differed substantially within this group. Therefore, post hoc comparisons were made between two subgroups of HSM: an FTND-high group and an FTND-low group. The differential findings in the FTND-high and FTND-low groups imply that it is important to include a measure of nicotine dependence severity in cue reactivity studies in smokers, in addition to selecting smokers based on the number of cigarettes they smoke. The group sizes of the FTND subgroups were small ( n = 10 and n = 8, respectively), and therefore the results regarding these subgroups have to be interpreted with caution. Studies in larger groups of smokers differing in FTND scores should be done to replicate these preliminary findings.
Conclusion
This study demonstrates that viewing gambling pictures (as opposed to neutral pictures) is related to greater brain activation in visual processing, emotion-motivation and attentional-control brain circuitry in treatment-seeking PRG, compared with HC and HSM, and that this activation is positively related to gambling urges. These effects are consistent with those observed in substance-dependent persons ( George et al . 2001 ; Myrick et al . 2004 ; Franklin et al . 2007 ). In the present study, we did observe increased brain reactivity to smoking cues in persons with FTND scores indicating moderate nicotine dependence compared with HC, but did not find differences in persons with an FTND score indicating low nicotine dependence. Higher smoking urge in HSM was associated with increased activity in reward and emotion-related brain areas. Future research needs to establish whether the long-term effects of gambling cues on brain activation in PRG in treatment are related to relapse in problem gambling. | Conclusion
This study demonstrates that viewing gambling pictures (as opposed to neutral pictures) is related to greater brain activation in visual processing, emotion-motivation and attentional-control brain circuitry in treatment-seeking PRG, compared with HC and HSM, and that this activation is positively related to gambling urges. These effects are consistent with those observed in substance-dependent persons ( George et al . 2001 ; Myrick et al . 2004 ; Franklin et al . 2007 ). In the present study, we did observe increased brain reactivity to smoking cues in persons with FTND scores indicating moderate nicotine dependence compared with HC, but did not find differences in persons with an FTND score indicating low nicotine dependence. Higher smoking urge in HSM was associated with increased activity in reward and emotion-related brain areas. Future research needs to establish whether the long-term effects of gambling cues on brain activation in PRG in treatment are related to relapse in problem gambling. | Disclosure/Conflict of interest: None
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms
Abnormal cue reactivity is a central characteristic of addiction, associated with increased activity in motivation, attention and memory related brain circuits. In this neuroimaging study, cue reactivity in problem gamblers (PRG) was compared with cue reactivity in heavy smokers (HSM) and healthy controls (HC). A functional magnetic resonance imaging event-related cue reactivity paradigm, consisting of gambling, smoking-related and neutral pictures, was employed in 17 treatment-seeking non-smoking PRG, 18 non-gambling HSM, and 17 non-gambling and non-smoking HC. Watching gambling pictures (relative to neutral pictures) was associated with higher brain activation in occipitotemporal areas, posterior cingulate cortex, parahippocampal gyrus and amygdala in PRG compared with HC and HSM. Subjective craving in PRG correlated positively with brain activation in left ventrolateral prefrontal cortex and left insula. When comparing the HSM group with the two other groups, no significant differences in brain activity induced by smoking cues were found. In a stratified analysis, the HSM subgroup with higher Fagerström Test for Nicotine Dependence scores (FTND M = 5.4) showed higher brain activation in ventromedial prefrontal cortex, rostral anterior cingulate cortex, insula and middle/superior temporal gyrus while watching smoking-related pictures (relative to neutral pictures) than the HSM subgroup with lower FTND scores (FTND M = 2.9) and than non-smoking HC. Nicotine craving correlated with activation in left prefrontal and left amygdala when viewing smoking-related pictures in HSM. Increased regional responsiveness to gambling pictures in brain regions linked to motivation and visual processing is present in PRG, similar to neural mechanisms underlying cue reactivity in substance dependence. Increased brain activation in related fronto-limbic brain areas was present in HSM with higher FTND scores compared with HSM with lower FTND scores. | This study was partly funded by a grant from the Netherlands Organization for Health Research and Development (#31000056) of the Netherlands Organization for Scientific Research (NWO) to A.G., D.V., J.O. and W.B, and by a New Investigator grant (A.G., Veni grant) from the Dutch Scientific Organization (NWO ZonMw, #91676084, 2007–10). Scanning costs were in part funded by the Amsterdam Brain Imaging Platform. A.G., M.R., D.V., J.O. and W.B. report no conflict of interests. We thank Jellinek Amsterdam for their help in recruitment of problem gamblers.
Authors Contribution
AG, MR, and DV take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have had full access to all the data in the study. AG, MR, JO, WB, and DV were responsible for the study concept and design. MR was responsible for data acquisition. MR, AG, and DV were responsible for statistical analysis and interpretation of data. AG drafted the manuscript. MR, JO, WB and DV provided critical revision of the manuscript for important intellectual content. All authors critically reviewed content and approved final version for publication. Preliminary data of this study were presented at the Human Brain Mapping Meeting June 15–19, 2008, Melbourne, Australia. | CC BY | no | 2022-01-12 15:46:43 | Addict Biol. 2010 Oct; 15(4):491-503 | oa_package/bd/a7/PMC3014110.tar.gz |
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PMC3014111 | 20678157 | .E-mail: [email protected] (malaria); [email protected] (imaging)
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/onlineopen.html | This Atomic Force Microscope image shows the surface of a Plasmodium malariae trophozoite-infected red blood cell isolated from a Burmese patient suffering from quartan malaria (top). Of note, the P. malariae -infected cell (bottom left) is covered with dense ‘spike-like’ excrescences (mean height: 7·59 nm; mean diameter: 52·95 nm), which are morphologically distinct from the larger, more rounded ‘knob’ structures found on a Plasmodium falciparum- infected red cell (mean height: 19·65 nm; mean diameter: 96·64 nm) (bottom right). The ‘knobs’ on red cells containing mature asexual forms of P. falciparum assist the infected cells to bind/sequester to the vascular endothelium under shear flow conditions and thus avoid splenic clearance. The function of P. malariae spikes (which we have observed on every sexual and asexual stage examined in isolates from Indonesia and Thailand) is not known. The sample from which the P. malariae pictured was isolated (Mae Sod, Tak, Thailand) had the diagnosis confirmed by polymerase chain reaction. This picture is artificially coloured. The horizontal black scale bars represent 1 μm. The two lower images are identical in scale and orientation. | CC BY | no | 2022-01-12 15:46:43 | Br J Haematol. 2010 Oct; 151(1):1 | oa_package/46/42/PMC3014111.tar.gz |
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PMC3014229 | 21217837 | DISCUSSION
The management of AVM remains a significant challenge, particularly those of the upper limb. Intervention should only be contemplated when the lesion becomes symptomatic. A variety of treatment strategies have been reported with varying success. 2 - 5
We believe that plastic surgeons should be an essential member of the multidisciplinary team treating such lesions. In some instances, surgical resection of the AVM with maximal preservation of upper-limb function may be required.
Clinical presentation of upper-limb AVMs can be diverse. Patients may suffer pain, nerve compression, ulceration, hand dysfunction, and spontaneous bleeding. 3 , 6 , 7 Shunting through proximal arteriovenous fistulae may result in distal steal phenomenon manifesting as severe pain, ischaemia, and discoloration of the digits. 5 , 9
Rarely, extensive arteriovenous shunting may lead to cardiac decompensation. 8 This is an absolute indication for surgery. The presence of an upper-limb AVM does not correlate with vascular anomalies of other organ systems, nor does the pattern predict skeletal overgrowth. 5 , 9
Management of AVM varies, with a conservative approach adopted for patients that are asymptomatic or have minor symptoms. If treatment is required, techniques that may be used include catheter embolization or direct percutaneous sclerotherapy. Embolization and sclerotherapy may be performed even after major surgery.
Surgical ligation of the main feeding vessels to the AVM often yields a poor result, as a collateral circulation subsequently develops. 10 In our patient, surgical ligation of the subclavian vessels was performed as part of the second stage. This measure was undertaken to prevent uncontrollable bleeding during the shoulder disarticulation.
With the advent of highly selective embolization techniques, a number of authors have reported safe and successful outcomes when treating AVM with this modality. 6 Indeed, it has been advocated as the treatment of choice by some. 8
Regardless of the embolic agent used, the underlying aim is to obliterate the nidus. However, embolotherapy must not be considered a definitive cure and can unintentionally worsen symptoms. Furthermore, repeat embolization may be necessary to deal with recurrences.
In our patient, embolotherapy would not have been appropriate. The diffuse nature of the AVM with multiple nidi wound render embolotherapy ineffective. Our patient had a life-threatening, symptomatic, nonfunctional upper-limb AVM. In this situation, we believe that amputation or disarticulation should be explained to the patient at the outset. Amputation should be viewed as the “reconstructive” first step in the patient's rehabilitation.
We favored glenohumeral disarticulation rather than amputation through the neck of the humerus for a number of reasons. First, the latter provides no functional advantage, as the patient cannot grip or stabilize large objects between the stump and thorax. Second, even though the MRI indicated no apparent involvement of the humerus, amputation through the bone may result in significant bleeding. Third, the patient was not keen for preservation of proximal humerus with associated soft tissue reconstruction. His concern was a protracted postoperative stay and rehabilitation. The cosmetic deformity with glenohumeral disarticulation, however, is greater. Shoulder width and axillary contour are both lost.
Our rationale for a 2-stage amputation was based on the following: the aim of the first stage was to control the risk of bleeding and infection at the hand. Elbow disarticulation could be easily performed as a tourniquet could be applied to the arm. After control of infection, the second stage could be performed with the reduced risk of wound infection and breakdown.
In one series of 33 patients with high-flow AVM of the upper limb, the majority of patients were treated by surgical excision. 5 However, in patients with diffuse lesions, 9 of 10 patients required major amputation despite repeated excision and reconstruction. White et al reported similar results, with 2 of 4 patients with high-flow diffuse AVM of the entire upper limb requiring amputation. 4 Dickey et al also reported a 28% complication rate in all patients with high-flow lesions that underwent treatment.
A multidisciplinary approach is central to the effective management of AVMs. These lesions are complex and difficult to treat. Despite advances in embolic agents and microcatheter techniques, outcomes for high-flow upper-limb AVM is far from satisfactory. For diffuse lesions that are life-threatening, amputation as the first step in the patient's rehabilitation may be appropriate. Indeed, it has been previously stated that amputation was unnecessarily delayed in such patients. 5
Surgical excision must be performed in a systematic manner, involving steps to minimize the risk of uncontrollable intraoperative bleeding and postoperative complications. In this case, successful amputation was achieved using temporary transcutaneous sutures to control bleeding from skin edges, ligation of the subclavian vessels, and glenohumeral disarticulation. | Arteriovenous malformations (AVM) are high-flow lesions having a direct connection between an artery and vein, with bypass of the capillary bed. 1 Those affecting the upper limb and causing cardiac decompensation are rare.
Treatment of such lesions remains a surgical challenge. Despite multiple procedures, total eradication is often not achieved. For some patients, limb amputation becomes necessary and carries significant risk.
We report a case of congenital AVM of the upper limb causing cardiac decompensation. The patient was safely and successfully treated by performing a 2-stage upper-limb amputation.
CASE REPORT
A 32-year-old man presented with a congenital high-flow AVM involving the left upper limb and anterior chest (Fig 1 ). In recent years, the AVM had undergone rapid expansion causing the patient significant pain, high-output cardiac failure, and dyspnea on minimal exertion. The pain was resolved by elevation of the arm.
In addition, there was a chronically infected wound on the dorsum of the left hand from which he suffered a massive bleed 6 years previously. On that occasion, he lost 3500 mL of blood. Amputation of the digits through the metacarpophalangeal joints had been previously performed at another hospital. At presentation, the left upper limb was warm and hypertrophic with pulsating vessels. Bruits were audible and a thrill palpable over the axillary artery. A dry eschar covered the chronic wound on the dorsum of the hand. The vascular malformation over the anterior chest manifested as an uncomplicated reddened patch.
Magnetic resonance imaging (MRI) was performed to determine extent of involved tissues. This revealed an extensive diffuse AVM involving all compartments of the left upper limb. There was no skeletal hypertrophy (Fig 2 ). Selective embolization was discounted for a number of reasons: the limb was nonfunctional; there was a risk of life-threatening hemorrhage from the chronic wound; embolization would not obliterate the multiple nidi; and partial excision following embolization would carry the risk of uncontrollable intraoperative bleeding, postoperative complications, and recurrence of the AVM. Hence, it was decided at the outset to perform amputation of the affected upper limb. To minimize the risk of uncontrollable intraoperative bleeding and wound-healing problems, amputation in 2 stages was planned.
The first stage comprised disarticulation through the elbow. We intentionally avoided amputation through bone to avoid the risk of uncontrollable bleeding from the bone end. To minimize intraoperative bleeding, a tourniquet was inflated above the elbow and 1/0 PDS (Polydioxanone, Ethicon) transcutaneous sutures placed proximal to the incision. The temporary sutures served to compress cutaneous and subcutaneous vessels thereby reducing bleeding. Consequently, it became easier to formally transfix these vessels during the amputation.
The second stage was performed 1 week later. Cardiovascular surgeons performed ligation of left subclavian vessels (Fig 3 ), immediately followed by disarticulation through the glenohumeral joint. Temporary transcutaneous sutures were used in a similar manner as in the first stage. Multiple, dilated vessels were encountered and ligated or transfixed. Some of the vessels matched diameter of the aorta.
A fasciocutaneous flap overlying the deltoid was raised and used for wound coverage. A suction drain was inserted and a soft, bulky dressing applied. Postoperative recovery, including wound healing, was unremarkable (Fig 4 ). The patient was discharged 2 weeks later. Cardiac failure soon resolved. One-year postsurgery, the patient remains well and has not encountered any wound problems. | CC BY | no | 2022-01-12 15:21:35 | Eplasty. 2010 Dec 22; 10:e71 | oa_package/84/0a/PMC3014229.tar.gz |
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PMC3014295 | 21209796 | Introduction
Neoplastic transformation is a gradual process, during which cells acquire successive mutations, which mainly cause the loss of proliferation control, the ability to divide indefinitely and invade other tissues. A critical step in the development of a malignant cancer is tumor cells' acquisition of the capacity to migrate and invade tissues [1] . Different molecular mechanisms are responsible for the acquisition of a migratory and invasive phenotype, such as changes in signal transduction pathways involving tyrosine kinases, changes in cytoskeletal organization and in cell adhesion.
The small GTPases of the Rho family, mainly RhoA, cdc42 and Rac [2] – [4] , play a pivotal role in regulating the actin cytoskeleton and cell movement. The RhoA/ROCK and Rac signalling pathways are required for respectively amoeboid and mesenchymal movements, which are the main types of movements adopted by tumor cells [5] , [6] . The mesenchymal movement is typical of cells displaying an elongated morphology in a 3D environment; it requires integrin attachment to the extracellular matrix, formation of focal contacts and pericellular proteolysis. Besides cancer cells of mesenchymal origin, carcinoma cells can adopt this type of migration, after undergoing an epithelial-mesenchymal transition (EMT) [7] .
In contrast, some carcinoma cells can move very fast with an amoeboid shape [8] , [9] ; in vitro studies in 3D environments have shown that this movement is typical of cells with a rounded morphology and is associated with the formation of actin cortical rings and membrane blebbings. The amoeboid movement, which is potentially faster than the mesenchymal one, does not rely on integrins, focal contacts and extracellular matrix degrading enzymes; it is mainly based on contraction of actomyosin filaments which enables the cells to squeeze through the extracellular matrix without the requirement of matrix degradation.
Acto-myosin contractility is strictly dependent on the activity of the RhoA-dependent kinase ROCK [9] – [11] and chemical blockade of ROCK inhibits amoeboid movement [10] . Cells can switch from one type of invasion mechanism to the other in response to changes in protein expression, or after treatment with certain compounds [5] , [12] – [18] . For example, inhibition of matrix proteases or integrins inhibits mesenchymal migration and promotes a transition towards amoeboid movement [19] , [20] . Identifying the factors and genes controlling the different types of motility could help direct therapeutic strategies aimed at reducing invasion.
Amoeboid migration has been recognized as an important mechanism of invasion and metastasis of carcinoma cells and, more recently, of sarcomas [21] . However, little is known about the possible association between sarcoma malignant progression and acquisition of the amoeboid phenotype. In this paper, we have exploited the cen3tel model of isogenic cells at different stages of transformation, from normal fibroblasts up to metastatic cells, to study changes in the migratory and invasive potential accompanying human fibroblast neoplastic transformation. The human fibroblast cell line cen3tel, obtained in our laboratory by telomerase immortalization, gradually underwent spontaneous neoplastic transformation [22] – [24] . Studying cells at different phases of transformation, we could show that an early event during transformation was the loss of expression of the CDKN2A locus, followed by inactivation of p53 and overexpression of c- myc . While CDKN2A downregulation was not sufficient to make cells tumorigenic, the ability to induce tumors in nude mice correlated with p53 inactivation and c- myc overexpression. During further culture propagation, cen3tel cells showed a shorter latency in inducing tumors, suggesting that they had acquired increased tumorigenicity and could be a useful tool for obtaining further information on molecular changes associated with tumor progression [23] .
In this study we found that, upon neoplastic transformation, cen3tel cells increased their migratory and invasive capacity by adopting a protease-independent/ROCK-dependent mechanism of invasion. We show here that Rnd3 (also known as RhoE), a cellular inhibitor of ROCK-I [25] , plays a relevant role in regulating invasion and metastasis formation of sarcoma cells. | Materials and Methods
Ethics Statement
Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Decreto Legge No. 116, Gazzetta Ufficiale, Suppl. 40, Feb. 18, 1992; Circolare No. 8, Gazzetta Ufficiale, July, 1994) and international laws and policies (European Economic Community Council Directive 86/609, Official Journal Legislation 358. 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, United States National Research Council, 1996). The study was reviewed and approved by the IRFMN Animal Care and Use Committee (IACUC), which includes “ad hoc” members for ethical issues. Aproval ID Frap1.
The cen3tel cellular system
The cen3tel telomerase immortalized cell line was obtained from primary cen3 fibroblasts, derived from a centenarian, by infection with an hTERT-containing retrovirus [22] . Cen3tel cells were used at different steps of propagation reflecting different phases of transformation [23] . In particular, we used cen3tel cells at five phases of propagation (up to around population doubling (PD) 1000): early cen3tel cells, these cells are at the initial passages after infection with the hTERT containing retrovirus (between PDs 34 and 45) and show a behaviour similar to that of primary cen3 fibroblasts; mid cen3tel cells (around PD 100), these cells are at an early phase of transformation, they are able to grow in the absence of solid support, but are not tumorigenic in nude mice; tumorigenic cen3tel cells, which induce tumors when inoculated subcutaneously into nude mice, were further subdivided in three groups, according to the time required for tumor formation, which decreases at increasing PDs (see first section of the Results ), tumorigenic cells of phase I (around PD 160), phase II (around PD 600) and phase III (around PD 1000).
Cell culture, transfection and plasmid
Primary and immortalized cells were grown in Dulbecco's modified Eagle's Medium (DMEM, Celbio) supplemented with 10% fetal bovine serum (Lonza), 2 mM glutammine, and 1% non-essential amino acids (Euroclone), 0.1 mg/ml penicillin (Euroclone), 100 U/ml streptomycin (Euroclone) at 37°C in an atmosphere containing 5% CO 2 . To analyze cellular morphology and organization of the actin cytoskeleton (see section “Immunofluorescence”) in a 3D environment, cells were plated on top of Matrigel (8 mg/ml) (BD Biosciences) in 8 chamber polystyrene vessels (BD Transduction Laboratories Biosciences Falcon) and incubated at 37°C with 5% CO 2 in complete DMEM to allow Matrigel invasion. Cells in Matrigel were treated with 10 μM Y27632 (Calbiochem) for 6 hours. Cell morphology was observed using an optical microscope Olympus IX71 equipped with a 4x objective (NA 0.13). Images were taken with a digital camera Cool SNAP ES (Photometrics) using the MetaMorph software.
Cells were transfected with the linearized plasmids EGFP-C1 (Clontech) or EGFP-C1-Rnd3 (kindly provided by Dr. Pierre Roux, CRBM-CNRS FRE2593, France) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfected cells were selected and expanded in complete medium containing 0.5 mg/ml G418 (Invitrogen).
Motility and Invasion Assays
Cell motility and invasiveness were assayed using modified Boyden chambers with polycarbonate PVP-free Nucleopore filters (8 μm pore size) [26] . Supernatant of NIH-3T3 cells was used as a reference attractant and was added to the lower compartment of the Boyden chamber. For motility, filters were coated with 0.1% gelatin. For invasion, filters were coated with a thick layer of the reconstituted basement membrane Matrigel (0.5 mg/ml; Becton Dickinson). Cells were detached, washed in DMEM containing 0.1% BSA, resuspended in the same medium at a concentration of 5×10 5 /ml, and added to the upper compartment of the chamber. When indicated, the ROCK inhibitor Y27632 (Calbiochem) or the MMP inhibitor Ro 28–2653 (kindly provided by H.W. Krell, Roche Diagnostics Gmbh, Penzberg, Germany) was added to the cells 1 hour before the assay and left throughout the assay. After 4 hours (motility) or 6 hours (invasion), filters were stained with Diff-Quik (Baxter), and migrated cells in 10 high-power fields were counted. We choose these times of analysis to avoid that possible proliferation differences between cell lines could affect the results. Results are expressed as the number of migrated or invaded cells or as the percentage of control invasion (inhibitor treated or transfected cells). Statistical differences between groups were evaluated by ANOVA followed by Bonferroni post hoc test.
Immunofluorescence
Cells were seeded at different density in 12 well plates (Corning) containing 19 mm diameter coverslips and incubated at 37°C for 24 hours. Filamentous actin (F-actin) was stained with phalloidin-TRITC (P1951, Sigma-Aldrich) diluted 1∶300 and nuclei with 2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 10 min. The phosphorylated form of myosin II regulatory light chain (p-MLC) was stained using the anti-pS19-MLC primary antibody (#3671, Cell Signaling) diluted 1∶50 and an anti-rabbit secondary antibody conjugated with FITC (Jackson Immunoresearch), after an overnight block in 0.3% BSA in PBS at 4°C. To detect F-actin in cells in Matrigel matrix, cells were fixed for 30 minutes with 4% paraformaldehyde-0.25% glutaraldheyde in PBS on ice and then treated for 30 minutes with 0.2% Triton-X 100 in PBS. Cells were then incubated with phalloidin-TRITC (P1951, Sigma-Aldrich) diluted 1∶1000 and with 0.2 μg/ml DAPI for 3 hours. Slides were analyzed with the Leica TCS SP2 confocal laser microscope using a 40x objective (NA 1.32). Figures were assembled using Adobe Photoshop and Adobe Illustrator.
Western blot analysis
Whole-cell lysates were prepared using the RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.1% DOC, 1x protease inhibitor cocktail (Roche) and 0.2% Na 3 VO 4 ) or the Laemmli buffer (60 mM Tris-HCl, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, 2% SDS, 0.02% bromophenol blue). The anti-Rnd3 antibody [R 6153, clone 4 (Sigma-Aldrich), dilution 1∶500] was used on extracts prepared with the RIPA buffer. The anti-pS19-MLC (1∶500) was used on extracts prepared with the Laemli buffer and blotted onto PVDF membranes (Biorads). The anti-γ-tubulin antibody (T6557, clone GTU-88, Sigma-Aldrich) was used as control for protein loading. Secondary Horseradish Peroxidase conjugated antibodies were from Jackson ImmunoResearch (anti-mouse IgG 115-035-146, anti-rabbit IgG 111-035-003). Chemiluminescent assays (Pierce) were used to detect the secondary antibody signal.
RNA extraction and microarray analysis
Total RNA was extracted using the Trizol reagent (Invitrogen) from actively dividing primary fibroblasts (PD 15) and from cen3tel cells representing the five phases of propagation (PD 37, 97, 167, 618, 1032 and 1042; cells at PDs 1032 and 1042 both represent cells of tumorigenic phase III). Microarray probe preparation, hybridization on Agilent Whole Genome 44k oligo microarrays and scanning were carried out as previously described [27] . Images were analyzed using Feature Extraction software (Agilent Technologies) version 8.1. Output files were then treated with the Limma (linear models for microarray data) package [28] available within Bioconductor ( http://www.bioconductor.org/ ). Linear models were fitted to the normalised data to compare cen3tel cells at each population doubling with control cen3 fibroblasts. The empirical Bayes method was used to compute the moderated t-statistic and F-statistic [29] . P-values were adjusted for multiple testing by the Benjamini-Hochberg approach [30] . To select differently expressed transcripts, a cut-off of 0.01 was applied to the adjusted p-value. Microarray data have been deposited in the NCBI database GEO (Gene Expression Omnibus), accession number GSE157442.
Animal models
The in vivo tumorigenic potential of cen3tel cells was evaluated by injecting 10 7 cells in the right flank of female athymic nude mice or female SCID mice (Harlan Italy, Bresso, Italy). Mice were monitored two/three times a week to assess tumor appearance and growth. To investigate the metastatic ability of cen3tel cells, 2×10 6 cells were injected i.v. in nude or SCID mice. Mice were monitored daily and sacrificed at the appearance of distress symptoms. Animals were autopsied to evaluate metastases. To establish the capacity of primary tumors to give spontaneous metastases, tumor masses obtained after s.c. injection of cen3tel cells were surgically removed under isoflorane anesthesia and the mice were kept alive until the appearance of distress symptoms. After sacrifice, animals were autopsied to evaluate metastases. For counting metastatic foci, tissues were collected and stored in BOUIN. For histopathologic examination, tumor masses and animal parenchymal organs were fixed in 10% buffered formalin, and paraffin embedded. 4-μm sections were cut and stained with hematoxylin and eosin. | Results
In vivo tumorigenicity and metastatic potential of cen3tel cells at different stages of propagation
Cen3tel cells at different PDs were inoculated subcutaneously in immunocompromised mice. Analysis of cen3tel cells at tumorigenic phase I and phase II (respectively around PD 160 and PD 600, see Material and Methods for the description of the cellular system) confirmed our previous results [23] , with tumors becoming detectable with latencies of about one month and eight days, respectively ( Fig. 1A ). Conversely, phase III tumorigenic cen3tel cells (PD 900–1020) generated tumors already evident two days after inoculation ( Fig. 1A ). Histological analysis revealed that the tumors developed by cen3tel cells at the first and second tumorigenic phases were pleomorphic sarcomas ( Fig. 1B and C ), those developed by phase III cen3tel cells showed a hemangiopericytoma-like vascular pattern, similar to human poorly differentiated, round-cell synovial sarcoma ( Fig. 1D ). Cen3tel cells of tumorigenic phase III recapitulated the histological features of synovial sarcomas even in the absence of the SYT-SSX fusion transcript (data not shown), which is diagnostic of this sarcoma subtype [31] , suggesting the involvement of other genetic or epigenetic mechanisms, possibly associated with the still poorly understood SYT-SSX downstream targets.
Cen3tel cells at different PDs were also injected intravenously to test whether they were able to give metastases. Both cen3tel cells at tumorigenic phases I and II did not induce metastases (in 3 mice each, Fig. 1E and F ). In contrast, in the lung of 10 out of 10 mice injected with cen3tel cells around PD 1000, a high number of metastases (always more than 50 metastatic foci per lung) were observed 4 weeks after injection ( Figure 1G ).
Tumorigenic cen3tel cells are more motile and invasive than non-tumorigenic cells
To study motility and invasiveness in cen3tel cells at different PDs, we used Boyden chambers with filters coated either with gelatin or with a thick layer of the reconstituted basement membrane Matrigel. The number of cells able to migrate through the porous membrane coated by gelatin was considered a measure of the migratory capacity, and the number of cells passing through Matrigel a measure of the invasive capacity. As illustrated in Figure 2A , which shows both spontaneous motility (black columns) and motility in response to a chemoattractant (grey columns), parental cen3 fibroblasts, as well as early and mid cen3tel cells, had a very limited migratory potential; in contrast, tumorigenic cen3tel cells were clearly able to migrate through the porous membrane. The same holds true for the ability to invade Matrigel ( Fig. 2B ). Interestingly, invasiveness was also high in the absence of the chemoattractant. Thus, cen3tel neoplastic transformation is associated with increased migratory and invasive potential. In the in vitro experiments, we could see no clear difference in the migratory or invasive potential between the cells in the three phases of tumorigenicity. In particular, in cen3tel cells around PD 1000, there was no clear increase in in vitro invasion, that could be related to the acquisition of the metastatic ability.
Tumorigenic cen3tel cells show actin cortical rings and a spherical morphology in a 3D environment
Cen3tel cells routinely grown on plastic showed a clear change in morphology during culture propagation. Cells switched from a typical elongated fibroblastic shape, distinctive of cells up to around PD 100, to a polygonal shape characteristic of cells that had become neoplastically transformed [23] . To test whether neoplastic transformation and the change in cell morphology were associated with changes in the actin cytoskeleton, we analyzed the organization of the actin filaments in cells at different stages of propagation. We stained F-actin using its interacting molecule phalloidin, conjugated to a fluorochrome. A clear difference in F-actin organization was observed between tumorigenic and non-tumorigenic cen3tel cells ( Fig. 3A–F ). As expected, F-actin was organized in stress fibers in primary cen3 fibroblasts ( Fig. 3A ) and the same organization was also observed in early and mid cen3tel cells ( Fig. 3B, C ). In tumorigenic cen3tel cells ( Fig. 3D–F ), phalloidin staining clearly highlighted the change in morphology with cortical rings formed along the inner cell periphery.
Together with the change in actin organization, there was a parallel re-distribution of p-MLC ( Fig. 3G–L ). In fact, while in non-tumorigenic cells, the phosphorylated form of myosin II was distributed in fibers along the cells ( Fig. 4G–I ), in tumorigenic cells it mainly formed a ring around the inner cell periphery ( Fig. 4J–L ).
The change in cytoskeletal organization observed in tumorigenic cen3tel cells suggests that the transformed cells could have switched to an amoeboid-type movement. Cells migrating through this movement have a characteristic rounded shape when grown in a deformable gel like Matrigel and show cortical actin rings with membrane blebbings [6] . To analyze cen3tel tumorigenic cell morphology and actin organization in 3D, we plated them on the top of Matrigel and allowed them to invade the gel. After 24 hours, cells were first analyzed by phase contrast microscope ( Fig. 4A, A '–C') and then fixed and stained with phalloidin conjugated with TRITC ( Fig. 4A, G '–I', M'–O'). Tumorigenic cells had a rounded morphology (A'–C'), with F–actin cortical rings (G'–I', M'–O') (see also Movie S1 ). It is worth noticing that the rounded morphology was also observed in cells of the sarcoma-like tumor masses generated by cen3tel cells in animal models.
ROCK inhibition leads to an elongated morphology and reduces cen3tel cell invasion
Rounded cell morphology and cortical actin rings depend on the activity of the Rho-effector kinase ROCK, which acts by increasing myosin-II-mediated actin filament stabilization and contraction [32] . Inhibition of ROCK causes the loss of the spherical cellular morphology [10] . To test whether this was the case in tumorigenic cen3tel cells, we exposed cells that had invaded Matrigel to the ROCK chemical inhibitor Y27632. Treatment with the inhibitor produced a more elongated morphology ( Fig. 4A, D'–F' ) and altered the actin cortical rings ( Fig. 4A, J'–L' , P'–R'), indicating that ROCK activity is involved in the change of shape of tumorigenic cen3tel cells.
It is known that the invasiveness of rounded tumor cells depends on ROCK activity, while it is independent of the activity of matrix proteases [10] , [19] . Global gene expression profiling of cen3tel cells at different PDs showed a decrease in the expression of different types of extra-cellular matrix proteases during cen3tel propagation in vitro , particularly of several matrix-metalloproteases (MMPs) ( Table 1 ), and zymographic analysis showed a decrease in the activity of MMP2 and MMP9 in tumorigenic cen3tel cells (data not shown), suggesting that increased motility in late cen3tel cells was not strictly dependent on pericellular matrix proteolysis.
To test the roles of ROCK and metalloproteases on cen3tel cell invasiveness, we analyzed the invasive ability of cen3tel cells at the three stages of tumorigenesis in the presence of the ROCK inhibitor Y27632 or in the presence of the metalloprotease inhibitor Ro 28–2653. As shown in Fig. 4B , Y27632 reduced the invasiveness of cen3tel cells at all three different stages (black columns), while the metalloprotease inhibitor did not affect it (grey columns). The opposite result was observed in HT1080 cells, which use mesenchymal invasion ( Fig. 4B ). These results indicate that tumorigenic cen3tel cells acquired a protease-independent type of invasion, which requires the activity of ROCK kinase.
Rnd3 expression inversely correlates with the invasive and metastatic properties of tumorigenic cen3tel cell
By microarray analysis and western blotting we did not observe any evident change in ROCK or RhoA expression in tumorigenic cen3tel cells (data not shown); however, microarray and western blot analyses revealed that the cellular inhibitor of ROCK-I, Rnd3 (Riento et al. 2003), was expressed at lower levels in tumorigenic cen3tel cells compared to non-tumorigenic ones ( Fig. 5A, B ), suggesting that Rnd3 downregulation might play a role in the transition to the ROCK dependent mode of invasion.
To test whether Rnd3 levels affected tumorigenic cen3tel cell invasiveness, we transfected phase II and phase III tumorigenic cells with an EGFP-Rnd3-expression vector and isolated clones with exogenous Rnd3 expression, three from phase II tumorigenic cen3tel cells and two from phase III cells ( Fig. 5C ). Compared to mock-transfected cells, in which the EGFP signal was, as expected, both nuclear and cytoplasmic ( Fig. 5D ), in the cells transfected with the recombinant vector, the EGFP-Rnd3 fusion protein showed the same subcellular localization described for the endogenous protein [33] : it was present in the cytoplasm, at the plasma membrane and showed a perinuclear accumulation suggestive of a localization in the Golgi apparatus ( Fig. 5E ). Rnd3 expressing clones showed proliferation rates similar to that of parental and mock transfected cells (data not shown).
Rnd3 overexpression induces actin fiber disassembly in several cell types [34] . In the clones stably expressing Rnd3, we found no major changes in cellular morphology compared to parental cells, or actin depolymerization (not shown). This might be related to the level of expression of the protein in the clones, which may not be high enough and, above all, to its stable expression. In fact, it has been shown that in mouse 3T3 fibroblasts, the strong effects induced by acute Rnd3 expression on the cytoskeleton are only transient and disappear within hours, even though the levels of the protein are still high [35] . In the Rnd3 expressing clones we did find lower level of p-MLC than in parental and mock-transfected cells, which could be due to an Rnd3-induced reduction in ROCK kinase activity ( Fig. 5F ).
We analyzed invasion in the five Rnd3 expressing clones and, in all of them, it was lower than in mock transfected cells ( Fig. 5G ), indicating that Rnd3 expression can modulate invasion of neoplastically transformed human fibroblasts.
We then investigated whether exogenous Rnd3 expression reduced the in vivo metastatic potential of phase III tumorigenic cen3tel cells. We analyzed the metastatic potential of Rnd3 clone 16, because of the greater stability of Rnd3 expression in this clone compared to that in clone 8 (not shown). We first inoculated mock-transfected control cells and Rnd3 clone16 cells subcutaneously in nude mice, to verify their tumorigenic potential. Cells from both transfected populations developed tumors; however, after about 10 days, when tumors were around 250 mm 3 , the tumors were rejected, probably because of an immune response to EGFP expressing cells [36] . To avoid rejection, we inoculated the cells into SCID (Severe Combined Immuno-Deficiency) mice, which are deficient in T and B cells. In these mice, both cell lines developed tumors, which showed similar growth rates, indicating that Rnd3 does not affect subcutaneous tumor growth ( Fig. 5H ). To analyse metastasis formation, cells were injected intravenously in SCID mice. Mice were sacrificed after about six weeks; metastatic masses were detected in the lungs (from 4 to 43 metastases/mouse with a median number of 9) and in the adrenal glands of the six mice inoculated with mock-transfected cells ( Fig. 5I ), while they were not observed in any of the eight mice inoculated with Rnd3 clone 16 cells.
Taken together, these results indicate a role for Rnd3 downregulation in promoting invasion and metastasis formation by mesenchymal tumor cells. | Discussion
The cen3tel cellular system [22] , [23] , derived from human telomerase immortalized fibroblasts, has allowed us to follow the stepwise process of cellular transformation, from normal fibroblasts up to tumorigenic cells with metastatic potential. In this paper, we focused on the study of molecular and biological features associated with changes in invasiveness during the progression from normal to tumorigenic and metastatic fibroblasts. During transformation, cen3tel cells increased their migratory and invasive potential and gradually acquired the ability to induce metastases in immunocompromised mice. We show here that the increased invasiveness does not depend on metalloprotease activity, as expected for cells of mesenchymal origin, but on the activity of the ROCK kinase; in addition, we show that Rnd3 negatively regulates invasion and metastasis formation.
Although ROCK-dependent invasion was first described in carcinoma and melanoma cell lines [10] , evidence has been recently reported that cells of mesenchymal origin can adopt a protease-independent amoeboid type of movement, making ROCK a possible therapeutic target for sarcomas [37] . In mouse 3T3 fibroblasts a switch to amoeboid movement was observed upon p53 inactivation [14] , while up-regulation of the Rho/ROCK signalling was found in highly metastatic rat sarcoma cells, together with the loss of MMP2 activity and an increased generation of protrusive forces, typical of the amoeboid movement [21] . Moreover, it has been shown that microtubule destabilization through stathmin overexpression can also lead to acquisition of amoeboid movement in sarcoma cells and that the tumor suppressor protein p27 kip1 is an important factor for the control of cellular morphology and motility of transformed fibroblasts by regulating microtubule stability [38] , [39] .
In cen3tel cells, neoplastic transformation was associated with an increasing invasive capacity, linked to a change in cell morphology from elongated to rounded, in the organization of the actin cytoskeleton from stress fibers to cortical rings, and in the subcellular distribution of p-MLC. Chemical inhibition of ROCK led to a reversal of the rounded morphology and to a decrease in the invasive capacity. Unlike in HT1080 fibrosarcoma cells, we found that invasion of tumorigenic cen3tel cells was not reduced upon treatment with a matrix protease inhibitor, indicating that cells of mesenchymal origin can spontaneously undergo a transition towards amoeboid movement during transformation.
At the molecular level, this switch could be linked to two main changes: the decrease in the expression of matrix protease genes and the reduced expression of the Rho GTP-binding protein Rnd3. Experimental inhibition of pericellular proteolysis can induce a transition from mesenchymal to amoeboid movement [19] ; in our cellular system, spontaneous downregulation of matrix protease genes, through a mechanism still to be clarified, might have contributed to the switch in the type of movement.
Rnd3, together with Rnd1 and Rnd2, belongs to the Rnd family of small GTP-binding proteins always bound to GTP, which is involved in the control of cytoskeletal organization [25] . Microarray analysis did not show any significant change in the expression of Rnd1 and Rnd2 during malignant transformation of cen3tel cells. Rnd3 is an inhibitor of ROCK-I [32] , [40] and evidence has been reported that it can play a role in controlling morphology and invasion of rounded tumor cells. Pinner and Sahai [41] showed that a fine balance between PDK1 and Rnd3 expression is required for the amoeboid movement of melanoma cells, because the two proteins have opposing roles in controlling ROCK-I activity. PDK1 drives ROCK-I to the plasma membrane where it stimulates actomyosin phosphorylation and contraction, while Rnd3 can bind to ROCK-I and inhibit its activity, reducing motility. Gadea et al. [14] showed that overexpression of Rnd3 in mouse embryonic fibroblasts in which amoeboid migration had been induced by p53 knockdown led to reduced invasion. Our observation that ectopic Rnd3 expression in phase II and III tumorigenic cen3tel cells reduced their invasive capacity points to Rnd3 as a possible regulator of invasion of cells of mesenchymal origin. In addition, the low levels of myosin phosphorylation in Rnd3 transfected clones is in agreement with the hypothesis that Rnd3 hampers amoeboid movement by inhibiting ROCK kinase activity. Finally, the low metastatic potential observed in phase III tumorigenic cen3tel cells that ectopically express Rnd3 suggests that Rnd3 exerts its effect also in vivo and that its downregulation can have a pro-metastatic effect.
Rnd3 shows reduced expression in prostate cancer and seems to have a protective role against breast cancer [25] ; however, increased Rnd3 expression has been found in other epithelial tumors and in metastatic melanoma cells [42] , suggesting that the genetic background of cancer cells can influence the role of Rnd3 in trasformation. Very little is known about Rnd3 expression in human sarcomas. Our results indicate that reduced Rnd3 expression in tumor cells of mesenchymal origin can be linked to the acquisition of a ROCK-dependent mode of invasion and can increase the metastatic potential. Thus, analysis of Rnd3 expression in human sarcomas might give information on the mechanism of invasion adopted by the tumor cells and help orientate the therapeutic strategy [37] .
It is worth noticing that the increase in the invasive potential and Rnd3 downregulation occur when cen3tel cells are tumorigenic but not yet metastatic. This suggests that the acquisition of invasive ability by tumor cells, although necessary, is not sufficient to give them full metastatic ability. Recent studies [43] – [47] suggest that even normal cells can disseminate and dissemination from primary tumors can actually precede the ability to form metastases; additional genetic or epigenetic changes are then required for the subsequent production of metastases. It is possible that cen3tel cells progressively acquired different and complementary malignant properties besides invasiveness that ultimately led to a fully metastatic cell population. In agreement with this, preliminary findings indicate a progressive increase in the expression of pro-angiogenic factors (such as VEGF and FGF2) and a concomitant decrease of angiogenesis-inhibitory factors (particularly TSP-1 and TSP-2) during cen3tel malignant progression, pointing to angiogenesis as a candidate pro-metastatic feature acquired by late-phase metastatic cen3tel cells.
In conclusion, the results presented here do suggest that sarcoma cells can adopt a protease-independent/ROCK-dependent mechanism of invasion and indicate that the Rnd3 gene can play a role in regulating the invasiveness and metastatic capacity of mesenchymal tumor cells. | Conceived and designed the experiments: CM. Performed the experiments: CB RF KB IC MMG APDT. Analyzed the data: FF. Wrote the paper: CM. Performed the bulk of the experiments: CB. Helped to draft the manuscript: CB IC EG GT MD. Carried out the in vivo experiments: RF. Performed the in vitro migration and invasion assays: KB. Conceived the transfection experiments: IC. Participated in the experimental work: IC. Analyzed microarray data: FF MMG. Performed the histological analysis: APDT. Discussed the results: EG. Participated in designing the study: GT MD.
Background
Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies.
Methodology and Principal Findings
We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo .
Conclusions
These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas. | Supporting Information | We are very grateful to Dr. Giovanna Chiorino (Fondazione Edo ed Elvo Tempia, Biella, Italy) for supervision of microarray analysis, to Dr. Patrizia Vaghi (University of Pavia, Italy) for confocal microscope analysis and to Dr. Pierre Roux (CNRS FRE2593, France) for the EGFP-C1-Rnd3 plasmid. We thank Dr. Valentina Di Felice (University of Palermo, Italy) for suggestions about immunofluorescence on cells in Matrigel and Ms Giudith Baggott for help in editing the English style. CB is a PhD student of the University of Pavia (Dottorato in Scienze Genetiche e Biomolecolari). | CC BY | no | 2022-01-13 08:08:43 | PLoS One. 2010 Nov 30; 5(11):e14154 | oa_package/31/5b/PMC3014295.tar.gz |
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PMC3014355 | 21209797 | Introduction
Species and subspecies are taxonomic levels generally considered as objective descriptors of biological reality, a mere fact at the origin of the numerous different studies proposed to date. Whereas long diverged lineages are generally easy to separate with various sets of characters, recently diverged lineages are usually much more difficult to address. In those cases, classical morphological approaches sometimes fail to provide consistent discrimination or hypotheses about evolutionary history and relationships, and benefit from the inclusion of other markers with more appropriate and objectively measurable rates of divergence [1] . Besides the most commonly used neutral genetic markers such as ISSR [2] , microsatellites [3] nuclear and mitochondrial sequences [4.5], allozymes [6] and karyotypes [7] , [8] , alternative sets of characters, such as cuticular compounds, have been proposed and tested with promising results [9] , [10] . These compounds are lipids present on the most external cuticular layer of all terrestrial arthropods: the epicuticular layer. This layer is composed of various categories of lipids. In most cases, the majority of these compounds are hydrocarbons but other types of compounds can be present such as fatty acids, ketones, alcohols and esters [11] , [12] . As the benefit of using the maximum number of compounds for population level studies has already been emphasized in the literature [10] , in the present study we consider the total composition of the cuticular profile regardless of families to which the chemical compounds belong. In insects, the different types of cuticular compounds are functionally involved, particularly in sexual communication [13] , [14] . In Collembola, intraspecific interactions such as reproductive attraction and recognition signalling [15] , [16] , aggregation [17] , [18] , [19] , [20] , [21] and alarm [22] , [23] , [24] rely heavily on olfactory interactions through chemical compounds.
As they also play a major role in sexual recognition [25] , [26] and thus in premating isolation mechanisms [27] , cuticular compounds, as a type of character, deserve a special attention in the study of groups of subspecific entities. This is especially relevant for Collembola since cuticular compounds were proven to play a major role in sexual recognition [16] suggesting that they could be potential key drivers of reproductive isolation, making them good candidates for infraspecific taxon discrimination in this group.
A previous population study of the wingless grasshopper Chorthippus parallelus from the Pyrenees revealed significant differences between subspecies cuticular profiles and suggested their major implication in specific sexual recognition [28] . A quantitative sexual dimorphism in cuticular blends was found in this grasshopper species [10] , [29] and subsequently those compounds were proven to be involved in reproductive isolation mechanisms [30] . A similar sexual dimorphism was also demonstrated in Collembola [31] .
In the present study, we tested the discriminating power of such characters at the infraspecific level for Collembola using the Deutonura deficiens complex as a model. This thoroughly studied group displays a very complex pattern of closely related subspecies and morphotypes living in parapatry within a relatively limited geographic range. Narrow overlapping areas and hybrid zones were documented in the Pyrenees region [32] , [33] . This complex includes three subspecies: D. d. deficiens , D. d. meridionalis and D. d. sylvatica . Each one splits into several morphotypes that are geographically well delimited. These infraspecific taxonomic categories defined by Deharveng [32] are based on their distribution and morphological similarity within populations (cephalic tuberculisation among subspecies and abdominal setae and body pigmentation for morphotypes within subspecies).
The extensive knowledge of the repartition areas of these different infraspecific entities allowed an overall assessment of the role that ecological factors played in producing the current biogeographical pattern and the position of the contact zones [32] . None of the ecological factors examined (vegetation, altitude, precipitation, temperature or hydrographic network) were found to be significantly related to the species boundaries within the mosaic pattern [33] . Concerning the origin of the pattern, these results suggest the involvement of inherent characteristics of the organisms and/or an historical inheritance.
In this context we address the utility of cuticular compounds as a set of characters for (1) separating infraspecific lineages in Collembola and (2) establishing phylogenetic relationships between them. We also discuss the origin of the parapatric pattern in the context of European glacial history. | Materials and Methods
Samples
Specimens were collected in the field, either by direct capture on substrate or by extraction from litter and decayed wood with Berlese funnels. They were freeze killed and stored at -28°C. In order to avoid the potential bias of variation of the cuticular profile with age [34] , only adult individuals were used. In this study, 21 individuals from the D. d. deficiens subspecies, 13 from D. d. meridionalis and 21 from D. d. sylvatica (with 12 individuals from morphotype C and 9 from morphotype A) were analysed ( Table 1 ). In order to infer phylogenetic relationships among these infraspecific entities, we used an external species, Deutonura monticola (17 individuals), to root the evolutionary hypothesis. Identifications to subspecies and morphotypes were carried out through morphological examination of specimens after microscopic preparation of all individuals.
Gas Chromatography
Cuticular lipids were extracted by soaking each individual for 5 minutes in 20 μl of hexane. Separation of compounds in the extracts was performed on a Hewlett Packard Chromatograph model 5890 equipped with a Flame Ionization Detector (320°C) and an automatic sampler (HP 6890). For each individual, 5 μl of extract was injected in an apolar capillary column (HT-5) of 25m long. Helium was used as the carrier gas (pressure 16 psi - injector temperature 280°C). The temperature program started from 150°C and reached 320°C with a rate of 5°C/min. Then a 20 minutes 320°C isotherm was used to clean up the column between two samples. Each compound was characterized with its Kováts index (KI) [35] calculated using the coelution of samples with a standard solution of linear alkanes (C16 to C40) ( Table S1 ). This homology hypothesis relying on KI brings limitations because compounds were not structurally identified. Some compounds even if structurally different can have the same KI. In some cases, this could lead to an underestimation of the variability of the profiles.
Statistical Analysis
The data were recovered with the Millennium 2.15 software (Waters). For each individual, the relative surfaces of each peak in the chromatogram matched the concentration of the corresponding cuticular compound in the blend. Those relative surfaces were calculated and the data were exported to Systat 10.0 for statistical treatment. A stepwise forward discriminant analysis was performed to explore infraspecific variation, and the “F-to-remove” value was retained for each discriminant peak. This value is a good indicator of peak importance for distinction between groups. But a discriminant analysis alone can eliminate highly correlated values and thus give a partial view of the data set separating different classes. For this reason we also performed Pearson Correlation Matrix with a Bonferroni correction (P<0.005) to recover the correlated peaks ( Table S1 - Supplementary material). A jackknife resampling method (default for discriminant analysis in the SYSTAT 10.0 software) was used to evaluate the discriminatory power of the cuticular compounds.
Phylogenetic Reconstruction
Distance relations were established between the different infraspecific entities through a Neighbor Joining calculation with MEGA 4 [36] . The Mahalanobis distances matrix obtained from the previous discriminant analysis (i.e. distances between centroïds of the different infra-specific entities) was used for this purpose ( Table 2 ). The external species D. monticola was used to root the phylogenetic hypothesis. | Results
A total of 128 distinct compounds were found for D. deficiens subspecies, morphotypes, and the external species Deutonura monticola . After the statistical analysis, we obtained a significant discrimination (P<0.00001, F = 27.6126) between the four infra-specific taxa and D. monticola ( Fig. 1 ). The Jacknife classification value was 100% for all taxa but D. d. sylvatica morphotype C with one individual misplaced ( Table 3 ). Thirty-five cuticular compounds were designated as discriminant. Eighty-two other compounds were recovered using the correlation matrix. Eventually, 117 products allowed separation of the groups ( Table S1 - Supplementary material). All variations were quantitative. Most compounds are implicated in the discrimination, indicating profile proximity between the different morphotypes and subspecies with numerous quantitative variations at a fine scale.
Phylogenetic relationships were established and rooted with Deutonura monticola ( Fig. 2 ). The subspecies D. d. sylvatica appeared as the first derived form of the lineage with its two morphotypes aggregated in basal position. Then the two other subspecies, D. d. deficiens and D. d. meridionalis , branched together suggesting their later individualisation. | Discussion
During the last twenty years, cuticular compounds have been investigated in chemotaxonomy for species specificity [37] , [38] , [39] , [40] , [41] . But the systematic use of those characters for taxonomy was challenged by some findings in termites: if cuticular compounds were proven useful in this group for species delimitation [40] , [42] , a strong variation of the profiles was found among several colonies of the same species [43] , [44] , [45] . But this is the peculiar case of social insect in which conspecific colonies are competing with each other and thus have a strong pressure for chemical recognition cues among colonies [13] , [46] , [47] even if a ‘dear enemy’ effect was demonstrated in termites [48] . It was suggested that a part of the cuticular compounds can be incorporated from some of the substances found in the alimentation as a possible explanation for the high variability of profiles among conspecific colonies of termites [48] , such a phenomenon was already proven to occur in ants [49] .
By contrast there is not such a competitive pressure among individuals in solitary animals like collembolans and thus a selection pressure toward a strong individualization of their cuticular profiles cannot be expected. Furthermore the repartition areas of the infraspecific entities of the species complex studied in this paper are very restricted and so the variations of feeding possibilities are expected to be quite narrow on such a range where the types of habitats are similar. The low variability of the collembolans cuticular profile within and among different populations of the species was documented in this respect [50] which allows to push the test of the taxonomic resolution further here.
In this study, cuticular compounds successfully discriminated all of the infraspecific taxa assigned by morphology within the Deutonura deficiens complex. The congruence between the two types of characters supports the biological reality of those entities. In addition, they brought further understanding of the phylogenetic relationships among these taxa. Those data confirmed that in Collembola, this character type is phylogenetically informative at low taxonomical levels [50] .
The phylogenetic relationships established from cuticular profiles support two hypotheses: (a) an early individualization of D. d. sylvatica morphotypes A and C, which is consistent with the current sympatry of some of their populations exhibiting a high density of both parental phenotypes and an absence of hybrids; such a pattern and their basal position in the chemical phylogeny support the hypothesis of an achieved speciation phase with pre-reproductive isolation completed as hypothesized by Deharveng [32] , (b) a more recent appearance of D. d. deficiens and D. d. meridionalis ; the strict parapatry and the occurrence of morphologically intermediate specimens (assumed to be hybrids) among mixed populations in narrow contact zones between those two subspecies provides further support for their recent individualization.
As these chemicals play a crucial role in sexual attraction and mate recognition in Collembola [16] , the discrepancies found among the cuticular profiles of all the infraspecific entities of the D. deficiens complex strongly suggest a status of an ongoing process of reproductive isolation involving those compounds. Clear divergence of cuticular profiles between sexes was demonstrated for other Collembola species [31] . Such sexual dimorphism exists also in D. deficiens , and all but one of the cuticular compounds involved in the sexual variation were implicated in the subspecies discrimination ( Table S2 - Supplementary material). This indicates that sexual selection is mostly responsible for the variation of these traits, implying a major role of the cuticular compounds in sexual and specific recognition, which fits with the last findings in this field [16] . Then the possibility of a reinforcement phenomenon weighting on these characters as described by Higgie and Blows [51] could be considered.
The elements brought by the analysis of cuticular profiles suggest that several events of allopatry could have led to different levels of reproductive isolation responsible for the appearance of the distinct intraspecific entities and the origin of the current parapatric pattern of the D. deficiens complex. Pleistocene glacial events could be at the origin of the occurrence of allopatric phases for several populations of this species. A comparable mosaic distribution of the subspecies of another Collembola species of the same subfamily ( Monobella grassei ) was uncovered in the same region [52] . One of the hypothetical causes examined for such a pattern was the major historical event of the Pleistocene glaciations. These key paleoclimatic episodes are mainly accountable for the current repartition of animal and plant species populations: a correlation was established between variation in vegetal cover and climatic osrelationships were established cillations in the last 135 000 years [53] , and Hewitt [54] proposed three European models for post-glacial recolonization in the Pleistocene based on plant and animal cases. The knowledge of the geographic distribution of the taxa studied here allows a comparison with Hewitt's recolonization models [54] in order to discuss the origin of the actual pattern (described in [33] ) in a historical perspective. One of the postglacial recolonization models described by Hewitt [54] matches the existing distribution of the D. deficiens subspecies and the location of the contact zones (the recent discovery of a D. d. sylvatica population in the Italian Peninsula strengthens this support). It implies a faster advance of the populations from eastern refuges (Balkan, southern Carpathians and Caucasus) by contrast with the populations from the Iberic and Italian Peninsula refuges. As a result, the northern zones of these southern refuges were colonized by eastern populations blocking the advance of the southern ones and leading to the occurrence of contact zones. The current repartition and the contact zones position observed in the D. deficiens complex is congruent with the scenario of post-glacial recolonization exhibited by several taxa [54] . This brings support to the involvement of the last Pleistocene glacial events in the establishment of this distribution pattern.
Though we acknowledge some limitations in the understanding of some aspects of the complex wider geographic repartition and history: the possible occurrence of populations belonging to D. deficiens complex in eastern refuges, or the role played by Atlantic and Mediterranean microhabitats near actual contact zones which could have interfered with this model at fine scales as described in the ‘refugia within refugia’ concept developed for the Balkan Peninsula [55] .
The successful use of a type of character directly involved in the mate choice on genetically distinct but closely related entities in a parapatric context, underlies their predominance in the origin and the maintenance of this parapatric pattern. The complexity of the whole pattern calls for a comparison of other markes types and a more exhaustive sampling through existing and potential repartition areas of the complex. The elucidation of the origin and the maintenance of such a pattern is crucial to understand what has driven the repartition and diversity of understudied soil microarthropods in this critical European zone. | Conceived and designed the experiments: DP. Performed the experiments: DP. Analyzed the data: DP. Contributed reagents/materials/analysis tools: DP AB LD. Wrote the paper: DP LD.
Current address: Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada
Background
In most Arthropod groups, the study of systematics and evolution rely mostly on neutral characters, in this context cuticular compounds, as non-neutral characters, represent an underexplored but potentially informative type of characters at the infraspecific level as they have been routinely proven to be involved in sexual attraction.
Methods and Findings
The collembolan species complex Deutonura deficiens was chosen as a model in order to test the utility of these characters for delineating four infraspecific entities of this group. Specimens were collected for three subspecies ( D. d. deficiens , D. d. meridionalis , D. d. sylvatica ) and two morphotypes ( D. d. sylvatica morphoype A and B) of the complex; an additional species D. monticola was added. Cuticular compounds were extracted and separated by gas chromatography for each individual. Our results demonstrate that cuticular compounds succeeded in separating the different elements of this complex. Those data allowed also the reconstruction of the phylogenetic relationships among them.
Conclusions
The discriminating power of cuticular compounds is directly related to their involvement in sexual attraction and mate recognition. These findings allowed a discussion on the potential involvement of intrinsic and paleoclimatic factors in the origin and the diversification of this complex in the Pyrenean zone. This character type brings the first advance from pattern to process concerning the origin of this species complex. | Supporting Information | The authors want to thank here André Fabres for his advice on statistical analyses, Sarah Adamowicz and Rodolphe Rougerie for their useful comments on the manuscript. | CC BY | no | 2022-01-13 08:09:45 | PLoS One. 2010 Dec 21; 5(12):e14405 | oa_package/d6/e2/PMC3014355.tar.gz |
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PMC3014473 | 21181226 | Introduction
The anterior cruciate ligament (ACL) plays a crucial role in knee stability, as it contrasts the combined movement of the tibia against the femur, anterior translation and internal rotation. ACL injuries can affect one or both strands (anterior-medial and postero-lateral) and, on the basis of individual characteristics, can affect ligament function and knee stability, raising the need for surgical reconstruction.
Defining the prevalence of this condition is not easy, as lesions are often asymptomatic; a study carried out on a large sample of students from a US college showed that the possibility of ACL injury may be over 3% in 4 years of physical activity, with a higher risk in female population [ 1 ].
Surgical reconstruction for primary isolated ACL lesions is performed using autograft (mostly patellar or hamstring tendons) or allograft (allogenic tissue from humans and of different sorts), while use of synthetic ligaments has recently attracted interest after being abandoned in the past due to a high failure rate.
The choice of technique is based on clinical and biomechanical factors, or on tradition and surgeon experience, or for reasons of context, as shown by various investigations carried out among surgeons from different countries [ 2 , 3 ].
The heterogeneity in the surgical management of this condition raised a need for clarity on the effectiveness of the different types of grafts, through a systematic, critical appraisal of the literature. The National Guidelines System (SNLG, Sistema Nazionale Linee Guida) of the Ministry of Health at the National Institute of Health (Istituto Superiore di Sanità, ISS) therefore engaged in the elaboration of a document aimed at guiding orthopaedic surgeons in the choice of best practice for primary anterior cruciate ligament reconstruction. The document does not evaluate the different systems for graft fixation, nor the different techniques for preoperative preparation or postoperative rehabilitation. | Materials and methods
The quick review document is an instrument designed to handle very specific clinical issues through a faster process than the one used to draw up guidelines. The panel created to carry out the activities needed to elaborate the quick review is monodisciplinary, in contrast to the one created for guidelines, and aims to answer a small number of clinical questions defined as crucial by the specialists, and to reduce all heterogeneous and sometimes inappropriate clinical practices.
Panel composition
The panel of experts who collaborated to draw up this document included 14 orthopaedic surgeons, 2 physiatrists, 1 physiotherapist and 2 epidemiologists familiar with evidence-based medicine and the methodology for guidelines development. The working group included representatives from all the main national scientific societies of reference [Gruppo di Lavoro Ortopedia Basata sulle Prove di Efficacia (GLOBE), Società Italiana di Artroscopia (SIA), Società Italiana di Chirurgia del Ginocchio, Artroscopia, Sport, Cartilagine e Tecnologie Ortopediche (SIGASCOT), Società Italiana di Medicina Fisica e Riabilitativa (SIMFER), and Società Italiana di Ortopedia e Traumatologia (SIOT)], supported by a balanced group of independent experts.
All participants signed a declaration of absence of conflict of interests and of acceptance of the methodology as explained during the first meeting.
The panel met twice (4 July 2008 and 6 February 2009), and all materials produced during the process for the elaboration of the document are available at: http://www.snlg-iss.it .
Definition of the clinical questions, bibliographic search and critical appraisal of literature
The objectives of the document, the clinical questions on the effectiveness and safety of the types of graft to be used for anterior cruciate ligament reconstruction, the inclusion and exclusion criteria for studies and the timeframe to be considered in the bibliographic search were defined by the panel during the first meeting at the Italian National Institute of Health (ISS).
Specific search strategies were defined in accordance with each established clinical question.
The following databases were searched to gather evidence: PubMed, Embase and Cochrane Library, including randomized clinical trials (RCTs) and systematic reviews (SRs) dated 2000–2008.
Observational studies dated 2000–2008 from the PubMed database were included for questions concerning safety. Figure 1 presents the search filters used for both questions (effectiveness and safety) and the main inclusion criteria.
Qualitative assessment of systematic reviews, RCTs and observational studies was carried out using a structured method [ 4 , 5 ].
Data extraction, summary of evidence and recommendations
The selection of studies, their methodological evaluation and the extraction of data were carried out by specifically trained personnel. The evidence gathered from each study was summarized in tables, each specific to a single question and type of study. The summary tables adopted in this document are those defined by the National Institute for Clinical Excellence (NICE), updated in 2007.
The recommendations were drawn up for each clinical question without adopting any specific grading system, that is, without using any structured system to grade the strength of recommendations. The intensity and certainty supporting all recommendations are reported in narrative form, without any symbol, graded score or hierarchy. Each recommendation is introduced by a description of the discussion that led to its definition, to make clear the level of agreement of the working group.
The panel adopted the GRADE system to carry out the critical appraisal of literature and to draw up the recommendations [ 6 – 11 ].
The critical appraisal of the literature was carried out for each outcome considered relevant by the panel, following the principles of this method. The quality of evidence, finally, was related to the assessment of all risks connected to adopting that specific procedure, thus reaching the definition of the recommendation.
External review
The final draft was shared by the panel in the second and last meeting, and then sent to four external referees, asking them to assess its readability and clarity, its clinical relevance and the feasibility of recommendations. The referee group included renowned orthopaedic surgeons with an interest in knee surgery and with active scientific production in the field. The full text of the document (currently available only in Italian), including all suggestions from the referees, is available on the SNLG website at: http://www.snlg-iss.it . | Results
The panel agreed on two clinical questions, one related to the effectiveness and the other to the safety of arthroscopic ACL reconstruction carried out using autograft (Table 1 ), allograft (Table 2 ) or synthetic graft (Table 3 ). The literature search gathered 489 titles and abstracts, among which 30 articles met the defined selection criteria.
Use of autograft in arthroscopic ACL reconstruction
Table 1 reports the question concerning autograft, the literature screening procedure and the recommendations as defined by the panel. Twelve SRs and seven RCTs were selected for the assessment of the effectiveness of autograft, comparing use of patellar tendon (PT) versus hamstring (HS), while five retrospective studies were chosen to define the recommendations concerning safety.
All selected reviews included mostly randomized or quasi-randomized prospective studies based on follow-ups of 2 or more years, and aimed at assessing the effectiveness of autograft using objectively measured or subjectively assessed mechanical or functional outcomes (laxity, stability, return to pre-injury activity and loss of flexibility).
The assessment of laxity and stability defined with various measures [Knee Test (KT), Lachman test, pivot shift test, International Knee Documentation Committee (IKDC) score], the frequency with which patients return to pre-injury activity and the loss of flexibility support the hypothesis that PT in several cases performs better than HS [ 12 – 19 ], while HS appears to reduce anterior pain and loss of extension.
The effect rates reported are often close to statistical significance (even if unable to prove superiority of one specific technique), confirmed by the results of the included RCTs. These [ 20 – 25 ] are not able to demonstrate differences between the two techniques due to the lower statistical power compared with the reviews of primary studies, and often show methodological flaws affecting the inferences.
Promising experiences using four-strand hamstring tendon have also been carried out [ 26 ], or using two-strand hamstring tendon associated to extra-articular plastic (2HS EP), to limit laxity in rotation [ 27 ].
Both seem to substantially improve HS graft performance in terms of stability, but require further investigation.
Evidence in relation to safety of autograft comes instead from uncontrolled observational studies and refers to infections, and in one case [ 28 ] to mechanical and functional side-effects of surgical procedures.
Clusters of joint infections are reported among subjects who underwent ACL reconstruction (1.6–2.6%), with slight predominance with HS use (5.7%) and an increase of risk, probably due to former ACL reconstruction [relative risk (RR) = 5.1] or knee surgery (RR = 1.90) and to the use of some fixation systems for femur (RR = 4.5 for Endobutton) or tibial (RR = 3.2 with metallic post and washers) fractures [ 29 , 30 ]. The infection rate, in the absence of clusters, results <1%, showing no differences in relation to the technique chosen [ 31 ].
Almazàn et al., finally [ 32 ], show that donor-site complications are more frequent in HS grafts (6.2% versus 0.6% in PT), as are complications due to complicated procedures.
PT graft appears therefore to be fairly superior to HS graft, in terms of stability, return to pre-injury activity and flexural strength, while use of HS can be reasonably restricted to specific situations, due to its effectiveness in reducing pain and loss of extension. Evidence on safety is scarce and fragmentary, and no inferences can be made apart from a few suggestions on infective complications.
The panel therefore decided to recommend use of PT due to its proven higher stability and to identify at the same time a possible subgroup of subjects for whom knee pain can represent a particular problem (e.g., some categories of workers), or for whom reducing length and intensity of pain as much as possible could be important, and define for this subgroup a specific indication for use of HS.
Use of allograft in arthroscopic ACL reconstruction
Table 2 summarizes the activities of the panel in relation to use of allograft. Only two SRs were selected at the end of the literature screening. These reviews include non-randomized primary studies aimed at comparing allograft versus autograft.
The study of Prodromos et al. [ 33 ] analyzes data from 20 case series from 18 studies on the stability of allograft, comparing them with data from a former meta-analysis on autograft [ 34 ].
The global stability rate indicates higher efficacy of autograft, with 72% normal stability (versus 59% registered in the allograft group) and 5.3% abnormal stability (versus 14% registered in the allograft group). The differences observed between the two types of grafts were statistically significant in both cases ( P < 0.001).
Moreover, higher efficacy of non-irradiated tissues (63%) versus irradiated tissues (43%, P < 0.001) has been observed, and of non-patellar tissue (64%) versus patellar tissue (57%, P < 0.001).
Krych et al.’s review [ 35 ] included one quasi-randomized study and five non-randomized studies comparing effectiveness between autogenic and allogenic patellar tendon graft. The follow-up was longer than 2 years, and the same rehabilitation protocols were adopted.
No statistically significant differences emerged between the two types of grafts apart from the worse performance of allograft in terms of graft failure [odds ratio (OR) = 5.03, 95% confidence interval (CI) 1.38–18.33] and of hop test results <90% versus healthy side (OR = 5.66, 95% CI 3.09–10.36).
The panel, in accordance with the GRADE methodology, was invited to vote on the relevance of the outcomes considered in the selected studies. Table 4 reports the assigned score, the quality score, the estimated effectiveness and the risk–benefit assessment for each outcome.
Graft rupture, re-operation rate, return to pre-injury activity and IKDC score were considered critical outcomes, and graft rupture in particular benefited from evidence much higher in quality than that gathered for other outcomes, supporting higher efficacy of autograft versus allograft. The other outcomes defined by the panel seemingly showed higher efficacy of autograft, even if the values did not reach statistical significance.
Evidence in relation to safety, on the other hand, relies on two studies and essentially concerns infective complications.
Centeno et al.’s study [ 36 ] was not assessed due to the inadequacy of its design and the irrelevance of the results. Crawford et al.’s study, on the other hand [ 37 ], reports a 3.3% (11/331) infection rate among 331 patients who underwent ACL reconstruction between 2000 and 2002. All infections were observed among the 250 patients treated with aseptic allograft (4.4%, 11/250), while no infections were observed among the 81 subjects treated with sterile allograft or autograft. The type of graft (allograft versus autograft, RR = 3.3, n.s.), the type of treatment adopted to process grafts (aseptic versus sterile, RR = 70.5, 95% CI 1.1–∝), use of supplemental tibial staples (use versus non-use, RR = 10, 95% CI 3.0–32.9) and use of a specific device (Intrafix versus no fixation, RR = 10.6, non-significant) resulted as the main risk factors.
Moderate superiority of autograft as ACL reconstruction technique is to be pointed out, on the basis of allograft performance (versus autograft), in relation to the outcomes considered relevant and the quality of selected evidence, subsequent to the risk–benefit assessment. Use of allograft, in fact, shows higher failure rate and higher risk of infective complications with aseptic tissue.
Use of synthetic grafts in ACL reconstruction
Table 3 reports the question concerning use of synthetic grafts and the scant evidence supporting the recommendations.
The full text of a systematic review identified by the literature search [ 38 ] resulted unavailable. The abstract stated that the study included 3 RCTs and 11 case series. Authors concluded that no indications could be stated due to the lack of evidence.
Two more studies (RCTs) gathered by the literature search compared patellar tendon autograft and synthetic graft. The full texts of both of these articles underwent critical appraisal [ 39 , 40 ], and the studies resulted of good quality, even if based on small populations (40 enrolled in Muren et al.’s study and 53 in Nau et al.’s study).
Muren et al.’s study investigated use of a polypropylene device, the Ligament Augmentation Device (LAD), stitched to the autograft, while Nau et al. investigated the use of the Ligament Advanced Reinforcement System (LARS), a device produced in France, fixed with titanium screws. This trial is included in Pichon Riviere’s systematic review.
Muren et al. follow the 40 randomized patients for 7 years (3 years for arthrometric assessment with KT 1000), reporting no significant differences between the two groups. The results of this study refer to patients with acute ACL injuries, with time of injury less than 3 weeks prior to enrolment being an inclusion criterion.
Nau et al., on the other hand, included patients who suffered ACL injury no less than 6 months before enrolment, and showed a substantial equivalence after arthrometric tests (2.38 mm in PT versus 4.86 mm in LARS, P < 0.05) and IKDC assessment. Patients expressed a slight preference [assessed using the Knee injury and Osteoarthritis Outcome Score (KOOS) score] for LARS treatment at 6 and 12 months, but not at 24 months.
No studies were identified investigating the safety of synthetic grafts in ACL reconstruction. The panel agreed on the potential benefits of synthetic grafts in ACL reconstruction; the lack of evidence, however, does not allow recommendation of use of such materials, and further investigations to assess their efficacy and safety are needed. | Discussion
The document on graft choice in primary ACL surgery is the first SNLG experience of a quick review ( documento di revisione rapida ). The main feature of this type of document, apart from some methodological issues, is the specificity of topics, being mainly monospecialistic.
The GRADE method was used to analyze the allograft question. Its strong structure allowed the collation of all information needed to draw up the recommendation, and highlighted the core points for discussion.
Two reviews [ 34 , 35 ] underwent critical appraisal in relation to the question concerning allograft. These two reviews included non-randomized studies (except for Gorschewsky’s quasi-randomized study). Kyrch et al.’s review, according to the GRADE method, started from a “low” quality level, while Prodromos et al.’s review, including case series with historic (non-concurrent) controls, started from a “very low” quality level.
The critical appraisal of this evidence raised some difficulties, as the quality resulted often below the “low” or “very low” level. Strongly recommending a specific procedure is embarrassing if the available evidence is of very low quality, even if the method states a certain independence between quality of evidence and strength of recommendation. The undertones needed in shaping recommendations and avoiding such embarrassment did not fit the “weak” and “strong” labels. The panel therefore decided to express recommendation strength in a narrative way, bringing together in the text both structured assessment of evidence and unstructured discussion.
Krych et al.’s study showed some heterogeneity between the included studies. The author stated that this was due to the presence of a single study [ 41 ] considering the type of preparation and sterilization used for patellar tissue in allograft. These treatments for sterilization with radiation and dehydration with acetone would have, in other words, decreased the efficacy of allograft, producing data against its use, and caused the heterogeneity of results.
The evaluation of the outcomes, as defined by the GRADE method, enabled verification that the statistical test used by the author highlighted no significant heterogeneity in relation to the outcomes considered relevant (ex. graft failure). The analysis of sensitivity, based on the inclusion/exclusion of Gorschewsky et al.’s study, did not substantially modify the results, causing only a loss of power that did non allow the results to reach statistical relevance. Results, however, showed a certain superiority of autograft.
The panel agreed that the sterilization procedure used in Gorschewsky et al.’s study is to be considered responsible for the lower efficacy of the allograft, but that it is not currently adopted. Therefore, the recommendation defined by the working group did not take into consideration the effects of this procedure on the effectiveness of the intervention.
Global agreement was reached for all questions and recommendations, irrespective of divergences arising in interpretation and assessment of some studies. Evidence, in fact, showed overall homogeneity, and the clinical opinions from each member converged without affecting the richness of information. The results of the review are therefore coherent with current trends in clinical practice, although they do supply robust scientific data to support the choice of graft in ACL primary surgery. | Conclusions
Available evidence allows recommendation of use of autograft over allograft in arthroscopic ACL reconstruction and to recognize, for autograft, better performance of PT over HS. It is therefore appropriate to select one of these two main choices (PT and HS), assessing the indication on a case-by-case basis. It is also appropriate to consider allograft and artificial ligaments only in very selected cases, discouraging widespread use, given the potential risks and paucity of well-performed, well-designed clinical studies. The indications for further research are also clear. Consolidation of the experience in use of two- and four-strand HS and in using specific techniques to contain laxity is suggested. Further investigations are also strongly suggested on use of synthetic grafts in studies comparing their effectiveness versus autograft. It is valuable to recall that stepwise introduction of new orthopaedic technologies should include preclinical testing, randomized clinical trials, multicenter studies and post-market surveillance, to provide surgeons with adequate information to make informed decisions regarding use of new technologies in their practice, including ACL reconstruction with synthetic ligaments [ 42 ].
Finally, this experience confirms the feasibility of practice guidelines to drive an evidence-based approach in orthopaedic surgery. In this particular case, representatives from the scientific societies with an interest in knee surgery (SIOT, SIA, SIGASCOT, SIMFER, and GLOBE) participated in collecting, analyzing and discussing the available data to develop evidence-based guidelines using a standardized and reliable methodology. The practice of evidence-based medicine can be conceptualized as the integration of the best available research evidence, clinical circumstances and patients’ values and preferences. Evidence-based practice guidelines allow practitioners to develop treatments for a specific patient, on the bases of not only his/her experience and personal knowledge, but also the most up-to-date scientific evidence, reviewed and evaluated using a structured, detailed and explicit approach. Through the process of guideline development, clinical and methodological experts evaluate and condense the universe of information available on a clinical issue into a useful set of parameters that the physician can complete with his/her own experience and knowledge in managing a patient. Guidelines are not a substitute for continuing study, rather they represent a tool for the practitioner to provide the best care for his/her patients [ 43 ]. | Background
Anterior cruciate ligament (ACL) surgical reconstruction is performed with the use of an autogenic, allogenic or synthetic graft. The document issued by the Italian National Guidelines System (SNLG, Sistema Nazionale Linee Guida) at the National Institute of Health aims to guide orthopaedic surgeons in selecting the optimal graft for ACL reconstruction using an evidence-based approach.
Materials and methods
A monodisciplinary panel was formed to define a restricted number of clinical questions, develop specific search strategies and critically appraise the literature using the grading of recommendations assessment, development, and evaluation (GRADE) method. The final draft was shared by the panel and then sent to four external referees to assess its readability and clarity, its clinical relevance and the feasibility of recommendations.
Results
Autograft shows moderate superiority compared with allograft, in relation to the relevant outcomes and the quality of selected evidence, after an appropriate risk–benefit assessment. Allograft shows higher failure rate and higher risk of infection. The panel recommends use of autografts; patellar tendon should be the first choice, due to its higher stability, while use of hamstring is indicated for subjects for whom knee pain can represent a particular problem (e.g., some categories of workers).
Conclusions
Autograft shows better performance compared with allograft and no significant heterogeneity in relation to relevant outcomes. The GRADE method allowed collation of all the information needed to draw up the recommendations, and to highlight the core points for discussion.
Keywords | Appendix
Ezio Adriani, Casa di Cura Mater Dei, Roma; Angelo Cacchio, Divisione di Medicina Riabilitativa, Università La Sapienza, Roma; Vincenzo Condello, Ospedale S. Cuore Don Calabria, Negrar (VR); Franca D’Angelo, Istituto Superiore di Sanità; Salvatore De Masi, Dipartimento Prevenzione, ASL 6, Livorno; Bernardino Di Paola, Casa di Cura Mater Dei, Roma; Andrea Foglia, Studi di Fisioterapia Riabilita, Civitanova Marche (MC); Piermaria Fornasari, Istituto Ortopedico Rizzoli, Bologna (Banche del Tessuto Muscolo-scheletrico); Giovanni Giordano, Istituto Ortopedico Rizzoli, Bologna; Roberto Iovine, Azienda U.S.L. di Bologna (Società Italiana di Medicina Fisica e Riabilitativa—SIMFER); Stefano Lupparelli, Università degli Studi, L’Aquila; Massimiliano Magaletti, Artrogruppo, Roma; Maurilio Marcacci, Università degli Studi, Bologna (Società Italiana di Chirurgia del Ginocchio, Artroscopia, Sport, Cartilagine e Tecnologie Ortopediche—SIGASCOT); Giuseppe Milano, Università Cattolica del Sacro Cuore, Roma; Riccardo Minola, Gruppo Policlinico, Monza (Società Italiana di Artroscopia—SIA); Roberto Padua, Ospedale Sandro Pertini, Roma; Giuseppe Rinonapoli, Clinica Ortopedica, Università degli Studi, Perugia; Emilio Romanini, Artrogruppo, Casa di Cura San Feliciano, Rome; Gustavo Zanoli, Casa di Cura S. Maria Maddalena, Rovigo (Gruppo di Lavoro Ortopedia Basata sulle Prove di Efficacia—GLOBE); Claudio Zorzi, Ospedale S. Cuore Don Calabria, Negrar—VR (Società Italiana di Ortopedia e Traumatologia—SIOT).
Conflict of interest
The authors declare that they have no conflicts of interest.
Open Access
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. | CC BY | no | 2022-01-15 01:14:04 | J Orthop Traumatol. 2010 Dec 23; 11(4):211-219 | oa_package/81/d3/PMC3014473.tar.gz |
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PMC3014569 | 21074407 | Introduction
Protein kinases are involved in orchestrating almost all aspects of cellular life by integrating cell signalling networks. A myriad of studies have described the molecular basis of protein kinase function. The first structure of a protein kinase, described by Knighton et al. , 1991 uncovered the architecture of the eukaryotic protein kinase domain and key elements of the enzyme Protein Kinase A (PKA) catalytic site together with the substrate binding mode [ 1,2 ]. Several structural and sequence homology studies of protein kinase domains have revealed a consensus of what are the common motifs that are required for catalytic activity [ 3,4,5 • ,6 • ,7 • ,8,9 ] ( Figure 1 a and b). These comprise residues that are required for nucleotide (ATP) binding, metal ion (Mg 2+ ) binding and residues required for phosphoryl group transfer. There are 518 known human protein kinases [ 10 •• ], representing approximately 2–2.5% of the estimated total number of genes in the human genome [ 11 ] and the third most common functional domain [ 12 ]. Intriguingly, ∼10% of the kinome appear to lack at least one of the motifs required for catalysis and have been termed pseudokinases [ 10 •• ,13 •• ]. | Protein kinases provide a platform for the integration of signal transduction networks. A key feature of transmitting these cellular signals is the ability of protein kinases to activate one another by phosphorylation. A number of kinases are predicted by sequence homology to be incapable of phosphoryl group transfer due to degradation of their catalytic motifs. These are termed pseudokinases and because of the assumed lack of phosphoryltransfer activity their biological role in cellular transduction has been mysterious. Recent structure–function studies have uncovered the molecular determinants for protein kinase inactivity and have shed light to the biological functions and evolution of this enigmatic subset of the human kinome. Pseudokinases act as signal transducers by bringing together components of signalling networks, as well as allosteric activators of active protein kinases. | Inactive pseudokinases or simply unusual active kinases?
The subject of pseudokinases has generated much attention recently [ 14–17 ] and remains controversial. For some proteins that were observed to lack catalytic residues and were thus originally classified as pseudokinases, protein kinase activity was subsequently reported. One of the best examples of this are the WNK ( W ith N o ( K ) Lys) protein kinases that activate SPAK and OSR1 kinases by phosphorylation, contributing to the regulation of ion transport and blood pressure (reviewed by Richardson and Alessi [ 18 ]). Interestingly, the WNK1 crystal structure revealed that although a key Lys residue was missing from the VAIK motif (found in subdomain II of the kinase domain), this was structurally compensated for by a Lys residue present in the neighbouring subdomain I [ 19 ]. Similar examples of apparent pseudokinases displaying the capacity for phosphoryl transfer have been reported recently (e.g. CASK [ 20 ], IRAK2 [ 21 ] and HER3 [ 22 • ]). Because of these recent findings, it appears that using variations in the primary sequences of the catalytic motifs to predict whether a kinase domain has catalytic activity is not always valid. This suggests that the bioinformatics prediction of ∼10% of the kinome being pseudokinases may be an overestimate. Predicted pseudokinases should thus be studied individually, their activity be probed with more direct methods and their structures determined.
Five ways of killing a kinase
A number of recent studies have provided structural information of pseudokinase domains that have extended our understanding of their biological functions and the mechanism of their inactivation. We summarise these examples below, thus explaining the molecular determinants for protein kinase inactivity and suggest a new set of criteria for cataloguing the currently predicted pseudokinases ( Box 1 ).
STRADα
The Ste20 related adaptor (STRADα and STRADβ) isoforms are part of the LKB1 heterotrimeric tumour suppressor complex [ 24,25 ]. Together with the adaptor protein MO25 [ 26 ], STRAD activates the LKB1 kinase through an allosteric mechanism that does not require LKB1 activation loop phosphorylation [ 27 •• ]. STRAD lacks six of the eleven catalytically important residues that are generally required for kinase activity ( Figure 1 b). Despite the changes in the glycine-rich loop, (the third consensus glycine being replaced by Met83 ( Figure 1 a, c, and d), the crystal structure of STRADα revealed that STRADα is capable of binding ATP with low nanomolar affinity, and retains a kinase fold that that is typical of the canonical ‘active’ kinase conformation [ 28 •• ]. Curiously, all catalytic motifs usually required for kinase activity ( Figure 1 c), adopt conformations compatible with phosphoryl transfer, yet the amino acids on these structural motifs lack the chemical properties required for catalysis ( Figure 1 d). For instance, instead of an Asp residue in the conserved Arg-Asp motif (Asp166 in PKA, Figure 1 c) that acts as a catalytic base [ 5 • ,29 ], a Ser residue (Ser195) is present in STRADα ( Figure 1 d). Interestingly, the Asp-Phe-Gly motif, crucial for binding Mg 2+ ions, is changed to Gly-Leu-Arg in STRADα. The Arg215 side chain coordinates the β-phosphate group of ATP and together with His200 they partially substitute the role of Mg 2+ ions ( Figure 1 c and d). The active conformation of STRADα was shown to be modulated by its binding partner MO25, as well as ATP [ 28 •• ]. The MO25 interaction is centered around the regulatory helix αC, which is analogous to the activation of CDKs by cyclins [ 30 • ,6 • ]. Loss of ATP and MO25 binding impinges on the ability of STRAD to activate the LKB1 kinase [ 28 •• ], suggesting that the ‘active’ conformation of STRADα plays a key role. When the structure of the full LKB1 heterotrimer became available, it was apparent that elements of the STRADα active and substrate binding sites such as the activation and substrate binding loops [ 2 ] and helix αG [ 31 • ,32,33 • ] were involved in binding and activating the LKB1 kinase ( Figure 2 a) [ 27 •• ]. The activation loop of STRADα adopts an extended conformation, reminiscent of active kinases and is involved in LKB1 binding ( Figure 2 a). These data suggest that STRADα engages LKB1 as a ‘pseudosubstrate’ and explain why STRADα must adopt an active conformation in order to activate LKB1 [ 27 •• ,28 •• ,16 ]. Therefore, STRADα appears to have evolved as a pseudokinase allosteric regulator of LKB1 no longer requiring the ability to catalyse phosphoryl transfer.
ILK
Integrin-linked kinase (ILK) is involved in signalling from the transmembrane integrin receptor to the actin cytoskeleton, and as such regulates many cell-adhesion-dependent processes [ 34 ]. ILK is part of a heterotrimeric complex together with PINCH and parvin (the so-called IPP complex) [ 34 ]. Although ILK lacks six of the eleven key residues required for catalysis ( Figure 1 a and b), a large number of studies have claimed that ILK phosphorylates several substrates (reviewed by Legate et al. , 2006). However, these data contradict reports from genetic studies suggesting that kinase catalytic activity per se may not be required for IPP complex function (reviewed by Wickström et al. , 2010 [ 35 ]. It is possible that ILK gains activity under very specific conditions (e.g. upon post-translational modification and/or allosteric activators), which may explain these contradictory reports. A recent crystal structure of ILK bound to α-Parvin [ 36 •• ] has uncovered the molecular basis of ILK function and explains why ILK is incapable of phosphorylating any substrates ( Figure 1 e). Inactivity is accounted for by substitutions of the catalytic base (PKA residue Asp166) with Ala319, the crucial lysine residue from the catalytic loop (PKA residue Lys168) with Asn321 and a Mg 2+ binding Asn residue (PKA residue Asn171) with Ser324 ( Figure 1 c and e). A striking feature of the ILK-α-parvin complex structure is the presence of ATP in the ILK nucleotide binding pocket, despite several non-conservative substitutions of crucial glycine residues in the glycine-rich loop ( Figure 1 a and e). The ATP γ-phosphate is also coordinated by a positively charged residue (Lys341), acquired by a substitution of the conserved glycine from the canonical DFG motif ( Figure 1 e), similar to the interaction between Arg215 of STRADα and the ATP γ-phosphate ( Figure 1 d). While it is unclear what the function of ATP binding in ILK is, one possible function is that ATP is required for the active-like conformational state of ILK to bind parvin molecules. Binding of parvins was wrongly assumed to activate ILK, as the recent structure by Fukuda et al. revealed that α-parvin makes use of the active site of ILK for binding [ 36 •• ]. Thus, α-parvin bound to the ILK pseudoactive site will sterically hinder any potential substrates of the ILK-α-parvin complex ( Figure 2 b). This is similar to the binding mode of STRAD and LKB1, where the pseudokinase (STRAD) makes use of its pseudoactive site and binds its partner (LKB1) as a pseudosubstrate ( Figure 2 a). Thus, structures of the ILK-α-parvin complex and the LKB1-STRAD-MO25 complex show a recognition mode between pseudokinases with their macromolecular partners that is similar to the known kinase–substrate interactions. Further examples of this will need to be uncovered to establish this as a general mechanism of interaction.
HER3
HER3/ErbB3 is a member of the human epidermal growth family (HER) of tyrosine kinase receptors that also includes HER1/ErbB1, HER2/ErbB2 and HER4/ErbB4. Of the four members, HER3 is classified as a pseudokinase because it lacks two of the eleven residues important for catalysis ( Figure 1 a, b and f). Upon ligand binding to the EGF receptor, the intracellular kinase domains undergo homodimerisation and heterodimerisation resulting in the formation of active asymmetric dimers ( Figure 2 c) [ 37 •• ,38 ]. The asymmetric dimers involve a kinase active component named ‘the receiver’ and ‘the activator’ kinase ( Figure 2 c). The activator binds via its C-lobe to the αC helix (N-lobe) of ‘the receiver’, thus activating ‘the receiver’ kinase in a manner that is reminiscent to the CDK2/cyclin mode of activation ( Figure 2 c). Curiously, residues involved in both ‘activator’ and ‘receiver’ interfaces (both N-lobe and C-lobe) are conserved among all active kinases HER1, 2 and 4 [ 39 • ], suggesting that these can act as both ‘activators’ and ‘receivers’. By contrast, only the C-lobe residues that are involved in the role of the ‘activator’ are conserved in HER3 [ 39 • ]. This suggests that the HER3 pseudokinase is an allosteric activator of ‘the receiver’ rather than catalyzing phosphoryltransfer ( Figure 2 c). Consistent with this, a recently published study of HER3 also revealed that the HER3 kinase domain attains a conformation common to inactive protein kinases [ 39 • ]. In addition, constructs comprising the tyrosine kinase domain and the intracellular kinase domain (ICD) are incapable of phosphoryltransfer [ 39 • ]. Intriguingly however, despite the relatively mild substitutions in the catalytic site ( Figure 1 f), a histidine-tagged HER3–ICD construct was reported to possess catalytic activity in the presence of vesicle lipids attached to NTA-Ni head groups [ 22 • ]. This measured HER3 activity is ∼1000 fold less than the active HER1 counterpart [ 22 • ,17 ] and it remains to be determined whether this trace level of phosphorylation is biologically relevant.
VRK3
VRK3 is a human vaccinia related kinase and lacks catalytic activity owing to the substitution of six out of eleven active site residues ( Figure 1 a and b). The structure of VRK3 explains how non-conservative substitutions of these catalytic motifs compromise VRK3 catalytic competence ( Figure 1 g) [ 40 •• ]. Of detrimental effect to ATP binding and hence catalytic activity, are the substitution of a small glycine residue from the glycine-rich loop (residue Asp175) and residue Gln177 that are predicted to clash with the phosphate moiety of ATP, although similar substitutions are tolerated in ILK. In addition, hydrophobic residues Leu180, Leu262 and Phe313 now fill the ATP binding pocket and complete the so-called ‘hydrophobic R-spine’ ( Figure 1 g) [ 41 ]. Consistent with these structural observations, VRK3 is incapable of binding nucleotides [ 40 •• ]. The VRK3 structure is similar to the structure of the closely related active kinase VRK2, although the inability to bind nucleotides renders VRK3 a truly ‘dead’ kinase. Recent studies suggest that VRK3 direct binding inhibits the vaccinia H1-related (VHR) phosphatase, a dual-specificity phosphatase that dephosphorylates and inactivates ERK [ 42,43 ]. Thus, VRK3 regulates MAP kinase signalling through inhibition of ERK activity, and VRK3 functions may be attributed to its interactions instead of functioning as a protein kinase.
ROP2 and ROP8
Two other pseudokinase structures, members of the Rhotropy (ROP) family, ROP2 and ROP8 from the intracellular parasite Toxoplasma gondii have been reported recently [ 44 •• ,45 ]. Of the eight members of the ROP family, five are predicted to be pseudokinases, namely ROP2, ROP4, ROP5, ROP7 and ROP8. It is not clear why so many members of the ROP family are pseudokinases, although it is possible that they are examples of evolutionary remnants of gene duplication, given that the genome of these parasites is more amenable to undergo rapid changes. ROP2 and ROP8 lack seven out of eleven conserved residues important for catalysis and are predicted to be inactive ( Figure 1 a and b). The structures of ROP2 and ROP8 adopt similar conformations, with the activation segment in the canonical conformation typical of active protein kinases [ 44 •• ,45 ]. One noticeable difference is the presence of a short insert within the αEF/αF loop, which may also account for specific binding of as yet unidentified macromolecular partners. Unlike VRK3, the ATP pocket is empty in ROP2 and ROP8 structures, and Labesse et al. reported that ROP2 does not interact with ATP [ 44 •• ]. Non-conservative substitutions from the N-lobe residues Glu275 and Tyr278, (glycine-rich loop) as well as residue Tyr280 (β2) are predicted to clash with a bound ATP molecule ( Figure 1 h), although the adenine pocket is not completely filled. Curiously, a Mg 2+ ion is present in the ROP2/8 active site ( Figure 1 h), and this is coordinated by an acquired Gly442Glu substitution in the conserved Asp-Phe-Gly motif ( Figure 1 a). Hence, it is still uncertain whether the nucleotide pocket of ROP pseudokinases harbours a true ATP or other ligand binding site. The catalytic base is also missing and is replaced by Tyr422 ( Figure 1 h), which further suggests ROP pseudokinases are devoid of catalytic activity. Instead, their role is predicted to be mainly of scaffolding nature.
Pseudokinases regulated by nucleotide binding
The link between nucleotide pocket occupancy of a kinase/pseudokinase domain and its conformation has not been fully appreciated, despite potential functional implications and examples in other classes of enzymes that bind nucleotides (e.g. G-proteins are regulated by guanine nucleotide-induced conformational changes). Pseudokinases like STRADα and HER3 bind ATP with low nanomolar affinity [ 28 •• ,22 • ]. Structural, biophysical and mutagenesis data demonstrate the importance of nucleotide binding to STRADα in influencing STRADα’s ability to interact with its biological partners (LKB1 and MO25), as well as the ability of STRADα to activate the LKB1 tumour suppressor kinase [ 28 •• ,27 •• ].
Recently, conformational regulation of a kinase domain through nucleotide pocket occupancy has also been described for catalytically active kinases. For instance in the case of IRE1 (Inositol-requiring enzyme 1), its phosphoryl transfer activity is functionally dispensable [ 46 ]. Instead, the nucleotide binding event serves to promote dimerisation that in turn composes a ribonuclease active site elsewhere on the protein. The kinase domain dimerisation occurs mainly through the N-lobe, including helix αC, and does not involve the activation segment [ 46 ]. The authors suggest, that the predicted pseudokinase RNaseL that is closely related to IRE1 but lacks its activation segment and hence phosphotransfer activity altogether, would similarly use nucleotide binding to promote RNaseL dimerisation and ribonuclease function [ 46 ].
Another prominent example of a pseudokinase influenced by ATP binding is the family of receptor guanylyl cyclases (RGC), that produce the second messenger cGMP in response to the binding of several natriuretic factors (reviewed in [ 47 ]). These lack the HRD motif that provides the catalytic base residue, although ANP-RGC receptors are able to bind ATP leading to further amplification of receptor activity. It is thought that both activity of the guanyl cyclase domain, C-terminal to the pseudokinase domain, as well as receptor ligand affinity are influenced by ATP binding [ 48 ]. The ATP-dependent activity of the receptor is not affected when non-hydrolysable forms of ATP (e.g. ATP-γ-S) are used, and is sensitive to point mutations in the glycine-rich loop and VAIK motifs that affect ATP interaction with the pseudokinase domain [ 49,47 ]. Moreover, Jaleel et al. , 2006 raised a monoclonal antibody that recognises the pseudokinase domain of guanylyl cyclase receptor 2C (GC-C) and showed that the immunoreactivity was compromised upon ATP binding, suggesting large conformational shifts between the ATP bound/unbound forms of this domain [ 50 ]. Fine epitope mapping revealed the epitope to be the region around the VAIK motif. In the absence of structural data, it is difficult to envisage the exact conformational changes that would occur owing to GC-C ATP binding. In the majority of kinases the VAIK motif (β3) is followed by helix αC, a well known region that undergoes large conformational changes upon formation of the conserved Lys(β3)/Glu(αC) ion bridge [ 6 • ]. Future studies in this area may demonstrate that the active/inactive conformations of the pseudokinase domain are indeed required for RGC activity via a regulatory mechanism that involves nucleotide binding.
Pseudokinases interacting with active kinases
EGF and Eph receptor tyrosine kinases
Interestingly, there are a number of membrane receptors that contain a predicted cytoplasmic pseudokinase domain, including members of the ephrin receptor EphB6 and EphB10 as well as members of the epidermal growth factor receptor family ErbB3/Her3. As already discussed above the four members of the EGF receptor kinases (ErbB1–4) are envisaged to trans-activate by forming so-called asymmetric heterodimers, upon ligand binding rather than phosphorylation [ 37 •• ,38 ]. This lack of evolutionary pressure to conserve phosphoryl transfer activity, owing to the presence of multiple ErbB genes coupled by the allosteric mechanism of activation rather than phosphorylation, may be the cause for the loss of catalytic activity of ErbB3. It is possible that other pseudokinases have also evolved in a similar way. Given the proximity to the EGF receptor tyrosine kinases in the human kinome, EphB6 and EphB10 may be regulating the active members of the Eph receptor tyrosine kinase family by using an as yet to be determined, analogous trans-activation mechanism to ErbB3.
KSR
Another example where a pseudokinase forms complexes with active kinases are members of the kinase suppressor of Ras 1 and 2 (KSR1/2), that are essential for Ras induced activation of the RAF-MEK-ERK module in MAP kinase signalling. These predicted pseudokinases act as scaffolds bringing together the three components of the MAP kinase pathway (MAPKKK, MAPKK and MAPK), thus regulating signalling output and potentiation [ 51–53 ]. The role of scaffold proteins in MAP kinase signalling is well studied in budding yeast and the importance of Ste5p adaptor protein for bringing together the MAPK components module is well established [ 54–56 ]. It appears the Ste5p scaffolding roles for MAP kinases in yeast, have been substituted by KSR1 and KSR2 in higher eukaryotes [ 57,51 ]. The pseudokinase domain of KSR1 binds MEK and RAF, whereas ERK is recruited to the signalling complex via a conserved domain N-terminal to the pseudokinase domain [ 51 ]. In addition, recent work that established KSR2 as an important scaffold (similar to KSR1) of MAP kinase signalling, reveals KSR2 can be regulated by dephosphorylation by calcineurin in response to changing Ca 2+ levels [ 53 ]. The lack of structural information makes it difficult to precisely understand the mechanism by which KSR1/2 contribute to these scaffolding complexes, and what conformation the KSR pseudokinase domain attains when acting as a scaffold. It will be interesting however to investigate ligand (ATP) binding capabilities of KSR1/2 and see if this is required for MAP kinase signalling, and whether an active conformation is required for KSR binding of macromolecular partners.
A prominent finding that suggests KSR may have functions beyond the scaffolding component, came from the recent revelation that RAF kinase domains form homodimers and heterodimers resulting in activated RAF kinase [ 58 •• ] ( Figure 2 d). This side-to-side dimerisation interface engages a region in close proximity to the regulatory helix αC and is required for RAF activation ( Figure 2 d). The residues involved in the dimer interface are also conserved in KSR, and Rajakulendran et al. demonstrate that KSR can function as an allosteric activator of RAF, without the requirement for KSR phosphoryltransfer activity [ 58 •• ].
JAKs
Perhaps the most studied pseudokinase domain belongs to the members of Janus tyrosine kinase (JAK) family. The JAK isoforms include JAK1, JAK2, JAK3 and TYK2 all of which contain an N-terminal pseudokinase domain (JH2) followed by a kinase domain (JH1). This characteristic feature of containing two kinase domains gives JAKs their name, referring to the two-faced Roman god Janus. JAKs respond upon receptor binding to cytokines and phosphorylate the cytoplasmic region of these cytokine receptors, thus creating sites of interaction for downstream signalling molecules. The pseudokinase domain is required for JAK2 auto-inhibition and is essential for JAK2 cytokine activation [ 59,60 ]. A gain-of-function mutation (Val617Phe) in the JAK2 JH2 domain is a cause of myeloproliferative disorders in humans [ 61–63 ]. Structural modelling and biochemical data suggest the N-lobe of JH2 domain where Val617 resides is in close proximity to the JH1 domain [ 60 ]. Recently, gain-of-function somatic mutations of another residue present in the JH2 domain (Arg683Gly/Ser/Lys) were found in 18% of patients suffering with Down's syndrome-associated acute lymphoblastic leukaemia [ 64–66 ]. The exact location of these disease causing mutations and how their position relates to the JH1 domain are not known. It will be interesting to see if structural studies of JH1/JH2 domains will reveal the molecular mechanism of action of these mutations, and whether these resemble any of the interactions that have been recently described for other pseudokinase–kinase interactions.
Evolution of pseudokinases
How did pseudokinases evolve? In general it is plausible to believe that kinases evolved from ATP binding enzymes, since nucleotide binding must have preceded catalytic function. Therefore it is not surprising that ligand (ATP) binding plays a key regulatory component for pseudokinase/kinase function. As a consequence there are two possibilities—either pseudokinases represent ‘would-be’ active kinases, or they represent kinases that have lost their catalytic activity.
The first scenario could be true for proteins that are well conserved as pseudokinases throughout eukaryotic kinase evolution such as SCYL1-3 and GCN2. For instance, ATP-binding precursors of yeast SCY1 and GCN2 may have failed to ‘mature’ as active protein kinases capable of phosphoryl transfer. Instead, important non-catalytic functions were conserved throughout evolution for these pseudokinases. In GCN2 this could have been aided by loss of evolutionary pressure for kinase maturation, since another kinase domain is present in the same polypeptide chain.
However, the reasoning above cannot account for a significant number of pseudokinases that do not have clear pseudokinase homologues in primitive species (e.g. STRADα, KSR1/2, HER3, etc...). Therefore these may have evolved via a different route. One possibility is that pseudokinases have evolved from active kinases once capable of phosphorylating and activating their substrates, but have lost their activity during evolution. The structures of STRADα, ILK and VRK3 show that despite being inactive, these pseudokinases are able to assume an active conformation with a highly organised active site poised for phosphoryl transfer. In addition, the structure of the LKB1 heterotrimer and ILK-α-parvin complex, reveal that STRAD and ILK can bind LKB1 and α-parvin respectively as pseudosubstrates [ 27 •• ,36 •• ]. This suggests that pseudokinases like STRADα and ILK were able to phosphorylate substrates at some stage during evolution, but changed their mechanism to favour regulation through binding, rather than post-translational modification. It is possible that other pseudokinases have evolved from active kinases in similar ways.
Going ‘pseudo’-loosing the will to catalyse
The process of using previously catalytically competent domains as scaffolds is plausible from an evolutionary perspective as enzymes have evolved to bind their substrates. This is also observed in other classes of enzymes such as pseudophosphatases [ 67 ], and similar to some pseudokinases, these enzymes lack catalytically important residues and act as scaffolds in signalling complexes. For instance the pseudophosphatase TAB1 is a scaffolding component of the TAK1-TAK2/3-TAB1 signalling complex [ 68 ]. In addition, a number of PTPs (protein tyrosine phosphatases) are predicted to be inactive [ 67,69 ]. Examples of predicted pseudophosphatases include STYX [ 67 ], EGG-4 and EGG-5 [ 70 ] that are thought to function by binding and ‘trapping’ phosphorylated tyrosine residues instead of phosphate removal. The exact mechanisms of action for these pseudophosphatases are as yet unclear, although engaging phosphotyrosine residues through this ‘substrate trapping’ mechanism will neutralise the effects of phosphorylation, and/or make them inaccessible for signal-transmitting phosphotyrosine binding domains (e.g. SH2 domains) [ 70 ].
The phenomenon of catalytically inactive members is not restricted to enzyme families involved in protein phosphorylation alone, but extends to other processes of postranslational modifications such as ubiquitinylation. A class of ubiquitin E2 ligases called Uev (ubiquitin E2 variant) domains have the same fold as E2 enzymes but lack certain catalytic residues and are hence devoid of catalytic activity [ 71 ]. Similar to the process by which some kinases require pseudokinases for full activity, some E2 enzymes also require their inactive counterparts (Uev domains) for ability to transfer ubiquitin to a substrate (e.g. Uev1a and Ubc13). By analogy to the existence of the pseudokinase–pseudophosphatase pair, a large number (∼12%) of ubiquitin specific proteases (DUSPs) lack conserved catalytic residues and are predicted to be inactive [ 72 ]. It is possible that the number of predicted inactive DUSPs may be an overestimate, and after further examination some of these DUSPs may indeed turn out to be active. However, important non-catalytic functions that utilise DUSPs’ ability to recognise ubiquitin and function through a similar ‘substrate trapping’ mechanism described for pseudophosphatases [ 70 ], are possible. The prevalence of non-catalytic functions may relieve the evolutionary pressure for conservation of key catalytic residues and may explain why some of these enzymes appear to be inactive. It also provides an example of nature's ability to repurpose already perfected molecular units in many different ways.
Concluding remarks
In recent years, a burst of studies have provided valuable insights into the biological functions and the mechanism of action of pseudokinases. An important revelation is that the initially predicted fraction of kinases that are truly ‘dead’, ∼10% of the human kinome, may have been an overestimate. Despite variations in the catalytic motifs of pseudokinases, nature has acquired alternative mechanisms to correct for the missing functions in phosphoryl transfer, by making use of the versatile kinase fold. Exhaustive structural studies of protein kinases have established that the kinase/pseudokinase domain is plastic, and attains many different conformations in response to binding of micromolecular and macromolecular ligands. Similarly, large conformational changes occur upon postranslational modifications such as phosphorylation. Therefore, pseudokinases can be envisaged as ‘elastic’ scaffolds, bringing together components of a particular signalling network, as well as being allosteric activators of protein kinases.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest | Acknowledgements
We thank Frank Sicheri and Thanashan Rajakulendran for critical reading of this manuscript and useful discussions. Our work on pseudokinases has been generously supported by TENOVUS Scotland, Cancer Research UK grant C33794/A10969a, a Wellcome Trust Senior Research Fellowship (DvA), Human Frontiers Science Program Organisation (EZ), a Sir Henry Wellcome Postdoctoral Fellowship (EZ), the Medical Research Council, and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck & Co. Inc, Merck KgaA and Pfizer). | CC BY | no | 2022-01-12 20:27:10 | Curr Opin Struct Biol. 2010 Dec; 20(6):772-781 | oa_package/35/91/PMC3014569.tar.gz |
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PMC3014591 | 21249121 | Introduction
Anautogenous mosquito females need vertebrate blood for reproduction. The nutrients taken up with the blood are used to synthesize large amounts of yolk proteins that are deposited in the eggs during a process called vitellogenesis. Yolk delivers the energy and building blocks for embryogenesis. This “need for blood” in order to reproduce makes anautogenous mosquitoes effective disease vectors because they require at least one insect-host contact for every batch of eggs they develop.
During feeding, adult female Aedes aegypti , can take up more than their own body weight in blood [1] . This severely impairs their mobility and puts them at risk to be killed by their host or other predators. In addition, mosquitoes face the problem of high sodium content of vertebrate blood plasma and high potassium content in red blood cells. Therefore, it is essential for mosquitoes to possess an efficient system for excretion of excess water and ions while retaining the nutrients contained in the blood meal. During and after a blood meal female Ae. aegypti secrete large amounts of urine through their Malpighian tubules (MT). Within the first hour after taking a blood meal (post blood meal - PBM) mosquito females can discharge more than 40% of water and sodium enclosed in the blood plasma [1] . Diuresis is under hormonal control by neuropeptide hormones secreted by the central nervous system [2] . In the current model, diuretic hormones, released seconds after start of the blood meal, stimulate the MT cells to produce the second messenger molecule cAMP which activates transcellular diuresis by increasing transepithelial cation (Na + , K + ) transport [2] . Another class of neuropeptide hormones, the kinins, increase intracellular calcium levels that regulate anion movement (Cl − ) into the MT lumen [3] , [4] . Urine produced by the MT is collected in the hind gut and subsequently forcefully ejected from the rectum in a process that involves rectal peristalsis and movement of the 7 th and 8 th abdominal segments. Females start expelling small urine droplets approximately 50–75 seconds after start of feeding. Urine droplets have a volume of about 10 to 12 nl and can fly up to 10 mm [1] , [5] .
Aquaporins (AQPs) are transport channels that make cell membranes permeable to water. They are found in all plant, animal, fungi, eubacteria, and archaea taxa studied [6] , [7] , [8] . In mammals, there are 13 AQPs and they form two subfamilies with different transport selectivity: orthodox aquaporins, which transport only water, and aquaglyceroporins, which transport glycerol, urea, small solutes, and water [9] . In the so-called hourglass model for AQP structure, the six transmembrane alpha helical domains (numbered 1 – 6) are connected by five loops termed A – E [10] , [11] , [12] . Both amino- and carboxy-terminus are located inside the cytoplasm. The transmembrane domains 2–3 and 5–6 are connected by loops B and E, both containing a highly conserved NPA (Asparagine-Proline-Alanine) motive and other conserved residues. These hydrophobic NPA loops form a ring as part of an hourglass-shaped pore within the center of the phospholipid bilayer membrane. This ring, with a diameter of 2.8 Å, is the primary filter that prevents protons from crossing through the AQP pore. Hg 2+ ions interact with a cysteine residue close to the NPA motive in the E loop and an alanine residue in the B loop of most AQPs and efficiently obstruct water transport through the pore [13] .
The activity of eukaryotic aquaporins is commonly regulated via three different mechanisms: translation, gating, or trafficking [13] . While regulation via translation is a relatively slow process, gating and trafficking can change water permeability of a membrane within seconds. Trafficking of aquaporins was first described in AQP2 in mammals where it is involved in concentrating urine in the kidneys [14] . AQP2-trafficking is controlled by a signaling cascade triggered by the neuropeptide arginine-vasopressin. The phosphorylation of conserved serine and theronine residues in the fourth loop region of AQP2 caused the redistribution of intracellular AQP storage vesicles to the plasma membrane. This resulted in a rapid increase of water permeability of the membrane. The mechanism of how AQP phosphorylation leads to the recognition of the flagged protein and subsequent vesicle movement and membrane fusion is still unknown. Gating refers to the ability of AQPs to control the flux of water by widening or constricting the channel. X-ray structures have revealed that the width of a fully opened channel can allow a single water molecule entry [13] .
While vertebrate AQPs are well studied, few studies have been conducted on invertebrate AQPs (reviewed by Spring et al., 2009 [15] ). DRIP (Genbank accession #: CG9023) is a partly characterized representative of Drosophila AQPs. It is expressed in embryonic and adult MTs of the fruit fly [16] . A mosquito AQP, a close homologue to Drosophila DRIP, has been cloned and characterized in the yellow fever mosquito Ae. aegypti . This AQP is localized in tracheolar cells associated with MTs of adult female Ae. aegypti mosquitoes [17] . Another mosquito aquaporin has recently been cloned and characterized from Anopheles gambiae. This aquaporin is a water transporter and expressed in multiple tissue and specifically in the stellate cells of MTs. It is important for water homeostasis in An. gambiae [18] .
In order to evaluate the potential of this class of molecule as targets for vector control strategies, we have surveyed the genome of Ae. aegypti and identified six genes encoding putative AQPs. We show that four AQPs are expressed in the Malpighian tubules of adult females and that knockdown of three of them affects diuresis. | Materials and Methods
Mosquito rearing
The Ae. aegypti mosquito strain UGAL was maintained in laboratory culture as has been previously described by Hays and Raikhel [33] . The strain was reared at a temperature of 28°C with 80% humidity and a photoperiod of 14 h light and 10 h dark. Larvae were fed on a diet of ground rat food, yeast and albumin (1∶1∶1 w/w).
Sequence Identification & Phylogenetic analysis
Predicted cDNA and deduced AA sequences of AQP family members were identified using BLAST at three databases: Ensembl [34] , Genbank [35] , and VectorBase [36] . A sequence alignment (supplemental online material: Figure 1 ) was performed using PROMALS3D software which considers structural constraints for the divergent AQPs protein sequences [37] . The reconstruction of the phylogenetic tree was performed using Mega 4 [38] . The tree was visualized using FigTree software [39] .
Microarray Expression Studies
Three biological samples composed of four day old mosquitoes were collected consisting of a pool of 20 insects. Four day old females were blood fed and three biological samples (20 females each) were collected at several times after the blood meal. Total RNA was extracted with TRIzol® (Invitrogen, Carlsbad, CA) and further purified with Qiagen RNAeasy columns (Qiagen,Valencia, CA) with DNase treatment according to the manufacturer's recommendations. RNA quality was assessed by capillary electrophoresis using the Agilent Bioanalyzer 2100 and spectrophotometric analysis. The RNA was reverse transcribed and amplified using WT Ovation Pico (Cat# 3300-60, NuGen, San Carlos, CA) and 2 g of this cDNA was labeled with Cy3 (Cat# 5190-1305, Agilent technologies, Santa Clara, CA) according to the Agilent Oligonucleotide Array-Based CGH For Genomic DNA Analysis Protocol (Cat# G4410-90010, Agilent technologies, Santa Clara, CA). Hybridization, washing, and scanning were processed as per the Agilent OneColor Microarray-Based Gene Expression Analysis Protocol (Cat# G4140-90040, Agilent technologies, Santa Clara, CA). Expression signals were normalized for background within chips with the Agilent spatial correction algorithm and were normalized between chips using quantile normalization. The data is available at Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo/ ) under series record GSE22339 [40] . Statistical analysis was performed with InStat (GraphPad Software, La Jolla, CA).
qRT-PCR Expression Studies
Gene-specific primers were developed using Primer BLAST [41] . Total RNA was obtained from different larval stages, pupae, and adult females with TRIzol® solution. Tissue-specific RNAs were isolated after dissection of samples from 30 individual mosquitoes including previtellogenic females 72 h after eclosion and female mosquitoes 3 h and 24 h post blood meal. Transcript was analyzed and quantified using quantitative RT-PCR (qPCR) using iQ Supermix (Biorad, Hercules, CA). Primers were as follows: AaAQP1f: ACC GGC ATC AGA AAG GAG AAG CG ; AaAQP1 r: GCC TGC TGT TTG ATG TGT TGT GCA ; AaAQP2 f: GCT CGC TCG TTT GGA ACG GC ; AaAQP2 r: CAC GGT AGC GCT CTG AGG CG ; AaAQP3 f: AGG TCC AGT GGG GAT GGC CC ; AaAQP3 r: CAG CTG AGG TGG TGG CGG TG ; AaAQP4 f: CTG CCG CCT GCA GTG TGG AA ; AaAQP4 r: TGT GGA AGT TCT CGT CGG AAG ACG T ; AaAQP5 f: CGG TGT TCA GGC GCG AGG TT ; AaAQP5 r: ATC CCG CGT TGG TGG AAC GG ; AaAQP6 f: CAG CTT GGT CGT GGC CGC AT ; AaAQP6 r: GCA CAG CTC CCG CAC ACG AT .
RNAi knockdown experiments
Generation of double-stranded RNAs (dsRNA) was performed as described earlier [42] using primers with the T7 primer sequences attached. PCR product was used as template for dsRNA synthesis with the MEGAscripts T7 Kit (Ambion, Austin, Tx). Approximately 500 ng of dsRNA in 268 nl of pyrogen-free H 2 O was injected into the thorax of CO 2 -anesthetized female mosquitoes three days after adult emergence. The injected mosquitoes were then allowed to recover for 3 days before diuresis assay. Knockdown effectiveness was confirmed with qPCR. Primers were as follows (The T7 sequence is omitted): AaAQP1f: TAA TAC GAC TCA CTA TAG GGA GCA CTA TGG GCT GGG GCG GAG ACT ; AaAQP1 r: TAA TAC GAC TCA CTA TAG GGA CGG CTG GTC CGA AAG AGC GAG CTG ; AaAQP2 f: TAA TAC GAC TCA CTA TAG GGA CTG CTG GCT TAC TTG CGG CTG GCA ; AaAQP2 r: TAA TAC GAC TCA CTA TAG GGG CTA CAC GGT AGC GCT CTG AGG CGG ; AaAQP3 f: TAA TAC GAC TCA CTA TAG GGC CCC ATC CCC AAG CGG GTG AAC CAC ; AaAQP3 r: TAA TAC GAC TCA CTA TAG GGT GGG CCA TTG GGTAGC CCC CTG GAT ; AaAQP4 f: TAA TAC GAC TCA CTA TAG GGG CGG CAT CGG GTT CGG CTT CAC AGT ; AaAQP4 r: TAA TAC GAC TCA CTA TAG GGT GGC CGG GTT CAT ACT CGC TCC GGT ; AaAQP5 f: TAA TAC GAC TCA CTA TAG GGT ACG TTG CGG CCC AGT GCA TCG GAG ; AaAQP5 r: TAA TAC GAC TCA CTA TAG GGG GAA CCT CGC GCC TGA ACA CCG TCT ; AaAQP6 f: TAA TAC GAC TCA CTA TAG GGG ATC GCG GCA GTT GCT CGC CGA GTG ; AaAQP6 r: TAA TAC GAC TCA CTA TAG GGA GGC CAA GCG ACA GCA CAG AGT GGC .
In vivo diuresis assay
Knockdown and control (eGFP dsRNA-injected) mosquitoes were anesthetized with CO 2 and pooled into groups of ten. Group weight was determined on a precision weighing balance. Each mosquito was subsequently injected with 1.25 ul of PBS and the groups were immediately weighed. Diuresis was monitored by weighing mosquitoes in 30 min intervals. Control mosquitoes were injected with 1.25 ul 200 uM HgCl 2 in PBS. After the last weighing the MTs were dissected and RNA was isolated in order to determine the knockdown efficiency. | Results
The AQP genes of Ae. aegypti
BLAST searches conducted with vertebrate and Drosophila AQPs as query sequences allowed the identification of six loci encoding putative AQPs in the genome of Ae. aegypti , which were denominated Ae. aegypti aquaporins 1–6 ( Table S1 ; AaAQP1–6). In addition we identified seven putative AQPs encoded in the genome of the malaria mosquito Anopheles gambiae [19] , eight in the Drosophila genome [20] , six in the genomic sequence of the head louse Pediculus humanus [21] , and seven in the genome of the red flour beetle Triboleum castaneum [22] , [23] . We also used five putative AQP of Leishmania major , six Plasmodium , and two fungal sequences of the yeast Pichia pastoris for our analysis.
A phylogenetic tree was constructed to indicate associations among the identified protein sequences ( Figure 1 ). AaAQP1 forms a clade with three proteins from the other insect species and with Drosophila DRIP. DRIP represents a typical water transporter [16] . AaAQP2 forms a well defined orthologous clusters with other insect AQPs. AaAQP1, 2, and 3 share a clade with well characterized vertebrate AQP subfamily members that mediate water transport. Within this clade AaAQP3 forms a separate cluster together with Drosophila BIB (CG4722), and two Anopheles AQPs.
AaAQP4 and AaAQP5 belong to two separate insect-specific clades. Mosquitoes have single representatives in each of these clusters, whereas other insects have apparent gene duplications. There are three relatives of AaAQP5 in fruit fly and four in the flour beetle. AaAQP6 is closely related to Drosophila CG12251 and vertebrate AQPs 11 and 12.
Expression of AQPs in adult female Ae. aegypti
First, we analyzed AQP expression data from microarray assays performed with probes from RNAs isolated from whole mosquito females ( Figure 2 ). As a general trend we found AQP expression down-regulated 12 and 24 h after a blood meal. At the later time points, expression returns to “non blood fed (NBF)” levels. An exception was AaAQP5 which was up regulated 12h PBM but is also down regulated at later time points.
Next, we determined AQP expression in selected organs and body parts of adult female mosquitoes before a blood meal, 3 and 24 h PBM. Using quantitative RT-PCR, we found that all six Ae. aegypti AQPs were expressed in adult female mosquitoes ( Figure 3 ). Different organs/body parts varied considerably in the assortment of AQPs they expressed. The observed patterns of AQP expression were as follows:
AaAQP1
This AQP is the mosquito homologue of Drosophila DRIP, a highly selective water-specific channel in the fruit fly [16] . AaAQP1 was expressed in all organs and body parts examined with highest expression levels in the MTs. It is significantly up regulated in the MTs 3 h after a blood meal.
AaAQP2
We found high AaAQP2 transcript levels it in all organs and body parts except the fat body. It was strongly expressed in the MTs, midgut, and ovaries at all time points. The overall expression pattern during vitellogenesis is similar to AaAQP1. It is significantly up regulated in the MTs 3 h after a blood meal.
AaAQP3
This AQP is the homologue of the Drosophila BIB. BIB does not function as a water channel in fruit flies but is involved in the regulation of cell adhesion [24] . AaAQP3 was weakly expressed in MTs, midgut, and ovaries and was significantly up regulated in ovaries and fat body PBM.
AaAQP4
This uncharacterized AQP was highly expressed in MTs and is significantly up regulated 3 h after a blood meal. It is down regulated in the midgut during early vitellogenesis.
AaAQP5
This uncharacterized AQP was highly expressed in all organs except the ovaries and was up regulated in most organs PBM.
AaAQP6
This uncharacterized AQP is predominantly expressed in the thorax. The highest mRNA expression levels were found in the foregut ( Figure 3B ).
Effect of AQP knockdown on diuresis after PBS injection
RNAi-mediated gene expression knockdown was determined in samples of total RNA isolated from MTs dissected three days after injection of AQP dsRNA. Knockdown was successful in the mosquito MTs with efficiencies between 95% (AaAQP5) and 60% (AaAQP2) reduction in transcript accumulation ( Figure 4A ).
In order to determine how fast PBS-injected mosquitoes start discharging urine, we injected female mosquitoes with 1.25 ul PBS and observed them under a stereomicroscope to determine the time period till the onset of urine discharge. Injected females started to discharge urine droplets on average at 157 s (SE = ±16 s) after injection, thus leaving a window of two minutes to perform the first weight measurement after injection. Excretion was strongest in the first hour after injection ( Figure 4B , control group, blue line).
To test the hypothesis that the AQP proteins are contributors to the function of the MT, we generated knockdown mosquitoes for each MT-expressed AQP transcript by dsRNA injection and tested for urine production using our in vivo diuresis assay ( Figure 4C ). Negative control mosquitoes (injected with a dsRNA directed against the unrelated jellyfish protein eGFP) excreted 39% of the injected 1.25 ul PBS within one hour. In contrast, positive control mosquitoes injected with 1.25 ul of 200 uM HgCl 2 in PBS (known to suppress AQP activity [13] ) excreted only 10% in the first hour after injection.
AaAQP1, 4, and 5 knockdown mosquitoes showed significantly reduced excretion rates compared to the eGFP dsRNA injected control ( Figure 4C ). Knockdown of AaAQP2 did not result in a significant effect. The strongest effect of a single gene knockdown was observed after knocking down AaAQP5 which resulted in a decline of excretion to only 22%. The combined knockdown of all four MT-expressed AQPs reduced excretion to only 18% of the injected PBS.
In order to visualize the effect of the combined knockdown on diuresis we injected control and combined knockdown mosquitoes with 2.5 ul PBS. Figure 4D shows two representatives of each group 3 h after injection. | Discussion
This report is the first comprehensive study on AQP genes and their expression in the adult yellow fever mosquito, Aedes aegypti . We specifically focused on AQPs expressed in the MTs, an insect organ, specialized in water and waste excretion.
An interesting result of our phylogenetic analysis (see Figure 1 ) is that we were unable to identify any typical aquaglyceroporin in dipteran insects. Human aquaglyceroporins, the Homo sapiens AQPs 3, 7, 9, and 10, form a separate clade with six Plasmodium , one Leishmania , one fungal and one louse AQP. A multiple sequence alignment of all AQPs used for our analysis ( Figure S1 ) revealed that all aquaglyceroporins share a cysteine residue at position six, C-terminal to the NPA motif in the B-loop. None of the dipteran AQPs in our analysis had a cysteine at this position. The fact that dipteran insects do not appear to possess an AQP-like glycerol transporter raises the question as to how they transport glycerol over cell membranes. Glycerol plays an important role in insect cold tolerance and diapause and is found in high concentration in the hemolymph of diapausing insects [25] .
The microarray expression data was created with RNA isolated from total mosquitoes ( Figure 2 ) and shows that AQP expression was generally down regulated after a blood meal, even at 3 h PBM. After taking a blood meal mosquitoes seek out a resting place to digest the blood and perform vitellogenesis and egg development. This resting phase extends over a period of approximately two days. During this time mosquitoes don't take up water, therefore high AQP expression levels are not necessary.
AQP expression show specific patterns in distinct organs/body parts ( Figure 3 ). All examined organs/body parts differ in their AQP expression patterns. We were specifically interested in patterns from midgut and MTs since blood meal-derived water has to cross the midgut epithelium and subsequently the MT epithelium for excretion. AQPs 1, 2, 4, and 5 were expressed in the midgut. Interestingly, three AQPs expressed in the midgut were down regulated at the 3 h PBM time point, corresponding to the time at which the bulk of blood meal-derived water has already been excreted. Our data suggests that AQPs 1, 4, and 5 are the principal AQPs in the MTs of adult females.
We developed an in vivo diuresis assay for adult yellow fever mosquito females based on PBS injection. PBS is a non-toxic isotonic buffer containing sodium chloride, sodium phosphate, potassium chloride and potassium phosphate. This novel assay has several advantages over classical blood meal-based assays. Firstly, mosquitoes receive a standardized amount of PBS, while blood meal sizes can vary significantly. Secondly, since Ae. aegypti mosquitoes start secreting urine about 30 s after starting a blood meal [1] , [26] , the determination of blood meal sizes is difficult. Applying our method, mosquitoes injected with PBS started secreting urine after 2 min, which allowed accurate measurement of their weight after injection. As mentioned above, mosquito diuresis is controlled by neuropeptide hormones that are secreted from the central nervous system in response to unknown stimuli associated with blood ingestion. PBS injection triggers this response efficiently. The rapid onset of diuresis after injection suggests that AQP activity in the MT is regulated by trafficking of AQP-containing vesicles to the plasma membrane, analogous to the processes described for the renal collection duct in human kidney [27] .
RNAi is a powerful tool used to study gene function in mosquitoes. We successfully employed RNAi-mediated knockdown for the analysis of AQP function in the MT. Knockdown rates, as determined by real time PCR, were between 60 and 95% and therefore well inside the range that can be achieved by dsRNA transfection in cell culture [28] . Similar knockdown efficiencies have been found in whole mosquitoes [29] .
A point of concern is that the AQP dsRNA injections likely resulted in AQP knockdown in tissues other than the MTs. However, excreted watery liquids have been shown to always pass through the MT in adult mosquitoes [1] , [5] , [30] . Therefore we expect the effects of AQP knockdown in other tissues to be negligible for the outcomes of our in vivo diuresis experiments.
As a positive control for successful AQP inhibition we used mercury ions (Hg 2+ ), which are capable of binding with a cysteine and an alanine within the AQP pore, thus obstructing water transport through the channel. Mosquitoes injected with 200 uM HgCl 2 were still able to excrete 10% of the injected fluid in one hour. This might be due to paracellular permeability, water transport through the space between the cells, which has been described in insect MTs and can be enhanced by kinins [26] .
Using RNAi knockdown mosquitoes we confirmed the function of three different AQPs in the MTs of Ae. aegypti . While RNAi control mosquitoes were able to excrete about 40% of the injected fluid in one hour, knockdown of single AQPs (AaAQP1, 4, or 5) resulted in a significant decrease in excretion. Simultanous knockdown of all four MT-expressed AQPs reduced excretion down to 18% of the injected fluid in one hour, indicating that a combination of AaAQPs 1, 4, and 5 performs water transport in the MTs of female Ae. aegypti .
The redundancy of function we have observed here has also been noted in human kidney where seven different AQP proteins are expressed [31] . One plausible explanation would be that several AQP genes with different promoters allow the fine regulation of AQP expression in stage- and tissue specific manner or after a range of stimuli.
There is a great need for the development of novel, effective insecticides to fight insect vectors, since the public health insecticides currently in use are based on only a limited number of active compounds [32] . Because of their vital importance in insect larvae and adult homeostasis, insect AQPs could become targets for the development of novel insecticides. The study presented here has identified six genes encoding putative AQP membrane transporters in Ae. aegypti and demonstrated the functional role of three of them in regulation of water transport. Further analysis of these AQPs and their regulation has the potential to contribute to the future development of novel anti-vector strategies. | Conceived and designed the experiments: IAH DYB OM. Performed the experiments: LLD VKC DYB OM. Analyzed the data: IAH LLD DYB OM ALD. Contributed reagents/materials/analysis tools: DYB OM ALD IAH. Wrote the paper: LLD IAH.
Background
The mosquito, Aedes aegypti , is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti .
Methodology/Principal Findings
Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti . Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis.
Conclusions/Significance
Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies. | Supporting Information | The authors thank the participants of the NMSU BIOL302, Molecular Biology Techniques Laboratory, Spring 2010 class: Natalie Aarons-Cooke, Jordan Basham, Hannah Cox-Jesko, Lynda Davis, Hui Fang, Elizabeth Gamez, Anna Garliss, David Hogan, Kelsey Kalbacher, Kevin Lee, Kaitlyn Norman, Shannon Pease, Katarina Provenghi, Marisol Resendiz, Cherish Skeen, Mathew Steritz, and Mary Topalovski for their help with sequence identification and developing primers used in this project. We thank Jessica Aguirre for technical support. | CC BY | no | 2022-01-13 08:09:45 | PLoS One. 2010 Dec 29; 5(12):e15578 | oa_package/93/15/PMC3014591.tar.gz |
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PMC3014602 | 21274339 | INTRODUCTION
Historically, over the last decades, growth disorders were managed on the basis of a growth hormone (GH)−oriented classification system. However, nowadays we are well aware that: a) GH is not the major mediator of skeletal growth; b) scepticism as well as criticisms are adequate and accepted while analyzing the variable results of the various GH stimulation tests; c) many genetic defects have been described and, therefore, have presented important insights into the molecular basis of also non−GH deficient growth failure.
Therefore, when a child is not following the normal, predicted growth curve, an evaluation for underlying illness and central nervous system abnormalities is required. Where appropriate, genetic defects causing GH deficiency (GHD) should be considered. Because Insulin−like Growth Factor−I (IGF−I) plays a pivotal role in growth, where it mediates most, if not all, of the effects of GH, in fact GHD could also be considered somehow as secondary IGF−I deficiency (IGFD). Although IGFD can develop at any level of the GH−releasing hormone (GHRH)−GH−IGF axis, we would like to differentiate, however, between GHD (absent to low GH in circulation) and IGFD (normal to high GH in circulation). The main focus of this review is on the GH gene cluster, the GHRH− as well as the GHRH−receptor− gene. | When a child is not following the normal, predicted growth curve, an evaluation for underlying illnesses and central nervous system abnormalities is required, and appropriate consideration should be given to genetic defects causing growth hormone (GH) deficiency (GHD). Because Insulin−like Growth Factor−I (IGF−I) plays a pivotal role, GHD could also be considered as a form of IGF−I deficiency (IGFD). Although IGFD can develop at any level of the GH−releasing hormone (GHRH)−GH−IGF axis, a differentiation should be made between GHD (absent to low GH in circulation) and IGFD (normal to high GH in circulation). The main focus of this review is on the GH gene, the various gene alterations and their possible impact on the pituitary gland. However, although transcription factors regulating the pituitary gland development may cause multiple pituitary hormone deficiency, they may present initially as GHD.
Conflict of interest: None declared. | CLASSIFICATION OF ISOLATED GROWTH HORMONE DEFICIENCY
Structure and Function of GH and CS Genes
The GH gene cluster consists of five structurally similar genes in the order 5' [GH−1, CSHP (chorionic somatomammotropin pseudogene), CSH−1 (chorionic somatomammotropin gene), GH−2, CSH−2] 3' encompassing a distance of about 65,000 bp (65 kb) on the long arm of chromosome 17 at bands q22−24(1). The GH−1 gene encodes the mature human GH, a 191−amino acid (aa) peptide, and consists of five exons and four introns ( 1 , 2 , 3 ). Approximately 75% of circulating GH is expressed in the anterior pituitary gland as a major 22−kDa product, whereas alternative splicing can give rise to minor forms ( 2 , 3 , 4 ). The most prominent minor form (5−10%) is a bioactive 20−kDa GH peptide isoform that results from the use of a cryptic 3’ splice site in E3, deleting aa 32−46 ( 4 , 5 , 6 , 7 ). The GH−2 gene encodes a protein (GH−V) that is expressed in the placenta rather than in the pituitary gland and differs from the primary sequence of GH−N (product of GH−1 gene) by 13 aa. This hormone replaces pituitary GH in the maternal circulation during the second half of pregnancy. The CSH−1, CSH−2 genes encode proteins of identical sequences, whereas the CSHP encodes a protein that differs by 13 aa and contains a mutation (donor splice site of its second intron) that should alter its pattern of mRNA splicing and, therefore, the primary sequence of the resulting protein. The extensive homology (92−98%) between the immediate flanking, intervening, and coding sequences of these 5 genes suggests that this multigene family arose through a series of duplicational events. With the exception of CSHP, each gene encodes a 217 aa pre−hormone that is cleaved to yield a mature hormone with 191−aa and a molecular weight of 22kDa. The expression of GH−1 gene is further controlled by cis− and trans−acting elements and −factors, respectively ( 2 , 3 , 4 , 5 , 6 , 7 , 8 ).
Familial Isolated GHD
Short stature associated with GHD has been estimated to occur in about 1/4,000 − 1/10,000 in various studies ( 9 , 10 , 11 ). While most cases are sporadic and are believed to result from environmental cerebral insults or developmental anomalies, 3−30% of cases have an affected first−degree relative suggesting a genetic aetiology. Since magnetic resonance examinations detect only about 12−20% anomalies of either hypothalamus or pituitary gland in isolated GHD (IGHD), it can be assumed that many genetic defects may not be diagnosed and a significantly higher proportion of sporadic cases may have indeed a genetic cause ( 12 ). Familial IGHD, however, is associated with at least four Mendelian disorders ( 2 , 3 , 4 , 5 , 6 , 7 , 8 ), including two forms that have autosomal recessive inheritance (IGHD type IA, IB) as well as autosomal dominant (IGHD type II) and X−linked (IGHD III) forms. Table 1 depicts the mutational spectrum of GHD, which is discussed in greater detail later in the review.
IGHD Type IA
In 1970, IGHD type IA was first described by Ruth Illig in three Swiss children with unusually severe growth impairment and apparent deficiency of GH ( 13 ). Affected individuals occasionally have short length at birth and hypoglycaemia in infancy, but uniformly develop severe growth retardation by the age of six months. Their initial good response to exogenous GH is hampered by the development of anti−GH−antibodies leading to dramatic slowing of growth ( 2 , 14 ).
GH−1 Gene Deletions
In 1981, Phillips et al ( 14 ) examined the genomic DNA from these Swiss children and discovered, using Southern blotting technique that the GH−1 gene was missing. Subsequently, additional cases of GH−1 gene deletions have been described responding well to the GH treatment. The development of anti−GH antibodies is an inconsistent finding in IGHD IA patients despite the presence of identical molecular defects (homozygosity for GH−1 gene deletions) ( 15 ). The frequency of GH−1 gene deletions as a cause of GHD varies among different populations and according to the chosen criteria and definition of short
stature ( 1 ). The sizes of the deletions are heterogeneous with the most frequent (70−80%) being 6.7kb ( 2 , 8 ). The remaining deletions described include 7.6, 7.0, 45 kb, as well as double deletions within the GH gene cluster ( 2 , 8 ). At the molecular level, these deletions involve unequal recombination and crossing over within the GH−gene cluster at meiosis ( 2 ).
GH−1 Gene Frameshift− and Nonsense Mutations
Single−base pair deletions and nonsense mutations of the signal peptide may result in an absent production of mature GH and in the production of anti−GH−antibodies on exogenous replacement therapy ( 8 , 16 , 17 , 18 , 19 ).
IGHD Type IB
Patients with IGHD type IB are characterized by low but detectable levels of GH (<7 mU/l; <2.5 ng/ml), short stature (<−2 SDS for age and sex), growth deceleration and height velocity less than 25th percentile for age and sex, significantly delayed bone age, an autosomal recessive inheritance (two parents of normal height; two sibs affected), no demonstrable direct and/or endocrine cause for IGHD, and a positive response and immunological tolerance to treatment with exogenous GH. This subgroup of IGHD has been broadened and reclassified on the basis of the nature of their GH gene defects and includes splice site mutations of the GH gene, even an apparent lack of GH has been found by RIA. The phenotype of IGHD type IB, therefore, is more variable than IA. In one family, the children may resemble IGHD type IA, whereas in other families, growth during infancy is relatively normal and growth failure is not noted until mid−childhood. Similarly, GH may be nearly lacking or simply low following stimulation test. This heterogeneous phenotype suggests that there is more than one candidate gene causing the disorder, as summarized recently.
Candidate Genes in IGHD Type IB
Some of the components of the GH pathway are unique to GH ( 18 , 20 ), whereas many others are shared. In patients with IGHD, mutational changes in genes specific to the GHRH−GH axis are of importance and there is a need to focus on them.
GHRH−Gene
Many laboratories put a lot of energy to define any GHRH gene alterations. To date, no GHRH gene mutations or deletions causing IGHD have been reported ( 8 , 21 , 22 ). This is, however, somewhat a surprising observation, and the GHRH gene must still be considered a candidate gene for familial forms of IGHD.
GHRH−Receptor (GHRHR) Gene
In 1992, Kelly Mayo cloned and sequenced the rat and human GHRH−receptor (GHRHR) gene that provided the opportunity to examine the role of GHRHR in growth abnormalities that involve the GH−axis ( 23 ). Sequencing of the GHRHR gene in the little−mouse (lit/lit) showed a single nucleotide substitution in codon 60 that changed aspartic acid to glycine (D60G) eliminating the binding of GHRH to its own receptor ( 24 ). As the phenotype of IGHD type IB in humans has much in common with the phenotype of homozygous lit/lit mice including autosomal recessive inheritance, time of onset of growth retardation, diminished secretion of GH and IGF−I, proportional reduction in weight and skeletal size, and delay in sexual maturation, the GHRHR gene was searched for alteration in these patients suffering from IGHD type IB ( 25 , 26 ). Wajnrajch et al ( 26 ) reported a nonsense mutation similar to the little mouse in an Indian Muslim kindred. Furthermore, in two villages in the Sindh area of Pakistan, Baumann ( 27 ) reported another form of severe short stature caused by a point mutation in the GHRHR gene resulting in a truncation of the extracellular domain of this receptor. Individuals who are homozygous for this mutation are very short (−7.4 SDS) but normally proportioned. They appear to be of normal intelligence, and at least some are fertile. Biochemical testing revealed that they have normal levels of GHRH and GH binding protein (GHBP), but undetectable levels of GH and extremely low levels of IGF−I. Later, families from Sri Lanka, Brazil, United States, Spain as well as Pakistan were reported ( 28 , 29 , 30 , 31 ). Mutations in the GHRHR gene have been described as the basis for a syndrome characterized by autosomal recessive IGHD and anterior pituitary hypoplasia, defined as pituitary height more than 2 SD below age−adjusted normal, which is likely due to depletion of the somatotrop cells (OMIM: 139190). In a most recent report, however, certain variability in anterior pituitary size even in siblings with the same mutation was described ( 32 ). This finding is of importance, as it was thought that patients with a GHRHR gene defect invariably have an anterior pituitary gland hypoplasia and that GHRHR gene mutations can be excluded in the absence of this pathological feature, because GHRHR may be critical for pituitary gland development and function of the somatotropes ( 33 , 34 ). Further, Hilal et al ( 35 ) discussed most interestingly the possible role of GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GHD.
Overall, mutations in the human GHRHR gene can impair ligand binding and signal transduction, and have been estimated to cause about 10% of autosomal recessive familial IGHD ( 36 ). Mutations reported to date include six splice donor site mutations, two microdeletions, two nonsense mutations, seven missense mutations, and one mutation in the promoter ( 35 , 36 ). These mutations have an autosomal recessive mode of inheritance, and heterozygous individuals do not show signs of IGHD, although the presence of an intermediate phenotype has been hypothesized. Conversely, patients with biallelic mutations have low serum IGF−1 and GH levels (with absent or reduced GH response to exogenous stimuli), resulting − if not treated− in proportionate dwarfism ( 36 , 37 ).
Muscarinic Acetylcholine Receptor (mAchR)
Acetylcholine, as a neurotransmitter, exerts many of its actions via interaction with one or more of the five mammalian muscarinic acetylcholine receptor (mAchR) subtypes, M1−M5. The importance of cholinergic pathways in the regulation of GH secretion in humans is well established. Central cholinergic stimulation gives rise to an increase in GH release, whereas cholinergic blockade is followed by a blunting in GH secretion ( 38 ). Acetylcholinesterase inhibitors, which indirectly activate cholinergic neurotransmission, are believed to act by reducing the release of somatostatin (SRIF), thus increasing spontaneous GH secretion, and potentiating GH responses to GHRH or to other stimuli. Conversely, muscarinic cholinergic receptor antagonist drugs reduce spontaneous GH release as well as GH responses to GHRH, sleep, exercise, L−dopa, glucagon, arginine, and clonidine. Mouse models have been generated, in which a specific subtype of mAchR has been ablated by genetic engineering ( 39 ). These animals have a wide variety of phenotypic abnormalities but not growth failure, seemingly showing, that at least in rodents, the lack of muscarinic receptor function would not cause a significant reduction in GH secretion. However, very recently a murine model was created, in which the function of the M3 receptor was ablated in both alleles exclusively in the central nervous system ( 40 ). In this model, body length is reduced, and this is associated with significantly reduced GH and IGF−I serum levels and a reduction in pituitary somatotroph cell mass. Although the degree of growth retardation and pituitary hypoplasia is not as marked, the phenotype of this animal has a striking similarity with the murine model of ablation of the GHRH gene ( 41 ), and with the naturally occurring mutation in the GHRHR gene that occurs in the little mouse ( 24 ). These observations are consistent with the hypothesis that the neuronal muscarinic receptors play an important role in controlling GH secretion. Based on all the above observations, we hypothesized that a subgroup of IGHD type IB families may have inactivating mutations in these receptors. To test this hypothesis, we analyzed the M1−M5 receptor genes in 39 of these families.
However, we concluded from this study that mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB ( 42 ).
Ghrelin Receptor, GH Secretagogue Receptor (GHSR)
To date, there is one recent report describing a loss of function of the constitutive activity of the GH secretagogue receptor (GHSR) in familial short stature ( 43 , 44 ). GHSR is highly expressed in the brain and in the pituitary gland. The first endogenous ligand of this receptor was discovered back in 1999 and was named ghrelin ( 45 ). Although pharmacological studies have demonstrated that this endogenous ligand stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR−dependent pathways remains an open question that gives rise to much controversy ( 43 ).
Homeobox Gene Expressed in ES Cells; HESX1
It has been shown that familial septo−optic dysplasia (SOD), a syndromic form of congenital hypopituitarism involving optic nerve hypoplasia and agenesis of midline brain structures, may be associated with homozygosity for an inactivating mutation in the homeobox gene hesx1/HESX1. Importantly, a small proportion of mice heterozygous for the hesx1 null allele show a milder form of SOD, implying that heterozygosity in human HESX1 gene alteration may lead to a mild phenotype of IGHD only ( 46 ). Therefore, actually the HESX1 gene has to be studied whenever looking for any molecular reason causing IGHD type IB ( 47 ).
SOX3 SRY (Sex Determining Region Y)−Box 3
SOX3 is located on the X−chromosome and both under− and overdosages of the gene lead to hypopituitarism ( 48 , 49 ). Male patients present with variable hypopituitarism (combined pituitary hormone deficiency (CPHD) or IGHD) and infundibular hypoplasia, an ectopic/undescended posterior pituitary and abnormalities of the corpus callosum with or without mental retardation, in other words, this gene needs a closer look as well while studying IGHD ( 47 , 50 ).
Specific Trans−Acting Factor to GH−Gene
Any alteration to the specific transcriptional regulation of the GH−1 gene may produce IGHD type IB. Mullis et al ( 51 ) have reported a heterozygous 211 base pairs (bp) deletion within the retinoic acid receptor a gene causing the phenotype of IGHD type IB.
Transcription Factors, Important for Pituitary Gland Development
In addition, as the occurrence of the various hormonal deficiencies caused by transcription factors important for the pituitary gland development may vary quite drastically also with a family presenting with an identical gene defect, GHD can be the only defect at the beginning. Therefore, the two most important transcription factors, namely POU1F1 (Pit−1) and PROP 1, are shortly discussed ( Table 2 ).
POU1F1 (PIT1)
The pituitary transcription factor PIT−1 is a member of the POU−family of homeoproteins, which regulates important differentiating steps during embryological development of the pituitary gland as well as target gene function within the postnatal life ( 8 ). Further, it is 291 aa in length, contains a transactivation domain and two conserved DNA−binding domains: the POU−homeodomain and the POU−specific domain. As PIT1 is confined to the nuclei of somatotropes, lactotrops and thyrotropes in the anterior pituitary gland, the target genes of PIT1 include the GH−, prolactin− (PRL) and the thyrotropin (TSH)−subunit−, and the POU1F1 gene itself. Therefore, the defects in the human POU1F1 gene known so far have all resulted in a total deficiency of GH and PRL, whereas a variable hypothyroidism due to an insufficient TSH secretion, at least during childhood, has been described ( Table 2 ). Although it is important to stress that the clinical variability is due to other factors than the exact location of the mutation reported, the type of inheritance, however, seems to correlate well with the genotype. The first mutation within the POU1F1 gene was identified by Tatsumi ( 52 ). The majority of the mutations reported so far are recessive, however, a number of heterozygous point mutations have been reported ( 53 ). Of those, the aa substitution R271W (Arg271Trp) seems to be a “hotspot”. Further, the dominant negative effect of the R271W POU1F1 form has been recently challenged by Kishimoto et al ( 54 ). Although most cases with R271W were sporadic and presenting with an autosomal dominant mode of inheritance, Okamoto et al ( 55 ) reported a family with normal family members, who were clearly heterozygous for that mutation. Therefore, further in vitro expression studies were performed that could not confirm its dominant negative effect, which is well in contrast with the original report using identical experimental conditions ( 8 , 54 ).
PROP1
Wu et al ( 56 ) described four families, in which CPHD was associated with homozygosity or compound heterozygosity for inactivating mutations of the PROP1 gene. PROP1 (prophet of Pit1) is a paired−like homeodomain transcription factor and, originally, a mutation in this gene (Ser83Pro) was found causing the Ames dwarf (df) mouse phenotype ( 57 ). In mice, Prop1 gene mutation primarily causes GH, PRL and TSH deficiency, and in humans, PROP1 gene defects also appear to be a major cause of CPHD. In agreement with the model of Prop1 playing a role in commitment of dorsal lineages (GH, PRL and TSH), Prop1 mutant mice exhibit a dorsal expansion of gonadotrophs that normally arise on the ventral side.
To date, many different missense, frameshift and splice site mutations, deletions, insertion have been reported and it has been realized that the clinical phenotype varied not only among the different gene mutations, but also among the affected siblings with the same mutation ( 58 , 59 ). In addition, although the occurrence of the hormonal deficiency varies from patient to patient ( 8 ), the affected patients as adults were not only GH, PRL and TSH deficient, but also gonadotropin deficient ( Table 2 ). The three tandem repeats of the dinucleotides GA at location 296−302 in the PROP1 gene represent a “hot−spot” for CPHD ( 58 , 59 , 60 ). Low levels of cortisol have also been described in some patients with PROP1 gene mutations ( 61 ). In addition, pituitary enlargement with subsequent involution has been reported in patients with PROP1 mutations ( 61 ). The mechanism, however, underlying this phenomenon remains still unknown.
IGHD Type II
Focusing on the autosomal dominant form of IGHD, type II (IGHD II) is mainly caused by mutations within the first six bp of intervening sequences 3 (5’IVS−3) ( 8 , 62 , 63 , 64 , 65 , 66 ), which result in a missplicing at the mRNA level and the subsequent loss of E3, producing a 17.5−kDa hGH isoform ( 8 , 65 ). This GH product lacks aa 32−71 (del32−71 GH), which is the entire loop that connects helix 1 and helix 2 in the tertiary structure of hGH ( 67 , 68 ). Skipping of E3 caused by GH−1 gene alterations other than those at the donor splice site in 5’IVS−3 has also been reported in other patients with IGHD II. These include mutations in exon 3 (E3) splice enhancer ESE1 (E3+1G−>T:ESE1m1; E3+2A−>C:ESE1m2, E3+5A−>G:ESE1m3) as well as ESE2 (downstream of the cryptic splice site in E3; ESE2: Δ721−735) and within
suggested intron splice enhancers (ISE) (IVS−3+28 G−>A: ISEm1; IVS−3del+28−45: ISEm2) sequences ( 8 , 64 , 69 , 70 , 71 , 72 , 73 , 74 , 75 ). Such mutations lie within purine−rich sequences and cause increased levels of E3 skipped transcripts ( 64 , 69 , 70 , 71 , 73 , 74 , 75 ), suggesting that the usage of the normal splicing elements (ESE1 at the 5’ end of E3 as well as ISE in intron 3) may be disrupted ( 73 , 74 , 75 ). Importantly, the first 7 nucleotides in E3 (ESE1) are crucial for the splicing of GH mRNA ( 75 ) such that some nonsense mutations might cause skipping of one or even more exons during mRNA splicing in the nucleus. This phenomenon is called nonsense−mediated altered splicing (NAS); its underlying mechanisms are still unknown ( 76 ). Furthermore, there is a recent report of Vivenza et al ( 77 ) presenting a patient with a specific deletion within intron 3 leading to E3 skipping, which underlines the importance of intron length on the splicing machinery, as it was previously suggested by the elegant work by Ryther et al ( 75 ). In addition to the above described splice site mutations that result in the production of del32−71 GH, three other mutations within the GH−1 gene (missense mutations) are reported to be responsible for IGHD II, namely, the substitution of leucine for proline, histidine for arginine and phenylalanine for valine at aa positions 89 (P89V), 183 (R183H) and 110 (V110F), respectively ( 66 , 78 , 79 ).
At the functional level, the 17.5−kDa isoform exhibits a dominant−negative effect on the secretion of the 22−kDa isoforms in both tissue cultures as well as in transgenic animals ( 80 , 81 , 82 ). The 17.5−kDa isoform is initially retained in the endoplasmic reticulum, disrupts the Golgi apparatus, impairs both GH and other hormonal trafficking ( 83 ), and partially reduces the stability of the 22−kDa isoform ( 80 ). Furthermore, transgenic mice overexpressing the 17.5−kDa isoform exhibit a defect in the maturation of GH secretory vesicles and the anterior pituitary gland is hypoplastic due to a loss of the majority of somatotropes (73,80,81). Trace amounts of the 17.5−kDa isoforms, however, are normally present in children and adults of normal growth and stature ( 84 ), and heterozygosity for A731G mutation (K41R) within the newly defined ESE2 (which is important for E3 inclusion) led to approximately 20% E3 skipping resulting in both normal as well as short stature ( 73 , 75 , 85 ).
From the clinical point of view, severe short stature (<−4.5 SDS) is not present in all affected individuals, indicating that in some forms of IGHD II, growth failure is less severe than one might expect ( 66 ). It has been hypothesized that children with splice site mutations may be younger and shorter at diagnosis than their counterparts with missense mutations ( 66 ). In addition, more recent in vitro and animal data suggest that both a quantitative and qualitative difference in phenotype may result from variable splice site mutations causing differing degrees of E3 skipping ( 8 , 85 , 86 , 87 , 88 , 89 ). In summary, these data suggest that the variable phenotype of autosomal dominant GHD may reflect a threshold and a dose dependency effect of the amount of 17.5−kDa relative to 22−kDa hGH ( 81 , 82 , 85 ). Specifically, this has a variable impact on pituitary size, as well as on onset and severity of GHD and, unexpectedly, the most severe, rapid onset forms of GHD might be subsequently associated with the evolution of other pituitary hormone deficiencies ( 90 , 91 ).
IGHD Type III
This reported type is X−linked, recessively inherited. In these families, the affected males were immunoglobulin−as well as GH−deficient ( 92 , 93 ). Recent studies have shown that the long arm of X−chromosome may be involved and that the disorder may be caused by mutations and/or deletions of a portion of the X−chromosome containing two loci, one necessary for normal immunoglobulin production, and the other for GH expression ( 94 ). In addition, Duriez et al ( 95 ) reported an exon−skipping mutation in the btk−gene of a patient with X−linked agammaglobulinemia and IGHD.
BIOINACTIVE GH
Short stature associated with bioinactive GH was first suggested and described by Kowarski and co−workers in 1978 ( 96 ). It is clinically characterized by normal or slightly increased GH secretion, pathologically low IGF−I levels, and normal catch−up growth on GH−replacement therapy. On a clinical basis, additional cases of bioinactive GH were described in the eighties ( 97 , 98 , 99 , 100 , 101 ). Chihara and co−workers ( 102 , 103 , 104 ) reported two missense mutations (R77C and D112G) in the GH−1 gene leading to Kowarski’s syndrome in two Japanese patients. However, these mutations were both found in the heterozygous state only, and furthermore, the mutation R77C was also identified in the normal−statured father. Further, six GH variants were suggested to be bioinactive by Millar and co−workers ( 85 ). Again, all these mutations were found in the heterozygous state and no clear correlation between laboratory/clinical phenotype and patient genotype was shown. Later, also our group described a heterozygous R77C mutation in the GH molecule in a patient with growth retardation and delayed pubertal development. However, no differences between wt−GH and GH−R77C were found by functional characterization of the GH−R77C through GHR binding, activation of JAK2/STAT5 pathway and additional secretion studies together with cell proliferation when stably GHR transfected cells (293GHR) were used ( 105 , 106 ). On the other hand, reduced capability of GH−R77C to directly induce GHR/GHBP gene transcription rate could indirectly affect the levels of GHBP in the circulation of our patient. In addition, this group of patients deserves further attention, because they could represent a distinct clinical entity underlining that an altered GH peptide may cause partial GH insensitivity through direct impact on GHR/GHBP gene expression leading to the delay of growth and pubertal development. Finally, GH−R77C is not invariably associated with short stature, although the serum IGF−I levels are low, the GH is elevated, and the GHBP levels are somewhat low, consistent with some degree of GH insensitivity, which is, presumably, compensated for by excess of GH production. Whether this is due to GH receptor transcription defects, remains unclear.
Furthermore, in one of the more convincing cases of bioinactive GH reported to date, a homozygous missense mutation (bp:G705C; aa:C53S) leading to disruption of the disulfide bond between Cys−53 and Cys−165 was found in a short (−3.6SDS) Serbian boy. Both GHR binding as well as JAK2/STAT5 signalling activities were markedly reduced ( 107 ).
FUNCTIONAL ANALYSIS OF ANY GENE VARIANT IS IMPORTANT
To make the story even more complicated, we reported a patient suffering from a specific form of IGHD II caused by a GH−1 gene alteration on a hypomorphic partial agonistic allele, emphasizing the importance of detailed functional analysis of GH variants. The patient was heterozygous for the GH−R178H mutation ( 108 ). Clinical findings combined with the experimental data of secretion studies confirmed the diagnosis of IGHD II. However, although the GH concentration after stimulation was reduced, admittedly supporting the diagnosis of GHD, neither the severity of short stature (−6.0 SDS at the chronological age of 5 years) nor the low IGF−1 concentrations could be fully explained. Therefore, further functional characterization of this GH mutant was performed through studies of GHR binding and activation of the JAK2/STAT5 pathway. Binding activity and the bioactivity of GH−R178H were investigated and compared with the wt−GH and revealed that GH−R178H by itself behaves more like a partial agonist. Therefore, phenotype and hormonal data underlined the fact that GH−R178H mutation expressed from a hypomorphic partial agonistic allele seems to functionally overlie IGHD II in our patient ( 108 ). | CC BY | no | 2022-01-12 15:22:47 | J Clin Res Pediatr Endocrinol. 2010 Jun 1; 2(2):52-62 | oa_package/17/88/PMC3014602.tar.gz |
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PMC3014618 | 21253011 | Introduction
Cryptococcal meningitis (CM), caused by the fungal organism Cryptococcus neoformans (commonly termed simply “cryptococcus”) is the most common fatal central nervous system (CNS) infection in persons with AIDS and is the initial AIDS-defining illness in 20%–30% of AIDS patients in Africa, causing 20%–40% of AIDS-attributable mortality [1] – [4] . The US Centers for Disease Control (CDC) estimates approximately 1 million CM cases occur annually, with 70% in sub-Saharan Africa [5] . The incidence of CM among Ugandans with CD4 counts <200 cells/μl is 10% annually without antiretroviral therapy (ART) [1] , and without ART, almost all persons with CM die within 6 mo [6] . Although ART availability has led to improved CM survival in Africa [7] – [9] , mortality after CM is still high, in part due to paradoxical HIV immune reconstitution inflammatory syndrome (IRIS), an exaggerated inflammatory response causing a subset of persons with recent CM to paradoxically deteriorate on ART in the presence of improving immune function [10] – [14] . Paradoxical CM-IRIS incidence has been reported as 10%–42% among ART-naïve persons with CM, with a pooled average of 18% (95% CI 14%–23%) [7] , [14] – [17] . CM-IRIS is of grave concern because of its high incidence and mortality (33%–66%) [7] , [14] , [17] , [18] . In many resource-limited regions, particularly sub-Saharan Africa where CM is the second most common AIDS-defining opportunistic infection [1] – [4] , CM-IRIS is a major clinical problem.
The pathophysiology of paradoxical IRIS is largely unknown, but IRIS is hypothesized to be due to dysregulated reconstitution of antigen-specific immunity, leading to an exaggerated immune response to persisting antigens despite microbiologic treatment success [19] – [22] . The antigens, which may be present as intact organisms, dead organisms, or debris [23] , evoke pathological inflammatory responses that cause paradoxical clinical deterioration [24] – [26] .
A healthy immune response in non-immunocompromised hosts to cryptococcal infection depends on coordinated interactions between antigen-presenting cells (APCs) and effector T cells, thereby generating an effective type-1 helper T cell (Th1) immune response that clears the infection. Interferon-gamma (IFN-γ) induces classical activation of macrophages to destroy phagocytosed cryptococcus ( Figure 1 ) [27] – [30] . In contrast, Th2 responses are not protective and allow intracellular proliferation of cryptococcus within macrophages, resulting in disseminated infection [31] , [32] . Colony-stimulating factors, such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), also up-regulate anti-cryptococcal activity of the innate immune system [33] – [36] . We hypothesize that an ineffectual immune response to cryptococcus occurs in a subset of AIDS patients, and these abnormal responses predispose to the development of IRIS.
We prospectively studied the incidence, clinical presentations, outcomes, and cytokine profiles of CM-IRIS in a cohort of HIV-infected Ugandans initiating ART in order to identify serum biomarkers of inflammation that can be used to predict and diagnose CM-IRIS. Characterizing the abnormal inflammatory responses associated with IRIS will enable the future selection of rational IRIS treatment strategies targeting specific inflammatory pathways. | Methods
Participants
One hundred two ART-naïve patients with AIDS and recent CM were enrolled for 12 mo of longitudinal evaluation after ART initiation. One participant was lost to follow up at <2 wk and excluded. Participants with CM were enrolled from May 2006 through September 2009 at Mulago Hospital in Kampala, Uganda [7] . Patients were treated with 0.7–1.0 mg/kg/day of amphotericin for 14 doses, then fluconazole 400 mg daily, and subsequently started on ART as outpatients at the Infectious Disease Institute clinic. ART was initiated at a median of 34 d (interquartile range [IQR] 24–41 d) after CM diagnosis and consisted of: zidovudine, lamivudine, and efavirenz or stavudine, lamivudine, and nevirapine. After ART initiation, participants attended clinic visits biweekly for 3 mo then monthly through 12 mo. Evaluation for IRIS was performed at each scheduled visit and when participants presented acutely. When IRIS was suspected, alternative diagnoses were evaluated by history, examination, laboratory, lumbar puncture, and/or radiological investigations. The International Network for the Study of HIV-associated IRIS (INSHI) consensus working case definition for CM-IRIS was used [37] . Suspected but unconfirmed IRIS events were considered non-IRIS. Each participant provided written informed consent for protocols approved by the institutional review boards at the University of Minnesota, Makerere University, and Uganda National Council for Science and Technology.
Laboratory Testing
CD4 + T cell and plasma HIV RNA measurements were made every 12 wk and at suspected IRIS events. From 2007 onward, CD4 + T cells were also measured at 4 and 8 wk of ART. Serum was frozen at −80°C at visits (0, 2, 4, 8, 12, 24 wk and suspected IRIS events). We measured 27 serum cytokines/chemokines in duplicate (Human 27-Plex Panel, Bio-Rad, Hercules, California) according to manufacturer protocol via a Luminex 100 system (Austin, Texas). Plasma D-dimer was measured in 1∶25 dilution by IMUCLONE D-Dimer ELISA (America Diagnostics, Stamford, Connecticut). Serum C-reactive protein (CRP) was measured by reflectance spectrophotometry at the University of Minnesota clinical laboratory. Baseline specimens, collected on the day of ART initiation, were missing from nine patients. Serum cryptococcal antigen (CRAG) titers were batched with each patient's specimens being tested simultaneously in parallel (Immuno-Mycologics, Norman, Oklahoma). At the time of CM-IRIS onset, cerebrospinal fluid (CSF) analysis excluded alternative diagnoses. Investigations included: Gram and acid-fast bacilli (AFB) stains, bacterial and fungal cultures, 16S ribosomal DNA amplification for bacteria [38] , and viral culture in Vero cells and RT-PCR amplification for arboviruses performed at the Centers for Disease Control–Fort Collins, Colorado. Patients presenting with pneumonitis were offered bronchoalveolar lavage with separate consent. Histopathology or autopsy specimens always included AFB staining to exclude tuberculosis (TB).
Statistical Analysis
The primary comparison was between CM patients developing IRIS and control CM patients without IRIS. Non-normally distributed variables (e.g., cytokines, CD4) were tested at baseline by Mann-Whitney U and presented as median with IQR. p -Values are two-sided with α<0.05 (SPSS 18.0.1, Chicago, IL). Cytokine p -values were adjusted for multiple comparisons via Benjamini-Hochberg methods [39] . To determine the risk of future CM-IRIS, pre-ART demographic variables, standard HIV parameters, and log 2 -transformed cytokines were analyzed. A training model was developed in October 2008 after the first 65 participants were enrolled [40] , which narrowed the selection from all 29 possible biomarkers to consideration of: CRP, interleukin (IL)-4, IL-8, IL-12, IL-17, G-CSF, GM-CSF, CCL2 (MCP-1), tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF). To evaluate the additive predictive power of the above cytokine set, we fitted penalized logistic regression models with penalty parameters specified by the Lasso method [41] . Cross-validation was used to estimate the penalty parameters in the model, with 10% of the data removed for each subset estimate. Models were fitted using the “penalized” package in R [42] . The unadjusted models look at the serum biomarkers alone. Six participants were a priori excluded during model development (four suspected IRIS, two TB-IRIS). We also considered an adjusted model including CD4 and the time from CM to ART initiation as unpenalized regression coefficients.
To describe the degree of inflammation occurring at CM-IRIS events, two methods were used. First, cytokine profiles at IRIS events were compared with CM controls time-matched from 4–12 wk of ART (corresponding to the approximate IQR of IRIS timing). Second, we modeled the longitudinal within-patient cytokine changes by Cox regression with a time-dependent covariate to represent each cytokine profile and the profile's evolution over time on ART [43] , [44] . The outcome variable in the Cox model was the time to first IRIS event. The “survival package” in R fitted the model on log 2 -transformed data [42] . Sandwich estimates determined the variance of regression coefficients, and the relative IRIS risk was portrayed as the hazard ratio (HR). The p -values resulting from each cytokine test were adjusted for multiple comparisons [39] . The Cox model allowed the use of the full longitudinal cytokine profile in order to test for association with the hazard of CM-IRIS events. The HR measures the relative risk of CM-IRIS for each two-fold change in the cytokine expression at any given time point through the first 6 mo of ART. | Results
Clinical Features of Cryptococcal IRIS
In this cohort, 57 paradoxical IRIS events occurred in 45 persons (45%; 95% CI 35%–55%; see Figure S1 ), of whom 35 persons had one event, nine persons had two events, and one person had three events. The median pre-ART CD4 count was 19 (IQR 7–38) cells/μl. Baseline demographic features and both virologic and CD4 + T cell responses to ART were similar in patients who did and did not develop cryptococcal IRIS ( Table 1 ). The most frequent clinical manifestation of paradoxical cryptococcal IRIS was aseptic meningitis, which occurred in 30% (30/101) after appropriate clinical responses to initial amphotericin therapy. In these 30 patients, CM-IRIS manifested as headache (87%), photophobia (33%), vomiting (30%), meningismus (27%), papilledema (27%) and seizures (20%). Analysis of CSF showed elevated WBCs in 75% (median 50, range 5–200/μl, lymphocytic predominance), elevated protein in 88% (median 80, range 40–150 mg/dl), and sterile cultures in 96% (27/28, missing two). Each variable was increased compared with pre-ART values ( p <0.001). Six patients with CM-IRIS presented with seizures that were accompanied by cryptococcoma(s) on CT and/or postmortem exam; three of these patients had concomitant aseptic meningitis. Other paradoxical cryptococcal IRIS events included: pneumonitis with nonproductive cough and radiologic infiltrate ( n = 14), CNS cryptococcoma without meningitis ( n = 3), sepsis syndrome with sterile cultures ( n = 2), and single episodes each of marked lymphadenopathy, retinitis, optic neuritis, and phlyctenular keratoconjunctivitis. CM-IRIS events are summarized and additional clinical details are provided in Text S1 . Overall, our results suggest that IRIS occurs in a large percentage of AIDS patients with CM who start ART, and the manifestations of IRIS can be severe, often involving aseptic meningitis and other CNS manifestations.
Association between IRIS and Death
In the absence of ART, CM in Africa is associated with high mortality [6] , [45] . We analyzed mortality data in our cohort to determine whether the development of IRIS was associated with increased mortality. Overall, 28 of 101 CM patients died (27%) after initiating ART. Mortality was 36% (16/45) in patients with IRIS and 21% (12/56) in those without IRIS ( Figure 2 ). Causes of death were cryptococcal IRIS ( n = 16), virologic failure and CM relapse ( n = 1), paradoxical TB-IRIS ( n = 1), aspiration after AZT-induced severe anemia and lactic acidosis (non-IRIS) ( n = 1), renal failure ( n = 1), cerebrovascular event ( n = 1), suspected but unverified cases of CM-IRIS ( n = 4), suspected pulmonary emboli ( n = 2), and one unknown cause of death occurring at home. By time-to-event analysis, mortality was higher among those experiencing cryptococcal IRIS (HR = 2.3, 95% CI 1.1–5.1, p = 0.04) than the remainder of the cohort, alone or after adjustment for baseline CD4 ( p = 0.11 for CD4). Our findings suggest that the development of IRIS is associated with an increased risk for death in patients with recent CM.
Demographic Risk Factors for IRIS
We sought to determine the risks for IRIS by analyzing demographic and standard HIV parameters. For participants with recent CM, we identified no statistically significant difference among those who did or did not develop IRIS in baseline number or change in CD4 + T cell number, in baseline plasma HIV RNA, nor in virologic suppression at 12, 24, 36, or 48 wk ( Table1 ). CD4 recovery is displayed in Figure S2 . The lack of differences in these variables pertained to univariate or multivariate analyses, controlling for CRP, body mass index (BMI), cytokine profiles, as well as time-to-event analysis or comparison of quartiles. Thus, the degree of immunosuppression, rate of immune recovery after ART, level of viremia, and degree of viral suppression on ART were not predictors of CM-IRIS in this prospective cohort with very severe immunosuppression.
Median serum CRAG titers when starting ART were 4-fold higher in participants with IRIS (1∶512 cases versus 1∶128 controls, p = 0.006). This result is in marked contrast to our previous CSF report from 85 members of this cohort, in whom there were no differences in CSF CRAG titer at time of their initial CM diagnosis between IRIS cases and controls (median CRAG 1∶1024 for both groups) [46] . A pre-ART serum CRAG ≥1∶512 was associated with later CM-IRIS (OR = 4.2, 95% CI 1.7–10.3; p = 0.002). Though serum was not available for direct comparison at time of their initial CM diagnosis, the implication is that patients with IRIS likely had poorer antigen clearance in the 5-wk median interval between CM diagnosis and ART initiation.
A concern among many clinicians is that early initiation of ART in patients with CM could lead to an increased incidence of IRIS and increased mortality [47] . We identified no statistical significant difference in the incidence of IRIS in those initiating ART 11–28 d from CM-diagnosis compared with those who waited ≥28 d, (44% versus 55% respectively; p = 0.4) nor a statistical difference in survival ( p = 0.9). Thus, earlier initiation of ART following the diagnosis of CM was not a risk factor for IRIS.
Predictive IRIS Model
Based on a training set derived from pilot data through 2008 [40] , we considered nine possible biomarkers to incorporate into a predictive IRIS model. We used penalized logistic regression with penalties defined by the lasso method to develop a classification model with the best overall fit. Applying this model to our cohort, we were able to stratify persons into high-, moderate-, and low-risks groups with IRIS incidences of 82%, 47%, and 22%, respectively ( Figure 3 ), based on seven serum biomarkers collected on the day of ART initiation (IL-4, IL-17, G-CSF, GM-CSF, CCL2 [MCP-1], TNF-α, and VEGF). The predictive performance (i.e. C-statistic) as quantified by the area under the curve (AUC) of a receiver operating characteristic (ROC) curve is 0.82 for correct prediction ( p <0.001).
The predictive model had better predictive performance than CRP (AUC = 0.58, p = 0.25) or CRAG titer (AUC = 0.67, p = 0.004). We considered an adjusted model including the pre-ART CD4 count and the delay from CM to ART initiation. Adjustment did not improve prediction (AUC = 0.83). We also considered the effect on predictive performance by using a more parsimonious model with fewer predictors ( Figure S3 ). The AUC decreased in a linear fashion with inclusion of fewer predictors, such that the AUC was 0.72 for a four-variable model of IL-17, GM-CSF, TNF-α, and VEGF. Although these biomarkers are associated with prediction of IRIS, these factors alone or in aggregate may not be causative of IRIS. However, the roles of these cytokines in immune responses to cryptococcus ( Figure 1 ) suggest that these cytokines may play a role in IRIS pathogenesis. Overall, our results suggest that the risk of IRIS could be stratified on the basis of pre-ART biomarker levels in persons with cryptococcosis.
Biomarkers Associated with Cryptococcal IRIS
Since predictive models have a different goal and interpretation than do models of association, we also tested for association. We considered the seven biomarkers identified above in a multivariate logistic model to determine their statistical association with IRIS ( Table 2 ). In a multivariate model, future CM-IRIS was associated with increased pre-ART levels of CRP, IL-4, and IL-17 (each p<0.05). Since Th1, rather than Th2 responses, are protective in cryptococcosis [28] , a Th2 response to cryptococcus characterized by increased IL-4 production by T cells might be associated with ineffective antigen clearance, leading to a predisposition to the subsequent development of IRIS. Notably, increased pre-ART levels of IL-17, often viewed as a proinflammatory cytokine, were also associated with future IRIS. Moreover, pre-ART levels of the inflammatory marker CRP were increased among patients who subsequently developed IRIS (mean 2.3-fold higher, 95% CI 1.2–3.5, p = 0.03). Persons with CRP >32 mg/l levels (highest quartile) experienced IRIS with an increased incidence of 74% (OR = 3.9, 95% CI 1.3-11.3, p = 0.01) and shorter time-to-event (HR = 3.4, 95% CI 1.8–6.6, p<0.001) compared with persons with CRP<32 mg/l, independent of CD4+ count. Our data suggest that not only is the presence of pre-ART inflammation associated with risk for IRIS, but the type of inflammation is important, with the closest association of IL-4 (Th2) and IL-17 (Th17) responses with a predisposition toward CM-IRIS development.
Although high pre-ART levels of IL-4, IL-17, and CRP were associated with increased risk of IRIS, lower pre-ART levels of VEGF, G-CSF, and TNF-α were also associated with increased risk ( p <0.05 each). IRIS developed in 79% (11/14) of patients with undetectable serum levels of TNF-α when starting ART, but only 35% (27/78) with detectable TNF-α (OR = 4.9, 95% CI 1.3 to 19.0, p = 0.019). TNF-α is involved in innate immunity by activating phagocytosis of cryptococcus by macrophages, antigen processing, and proinflammatory recruitment of additional leukocytes [48] , [49] , resulting in improved clearance of the organism. Thus, lack of these protective proinflammatory responses at baseline may be associated with subsequent IRIS. In summary, a predisposition to developing IRIS is associated with a paucity of cytokines involved in antigen processing and macrophage function, such as TNF-α, coupled with increased generalized inflammation (e.g., increased CRP, IL-17), lack of antigen recognition by CD4 + T cells (e.g., decreased VEGF), and inappropriate Th2 responses (e.g., increased IL-4) before ART.
Biomarkers on ART
In addition to identifying pre-ART predictors of CM-IRIS, we performed a time-to-event analysis to identify additional predictors of IRIS that evolved in patients while on ART. After ART initiation, analysis of changes in biomarkers during all time points through the first 24 wk of ART showed a significant divergence in inflammatory profiles associated with immune reconstitution between cases and controls that was evident in blood weeks before IRIS events. Increasing levels of CRP, D-dimer, IL-1RA, IL-6, IL-7, IL-13, or G-CSF following initiation of ART were associated with increasing hazard of IRIS (each p ≤0.001) ( Table 3 ). The pattern of inflammation indicated increasing levels of biomarkers of generalized inflammation such as CRP, IL-6, and D-dimer; inappropriate Th2 responses characterized by elevated IL-13 levels; and elevated levels of growth factors such as G-CSF and the T cell growth and differentiation factor IL-7. The anti-inflammatory mediator IL-1RA increased following ART initiation and before IRIS. The clinical implication of these findings is that these biomarkers could be used to indicate risk for CM-IRIS before the development of IRIS events, providing an opportunity for intervention before clinical deterioration occurs.
Biomarkers of Inflammation at IRIS Events
At time of cryptococcal IRIS, serum biomarkers of inflammation were elevated in almost all IRIS patients over those in time-matched controls ( Table 4 ). However, the pattern of inflammation was heterogeneous with individual variation ( Table S1 ). Early IRIS events were associated with more systemic inflammation than were events occurring after 24 wk. IL-6 was most frequently elevated at time of IRIS events (median 5-fold increase compared with time-matched control participants). CRP levels often correlated with IL-6 levels, as CRP is stimulated by IL-6; and CRP levels were most often increased at IRIS diagnosis compared with levels pre-ART or pre-event ( Figure 4 ; p <0.001). In comparing IRIS events with CNS manifestations versus non-CNS IRIS events, there were no statistical differences in any biomarker (each p >0.1) with the possible exception of D-dimer being more elevated in non-CNS events (median 3.68 versus 2.22 μg/ml, unadjusted p = 0.031). Interestingly, the inflammatory profile distinguished relapse from CM-IRIS in the four cases of culture-positive CM relapse. In the relapse cases, CRP was normal (<8 mg/l), whereas CRP was elevated in 76% of participants with cryptococcal IRIS. These results confirm that IRIS is associated with elevations in markers of inflammation that are large enough to be measured in the serum and can help distinguish patients with CM-IRIS from those with CM relapse before culture results are available.
Biomarkers Predictive of or Associated with Mortality
In addition to evaluating predictors of IRIS, we performed analyses to determine if inflammatory profiles could predict death. Using penalized logistic regression with Lasso estimates, we identified baseline pre-ART serum biomarkers that were associated with subsequent mortality on ART. The best fit model consisted of: IL-17, GM-CSF, IL-8, CXCL10, TNF-α, VEGF, and CRP >32 mg/l with an AUC of 0.84 for correct classification with 78% sensitivity, 81% specificity, 60% positive predictive value, and 91% negative predictive value. A more parsimonious three-biomarker model of increasing IL-17 or CRP >32 mg/l, and decreasing GM-CSF, predicted increasing mortality (AUC = 0.76). A weighted score based on these three biomarkers was used to classify high- and low-risk mortality groups ( Figure 5 ). Persons scored as high risk had 69% mortality (i.e., 69% positive predictive value) whereas those with low risk had 83% survival (i.e. 83% negative predictive value), demonstrating the potential clinical utility of the tool to identify patients at increased and decreased risk for death. CRP alone was also clinically useful for predicting risk of death ( Figure 5 ). Participants with a CRP >32 mg/l had 8-fold higher odds of all-cause mortality compared to those with CRP<32 mg/l (OR = 8.3, 95% CI 2.7–25.6, p <0.001). Using these biomarkers as clinical tools at the time of ARV initiation could be useful to identify high-risk patients and to monitor them for early therapeutic intervention.
We also performed analyses to determine if levels of biomarkers at time of IRIS events were associated with mortality. Among persons experiencing CM-IRIS, mortality after an IRIS event was associated with increased IL-10 levels (OR = 2.8 per 2-fold change, 95% CI 1.3–5.9, p = 0.007) and decreased IL-17 levels (OR = 0.59 per 2-fold change, 95% CI 0.40–0.86, p = 0.006) at event compared to persons with IRIS who survived. | Discussion
In this prospective cohort study of patients with AIDS and recent CM in Uganda, we characterized the incidence and clinical features of CM-IRIS and identified serum biomarkers that can diagnose IRIS and can stratify the risk for IRIS or death. CM-IRIS is a common complication of ART in sub-Saharan Africa, with an incidence reported in the literature ranging from 10% to 31% [11] , [14] – [18] , [50] . In our cohort with <1% loss to follow up, the incidence of CM-IRIS was 45%, two-thirds of whom experienced characteristic CNS manifestations (30%). Unlike many other forms of IRIS, which produce less dramatic consequences, CM-IRIS is exceptional for its substantial morbidity and mortality [11] , [50] – [52] . The 36% CM-IRIS mortality in our study is consistent with other CM-IRIS studies [14] , [15] , [17] , [18] , [52] , [53] . Such dire outcomes may derive from the increased inflammation that accompanies CM-IRIS in the anatomically constrained compartment of the CNS.
Before this study, it was not possible to predict risk for CM-IRIS. Epidemiologic factors, that were identified in retrospective studies, were not predictive of CM-IRIS in our study, and have not been found consistently across other studies [14] , [15] , [17] , [52] . Similar to a recent ACTG (AIDS Clinical Trial Group) a5164 trial, in our study timing of ART initiation was not associated with IRIS [54] . In contrast, however, we identified specific patterns of biomarkers that were predictive of the development of IRIS and death at initiation of ART, after initiation of ART, and at IRIS event.
Based on our identification of biomarkers that predict CM-IRIS, our theoretical model is one whereby persons predisposed to IRIS have more dysfunctional immune systems that fail to effectively clear antigens from cryptococcus before ART-associated immune restoration. Indeed, persons with IRIS had higher median serum CRAG titers when initiating ART than controls without IRIS ( p = 0.006), but they had identical 1∶1024 median CSF CRAG titers at their initial CM diagnosis [46] . This result may have implications for customization of timing of ART based on initial induction antifungal therapy prescribed with a longer delay required for less active antifungal therapy (e.g., fluconazole monotherapy).
Further failures of antigen clearance may result from an imbalance in immune regulation, initially characterized by increased production of Th2 cytokines (IL-4, IL-13) and a lack of the proinflammatory mediators (VEGF, TNF-α, G-CSF, GM-CSF) that are required to recruit lymphocytes and activate macrophages and other innate cells to clear C. neoformans . Specifically, in neutrophils and macrophages, G-CSF and GM-CSF promote phagocytosis, up-regulation of reactive oxygen species, and intracellular destruction of cryptococcus [33] – [36] . Decreased pre-ART levels of G-CSF, GM-CSF, and TNF-α should result in decreased phagocytosis and promotion of a fertile intracellular environment within macrophages for cryptococcus [36] , [49] . Based on our model of CM-IRIS pathogenesis, this inappropriate or ineffective macrophage activation leads to failure to clear cryptococcal organisms and predisposes toward the development of CM-IRIS. In this respect, CM-IRIS pathogenesis may parallel the proposed mechanism of TB-IRIS [55] .
A failure of antigen clearance promotes generalized inflammatory signaling to then occur, specifically with IL-6. Compared with time-matched control participants, persons who developed IRIS had elevated levels of CRP before ART initiation, and elevated levels of CRP and IL-6 before and at the time of IRIS events. Our results indicate that the inflammatory signaling response involving IL-6 progressively increased on ART until the development of IRIS. Since IL-6 is the primary stimulant of hepatic CRP production, it is unsurprising that these biomarkers had similar patterns. French and colleagues reported similar increases in plasma IL-6 after a variety of IRIS events or before herpesvirus-related IRIS events, and they verified by flow cytometry experiments that activated macrophages were the primary source of IL-6 [56] , [57] .
We hypothesize that the abnormal cytokine profiles we observed before CM-IRIS, including elevations of Th2 cytokine (e.g., IL-4, IL-13) and lack of proinflammatory cytokines such as TNF-α, indicate ineffective or inappropriate alternative macrophage activation. In contrast to Th1 responses, which promote IFN-γ–mediated killing of cryptococcus by macrophages [28] , [32] , IL-4 produced in Th2 cells causes alternative activation of macrophages—typically a response to parasite infections—promoting fibrosis [58] . Alternative activation of macrophages allows for intracellular growth, proliferation, and dissemination of C. neoformans [28] , [29] . In other fungal processes, IL-4 reverses GM-CSF–mediated macrophage killing of yeast in vitro [59] . In our results, small quantitative increases in IL-4 were associated with large statistical increases in IRIS risk. Since Th1 and Th2 responses are often mutually exclusive, inappropriate increases in Th2 responses (e.g., IL-4, IL-10, IL-13) generate ineffective inflammation and inhibit the protective Th1 responses necessary for efficient antigen clearance. The association of a Th2 bias and risk for IRIS was evident with elevated IL-4 levels at baseline, elevated IL-13 levels on ART before IRIS events, and elevated IL-10 levels at IRIS events as well as an association between increased IL-10 levels at IRIS and subsequent death. The Th2 responses that limit clearance of C. neoformans may be driven by preexisting Th2 biases to other concomitant coinfections, such as intestinal helminths or schistosomiasis. This phenomenon might underlie the higher overall incidence of IRIS in our cohort compared to cohorts from temperate regions [14] , [15] , [17] , [50] . Unexplained absolute eosinophilia (>500 cells/μl) was present in over one-quarter of the cohort; however there were not statistical differences in eosinophils or total serum IgE between cases and controls. Overall, our data suggest that Th2 responses to cryptococcus are associated with poor outcomes, including increased risk for IRIS and death.
Risk factors for IRIS extend beyond the Th1:Th2 paradigm. Increased Th17 responses (i.e. IL-17) were a pre-ART risk for both IRIS and mortality on ART. Proinflammatory Th17 cells have been previously hypothesized as important in IRIS pathogenesis [60] , [61] . In normal immune homeostasis, a balance between Th17 cells and regulatory T cells (Treg) is crucial. A Th17 imbalance can cause autoimmune diseases. In this balance, IL-6 plays a key role in naïve T cell differentiation inducing Th17 differentiation and inhibiting Treg differentiation in the presence of TGF-β [62] . The copious IL-6 present before IRIS events may alter the balance between Treg and Th17 cells, suppressing Treg differentiation or function during ART-related immune reconstitution.
Additionally, other cytokines that we identified may play a role in IRIS pathogenesis by contributing to ineffective macrophage activation. For example, low pre-ART levels of TNF-α or VEGF were associated with increased IRIS risk. TNF-α, secreted by macrophages and T cells, is particularly important in activating antigen-presenting cells. Absence of TNF-α causes failure of mature dendritic cell activation and recruitment, thereby blunting further recruitment of T cells [63] . Lack of TNF-α could indicate failure to present and/or process antigen and could contribute to ineffective macrophage activation in patients at risk for IRIS. In cryptococcosis, VEGF is secreted by a variety of leukocytes but especially by CD4 + T cells during antigen-specific responses to cryptococcal mannoprotein being presented by MHC-II molecules [64] . Decreased VEGF may reflect greater immune dysfunction due to failure of CD4 + T cell antigen recognition from antigen-presenting cells. The downstream effects of decreased VEGF would further diminish both chemotaxis and the costimulatory activity of VEGF on IFN-γ–secreting Th1 memory T cells [65] . Thus, decreased VEGF may then diminish the Th1 response and shift the Th1:Th2 balance toward Th2 responses. The decreased levels of TNF-α and VEGF in patients at risk for IRIS may be causes or reflections of impaired macrophage function. Overall, the inappropriate Th2 responses and IL-4 production, coupled with ineffective macrophage activation, suggest that a failure in pathogen recognition and clearance could set the stage for paradoxical IRIS by promoting antigen persistence. Upon eventual immune restoration of more appropriate antigen-specific responses, these responses are exaggerated because of the abundance of uncleared foreign antigen and promotion by IL-6 of a proinflammatory state.
Study Strengths
Strengths of this study include its prospective design, careful and complete follow-up, and integration of pathophysiologic analyses in the context of characterizing clinical phenotypes. As such, we believe the results reported here are generalizable to cryptococcal IRIS as a pathophysiologic entity. In general, the diagnoses of other forms of IRIS are somewhat subjective, but in CM-IRIS, the identification of IRIS in the CNS is very objective due to the ability to examine the CSF, where the inflammation occurs. A problem arises with non-CNS manifestations of IRIS, which often represent diagnoses of exclusion and depend on the diagnostic capabilities available. In this cohort, we identified several ( n = 20) non-CNS IRIS events, of which 60% were associated with later CNS-IRIS events or with supportive/definitive histopathology ( Text S1 ). Eight cases (retinitis, optic neuritis, keratoconjunctivitis, pneumonitis; n = 5) were probable IRIS diagnoses and unsupported by histopathology or culture (17% of total IRIS cases). Although cryptococcus most often causes meningitis, cryptococcosis is a disseminated disease with systemic CRAG measurable for months in peripheral blood [66] . In our experience here, dead C. neoformans causing IRIS reactions were identified in tissue by histopathology of brain, lymphatics, gut, skin, and tongue and were isolated via bronchoalveolar lavage. Thus, non-CNS cryptococcal IRIS events do occur, allowing these “probable events” as likely, but not definitely, attributable to IRIS. In separately considering these non-CNS IRIS events, there were no statistical differences in the inflammatory profiles observed at time of IRIS as compared to CNS-IRIS events, except for possibly D-dimer. In Uganda, there is likely high and ongoing environmental exposure to cryptococcus [1] , [4] , thus pneumonitis-IRIS could equally be triggered by persisting original antigen or by environmental re-exposure manifesting effectively as an acute hypersensitivity pneumonitis reaction. Pulmonary cryptococcosis often goes unrecognized and/or is suspected as smear-negative TB [67] .
Study Limitations
This prospective observational study has allowed us to characterize the statistical association between cytokine profiles and IRIS. Of course, this does not prove causality; however, the anomalies we report are biologically plausible within the known immunology of responses to cryptococcus. A challenge in pathogenesis studies such as ours is that the inflammatory profiles associated with IRIS are heterogeneous, representing a continuum that is likely dependent on the duration of symptoms, the antigen, and the robustness of the event. In cytokine profiling, six of our participants had minimal immunologic perturbation in peripheral blood even though they clinically deteriorated, presenting with headache and elevated intracranial pressure several months into ART. Thus, some patients fulfilling the IRIS clinical case definition either have localized immune responses that were not detectable in peripheral blood, or they may not have had true immunologic IRIS but instead had delayed complications of CM that were clinically indistinguishable from CM-IRIS. This heterogeneity may have implications for IRIS management and the response to anti-inflammatory therapies. Although this prospective study of CM-IRIS is the largest to date, our biomarker data should be viewed as hypothesis-generating and will require validation in future cohorts. We plan to validate these biomarkers in subjects enrolled in the multi-site Cryptococcal Optimal ART Timing (COAT) trial (NCT 01075152; http://clinicaltrials.gov/ct2/show/NCT01075152 ) and investigate these biomarkers with regard to timing of ART initiation after CM diagnosis. This validation will give insight on whether altering the timing of ART initiation is a potential intervention to alter high risk or whether other strategies should be pursued. Once validated, creating diagnostics such as multiplex ELISAs or bead-based techniques could move this into clinical use.
Conclusions
Given the high incidence, morbidity, and mortality associated with CM-IRIS, identifying patients at risk for IRIS may enable interventions to improve management. Three distinct phases of IRIS pathogenesis can be identified ( Table 5 ). First, before ART, a paucity of innate inflammatory responses or inappropriate (Th2) responses promote ineffective antigen clearance. Second, after ART initiation, copious antigen presentation promotes proinflammatory signaling (e.g., IL-6, CRP, IL-7). Third, at the time of IRIS, a generalized cytokine storm occurs. The biomarkers identified here, although requiring validation, may help target interventions to decrease IRIS risk, such as (1) early adjunctive GM-CSF or IFN-γ to increase macrophage function, (2) antiparasitic therapy to eliminate coinfections that promote inappropriate Th2 bias, (3) anti-IL-6 receptor antibody (tocilizumab) therapy to blunt inflammatory signaling, or (4) delaying ART initiation. This study suggests that prediction of IRIS or death may be possible with measurement of pre-ART serum biomarkers. | Conclusions
Given the high incidence, morbidity, and mortality associated with CM-IRIS, identifying patients at risk for IRIS may enable interventions to improve management. Three distinct phases of IRIS pathogenesis can be identified ( Table 5 ). First, before ART, a paucity of innate inflammatory responses or inappropriate (Th2) responses promote ineffective antigen clearance. Second, after ART initiation, copious antigen presentation promotes proinflammatory signaling (e.g., IL-6, CRP, IL-7). Third, at the time of IRIS, a generalized cytokine storm occurs. The biomarkers identified here, although requiring validation, may help target interventions to decrease IRIS risk, such as (1) early adjunctive GM-CSF or IFN-γ to increase macrophage function, (2) antiparasitic therapy to eliminate coinfections that promote inappropriate Th2 bias, (3) anti-IL-6 receptor antibody (tocilizumab) therapy to blunt inflammatory signaling, or (4) delaying ART initiation. This study suggests that prediction of IRIS or death may be possible with measurement of pre-ART serum biomarkers. | ICMJE criteria for authorship read and met: DRB DBM TLB DLW JR AM SJL AK ENJ PRB. Agree with the manuscript's results and conclusions: DRB DBM TLB DLW JR AM SJL AK ENJ PRB. Designed the experiments/the study: DRB DBM JR AK ENJ PRB. Analyzed the data: DRB TLB SJL ENJ PRB. Collected data/did experiments for the study: DRB DBM DLW JR AM SJL. Enrolled patients: DBM JR AM. Wrote the first draft of the paper: DRB DBM. Contributed to the writing of the paper: DRB DBM TLB DLW JR AM ENJ PRB. Primary statistician: TLB. Substantial analytic contributions and model development: ENJ.
David Boulware and colleagues investigate clinical features in a prospective cohort with AIDS and recent cryptococcal meningitis after initiation of antiretroviral therapy to identify biomarkers for prediction and diagnosis of CM-IRIS (cryptococcal meninigitis-related immune reconstitution inflammatory syndrome).
Background
Although antiretroviral therapy (ART) improves survival in persons with cryptococcal meningitis (CM) and AIDS, ART frequently elicits HIV immune reconstitution inflammatory syndrome (IRIS), an exaggerated and frequently deadly inflammatory reaction that complicates recovery from immunodeficiency. The pathogenesis of IRIS is poorly understood and prediction of IRIS is not possible.
Methods and Findings
We prospectively followed 101 ART-naïve Ugandans with AIDS and recent CM for one year after initiating ART, and used Luminex multiplex assays to compare serum cytokine levels in participants who did or did not develop IRIS. IRIS occurred in 45% of participants with recent CM on ART, including 30% with central nervous system (CNS) manifestations. The median time to CM-IRIS was 8.8 wk on ART. Overall mortality on ART was 36% with IRIS and 21% without IRIS. CM-IRIS was independently associated with death (HR = 2.3, 95% CI 1.1–5.1, p = 0.04). Patients experiencing subsequent CM-IRIS had 4-fold higher median serum cryptococcal antigen (CRAG) levels pre-ART ( p = 0.006). Higher pre-ART levels of interleukin (IL)-4 and IL-17 as well as lower tumor necrosis factor (TNF)-α, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF) predicted future IRIS in multivariate analyses (area under the curve [AUC] = 0.82). An algorithm based on seven pre-ART serum biomarkers was a robust tool for stratifying high (83%), moderate (48%), and low risk (23%) for IRIS in the cohort. After ART was initiated, increasing levels of C-reactive protein (CRP), D-dimer, IL-6, IL-7, IL-13, G-CSF, or IL-1RA were associated with increasing hazard of IRIS by time-to-event analysis (each p ≤0.001). At the time of IRIS onset, multiple proinflammatory cytokine responses were present, including CRP and IL-6. Mortality was predicted by pre-ART increasing IL-17, decreasing GM-CSF, and CRP level >32 mg/l (highest quartile). Pre-ART CRP level >32 mg/l alone was associated with future death (OR = 8.3, 95% CI 2.7–25.6, p <0.001).
Conclusions
Pre-ART increases in Th 17 and Th 2 responses (e.g., IL-17, IL-4) and lack of proinflammatory cytokine responses (e.g., TNF-α, G-CSF, GM-CSF, VEGF) predispose individuals to subsequent IRIS, perhaps as biomarkers of immune dysfunction and poor initial clearance of CRAG. Although requiring validation, these biomarkers might be an objective tool to stratify the risk of CM-IRIS and death, and could be used clinically to guide when to start ART or use prophylactic interventions.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Since 1981, AIDS has killed more than 25 million people and about 33 million people are now infected with HIV, which causes AIDS. HIV, which is most often transmitted through unprotected sex with an HIV-infected partner, infects and kills immune system cells. Eventually, the immune system becomes so weak that unusual infections begin to occur. These “opportunistic” infections are infections that take advantage of the opportunity offered by a weakened immune system. One common and deadly opportunistic infection in people affected by AIDS is cryptococcal meningitis (CM), an infection around the brain that is caused by the fungus Cryptococcus neoformans. About one million cases of CM occur every year. CM can be treated with a drug called amphotericin but usually recurs unless another drug called fluconazole is taken daily thereafter. HIV therapy is lifesaving by suppressing the HIV virus and allowing immune system recovery. This immune recovery also helps to prevent the recurrence of CM.
Why Was This Study Done?
Unfortunately, HIV therapy can also elicit a serious condition called immune reconstitution inflammatory syndrome (IRIS) in people with CM and AIDS. IRIS is an exaggerated inflammatory immune response that kills up to one-third of affected people. Inflammation, which is characterized by swelling and redness, is the body's first defense against infection, but uncontrolled inflammation causes widespread tissue damage. Experts think that CM-IRIS may be the result of an unbalanced recovery of the immune system leading to an inappropriate immune response to persisting C. neoformans fragments and proteins that are slowly cleared from the body over months. Unfortunately, it is impossible to predict which individuals with CM and AIDS will develop IRIS when they are given HIV therapy. In this prospective study, the researchers investigated clinical features and cytokine profiles in a group of Ugandans with AIDS and recent CM for one year after starting HIV therapy to identify biomarkers that could be used to predict and diagnose CM-IRIS. Cytokines are proteins secreted by immune system cells that regulate the immune response; biomarkers are proteins found in the blood that indicate specific diseases.
What Did the Researchers Do and Find?
The researchers enrolled 101 Ugandans with AIDS and recent CM who had not previously received HIV therapy. They compared cytokine patterns in individuals who did and did not subsequently develop IRIS after starting HIV therapy. Overall, 45% of the patients developed IRIS. Deaths occurred in 36% of the patients who developed IRIS and in 21% of those who did not develop IRIS. Patients who developed CM-IRIS after starting HIV therapy had 4-fold higher baseline concentrations of cryptococcal antigen in their blood than patients who did not develop CM-IRIS. Prior to starting HIV therapy, higher levels of the cytokines IL-4 and IL-17 and lower levels of four cytokines—TNF-α, G-CSF, GM-CSF, and VEGF—predicted IRIS development, and an algorithm (formula) based on the baseline levels of seven serum biomarkers was able to group the patients into high, moderate, and low risk of IRIS. After starting HIV therapy, increasing levels of the inflammatory proteins C-reactive protein and D-dimer, and of several cytokines, were associated with an increased risk of IRIS. At the time of IRIS onset, the levels of many proinflammatory cytokine increased. Biomarkers also predicted death after starting HIV therapy with increasing levels of IL-17, decreasing levels of GM-CSF, and a C-reactive protein level of more than 32 mg/l (four times higher than normal) predicted death within one year.
What Do These Findings Mean?
These findings support the hypothesis that some AIDS patients who have a very damaged immune system have a very poor initial immune response and poor clearance of cryptococcus, which predisposes them to IRIS. The findings also identify three distinct phases of IRIS development. Before HIV therapy, a very damaged immune system with a lack of inflammatory responses to infection or inappropriate responses leads to ineffective clearance of the organism and its antigens. After HIV therapy is started, the presence of copious antigens promotes proinflammatory signaling to the immune system. As the immune system recovers proinflammatory immune cells are promoted. Finally, at the time of IRIS, a generalized “cytokine storm” occurs, which is potentially fatal when this inflammation occurs in the brain. The biomarkers identified here as indicators of a predisposition to IRIS need to be validated in more patients in more countries before they can be used as a clinical tool for predicting the risk of IRIS. If they are validated, they could help clinicians decide when to start HIV therapy in patients with AIDS and recent CM, and could guide the use of therapies that could help prevent the abnormal inflammatory responses.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000384 .
The US National Institute of Allergy and Infectious Diseases provides information on HIV infection and AIDS
HIV InSite has information on all aspects of HIV/AIDS, including Knowledge Base Chapters on cryptococcosis and HIV and on the clinical implications of IRIS
Information is available from Avert , an international AIDS charity on all aspects of HIV/AIDS, including HIV-related opportunistic infections (in English and Spanish)
The MedlinePlus encyclopedia has a page on cryptococcal meningitis (in English and Spanish)
AIDS InfoNet provides fact sheets on many HIV/AIDS topics, including a fact sheet on cryptococcal meningitis (in several languages) and treatment guidelines for opportunistic infections | Supporting Information | We thank Ms. Irene Namugga and Jane Frances Ndyetukira for patient care. We wish to thank Ms. Darlisha Williams MPH, Drs. Danny Muskardin, Courtney McElroy, Radha Rajasingham, Ann Vogt, Kristina Krohn, Theresa Wampler, and Vivian Cox for assistance with data collection; Mr. Emmanuel Okiring, Ryan Flynn, and Dr. Henry Kajumbula for laboratory support, and Drs. Greg Park and Chandy John for assistance with cytokine profiling. We wish to thank Drs. Keith McAdam, Allan Ronald, and Merle Sande for conceptual discussions and institutional support. We thank Dr. Barry Miller and the U.S. Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases for laboratory investigations for arboviral etiologies of aseptic meningitis, and Dr. Bazak Sharon for 16S ribosomal amplification.
Abbreviations
acid-fast bacilli
antigen-presenting cell
antiretroviral therapy
area under the curve
cryptococcal meningitis
central nervous system
cryptococcal antigen
C-reactive protein
cerebrospinal fluid
fibroblast growth factor
granulocyte colony-stimulating factor
granulocyte-macrophage colony-stimulating factor
hazard ratio
interleukin
immune reconstitution inflammatory syndrome
receiver operating characteristic
reactive oxygen species
tuberculosis
tumor necrosis factor
regulatory T cells
vascular endothelial growth factor | CC BY | no | 2022-01-13 02:58:35 | PLoS Med. 2010 Dec 21; 7(12):e1000384 | oa_package/70/35/PMC3014618.tar.gz |
PMC3014652 | 20569139 | Introduction
Seasonal invasion by the litter dwelling darkling beetle, Luprops tristis Fabricius 1801 (Coleoptera: Tenebrionidae) (locally called ‘ mupli ’), of between 0.5 and 4 million/building immediately after summer rains is a regular event near and within rubber plantation tracts in the western slopes of South-Western Ghats. The continued presence of these nocturnally active beetles inside living areas is a nuisance. Although, these beetles neither sting nor bite, when disturbed by hand-picking them from walls or when they are squashed, they release an irritating, strong-smelling secretion that causes blisters to humans. Observations of L. tristis revealed neither any vertebrate nor invertebrate predators prevalent in rubber litter floor or in the buildings fed on L. tristis (personal observations). It has been suggested by Sabu et al. ( 2008 ) that this lack of predation could be a key reason for the massive population build up of L. tristis in rubber plantation belts. Moreover, the larvae, on being disturbed, release a strong smelling secretion indicating the presence of defensive glands that may deter predators. Defensive-compound secreting glands in Tenebrionidae are everted by haemolymph pressure ( Tschinkel 1975 ) and the secretion is released involuntarily during stress when being predated upon ( Kendall 1968 , 1974 ). As these glands have not been described in larvae of L. tristis , the present study was undertaken to analyze the structure of defensive glands in adult and larval stages of L. tristis . | Methods and Materials
A total of 20 freshly collected adult beetles that were sexed based on Vinod et al. ( 2008 ), and 20 fourth and fifth instar larvae, were killed using diethyl ether. Killed adults were pinned to a wax tray, the elytra and terga were removed exposing internal structures. Reproductive and digestive structures and fat reserves were removed to expose defensive glands. After washing in water followed by 70% alcohol, the sixth and seventh sternites with attached glands were separated from the rest of the sternites. The defensive glands were separated from the sterna by cutting along the posterior margin of the seventh sternum.
Killed larvae were pinned on a wax tray and were cut along the mid—dorsal side to expose the glands. The second and fourth sternites with attached glands were separated. Glands from both adults and larvae were dehydrated in graded series of ethyl alcohol, and brought to xylene through alcohol: xylene (1:1) mixture and they were mounted on glass slides in Canada balsam.
Discharge of gland secretions was observed by gently pressing the abdomen of live larvae and adults. The adult or larva was held between the left thumb and index finger, placed on the stage of a stereo zoom microscope (Labomed CZ 70; Labomed India Ltd, http://www.labomed.in ) with the ventral surface of the insect facing up and keeping the posterior end away from the observer. When pressed, usually both larvae and adults discharged secretions and on pressing with modest pressure, glands were extruded in adults alone. Care was taken to immobilize the hind legs of adults with forceps, to avoid rupturing the gland reservoirs as the release of the oily secretion renders further observations difficult. Line drawings of the adult and larval gland were reconstructed from digital images. | Results and discussion
The structure of defensive glands is same in larvae and adults, but the location, alignment, number of glands and pattern of discharge of secretion vary. The gland in adults consists of a pair of small (0.8–0.9 mm), strongly wrinkled conical pouches that open independently; these glands occur parallel to the long axis of the body ( Figure 1a , 2a ). Glands in adults are evaginations of the intersegmental membrane between the seventh and eighth sternites and occur on either side of the hind gut immersed in a thick matrix of fat reserves. The secretion is produced by gland cells that cover the dorsal surface of the reservoirs. The opening of the glands is directed backwards and on pressing the abdomen the glands are everted ( Figure 2a ). In addition, when strongly disturbed beetles rupture the gland by rubbing with hind tarsus, leading to the release of the secretion.
In the larvae, defensive glands consist of a pair of pouches placed in a conical depression on the second and third sternites ( Figure 1b ). Both pair of glands are evaginations of the sternal membrane of the second and third sternites. Each gland opens to the exterior dorsolaterally through a long and narrow channel. Glands are non-eversible and upon disturbance the secretion is discharged without eversion. The secretion is produced by gland cells that are dispersed over the entire dorsal surface of the reservoirs. Glands occur in parallel rows along the segment and at right angles to the long axis of the body. The glands in larvae are visible externally as a pair of conical lateral swellings on either side of midline in second and third abdominal segments ( Figure 2b ). These paired lateral swellings correspond to the structures described as of unknown function by Hayashi ( 1964 ).
Classifying the adult tenebrionid defensive glands, Tschinkel and Doyen ( 1980 ) indicate that Luprops deviates from the typical Lagriine type. The present study shows that the gland is more of Tenebrio type with a pair of conical reservoirs, opening to a common area of discharge and are devoid of exit ducts. | Results and discussion
The structure of defensive glands is same in larvae and adults, but the location, alignment, number of glands and pattern of discharge of secretion vary. The gland in adults consists of a pair of small (0.8–0.9 mm), strongly wrinkled conical pouches that open independently; these glands occur parallel to the long axis of the body ( Figure 1a , 2a ). Glands in adults are evaginations of the intersegmental membrane between the seventh and eighth sternites and occur on either side of the hind gut immersed in a thick matrix of fat reserves. The secretion is produced by gland cells that cover the dorsal surface of the reservoirs. The opening of the glands is directed backwards and on pressing the abdomen the glands are everted ( Figure 2a ). In addition, when strongly disturbed beetles rupture the gland by rubbing with hind tarsus, leading to the release of the secretion.
In the larvae, defensive glands consist of a pair of pouches placed in a conical depression on the second and third sternites ( Figure 1b ). Both pair of glands are evaginations of the sternal membrane of the second and third sternites. Each gland opens to the exterior dorsolaterally through a long and narrow channel. Glands are non-eversible and upon disturbance the secretion is discharged without eversion. The secretion is produced by gland cells that are dispersed over the entire dorsal surface of the reservoirs. Glands occur in parallel rows along the segment and at right angles to the long axis of the body. The glands in larvae are visible externally as a pair of conical lateral swellings on either side of midline in second and third abdominal segments ( Figure 2b ). These paired lateral swellings correspond to the structures described as of unknown function by Hayashi ( 1964 ).
Classifying the adult tenebrionid defensive glands, Tschinkel and Doyen ( 1980 ) indicate that Luprops deviates from the typical Lagriine type. The present study shows that the gland is more of Tenebrio type with a pair of conical reservoirs, opening to a common area of discharge and are devoid of exit ducts. | Invasion by large populations of the litter-dwelling darkling beetle Luprops tristis Fabricius (Coleoptera: Tenebrionidae) following the short spell of summer rains during April, and their extended state of dormancy is a regular event in rubber plantation habitats in south-western India. Strong smelling secretions of the beetle cause blisters on skin of human beings. Such secretions appear defensive because they appear to facilitate their avoidance by other predatory organisms. Defensive glands in the larvae and adults of L. tristis are described, as well as the mode of eversion of the glands. The glands in larvae consist of two pairs of noneversible glands in a conical depression on the 2 nd and 3 rd sternites, whereas in adults only one pair occurs between 7 th and 8 th sternal segments. These glands may be a major reason for avoidance of larvae and adults by their natural enemies and their very high numbers in the litter of rubber plantations.
Key words | Acknowledgements
We thank the Kerala State Council for Science, Technology and Environment (KSCSTE), Government of Kerala for financial assistance. We also thank Wolfgang Schawaller (Staatliches Museum für Naturkunde, Stuttgart, Germany), Otto Merkl (Hungarian Natural History Museum, Budapest, Hungary), Maxwell Barclay (Natural History Museum, London) and Eric G. Matthews (South Australian Museum, Adelaide, Australia) for literature and advice; A. Raman (Charles Sturt University, Australia) and H. F. Nahrung (Department of Primary Industries and Fisheries, Queensland, Australia) for critical review of the article and to Paul Pallickaparambil and Shaji Kallarackal for their kind help during the field studies. | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Feb 25; 10:7 | oa_package/b3/8c/PMC3014652.tar.gz |
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PMC3014653 | 20569140 | Introduction
The sterile insect technique is an important component of many area-wide integrated pest management programs against tephritid fruit flies ( Enkerlin 2005 ). Regardless of the strategic goal of the control program (e.g., eradication, suppression, etc.), the success of the sterile insect technique hinges, to a large extent, on the field performance of the mass-reared, sterilized, and released insects ( Calkins et al. 1994 ). For certain fruit fly species, most notably the Mediterranean fruit fly, Ceratitis capitata , released males must have the capability to locate mating aggregations (leks), synthesize and broadcast attractive pheromones, and perform complex courtship displays involving olfactory, visual, and acoustic signals ( Hendrichs et al. 2002 ; Robinson et al. 2002 ). Unfortunately, processes inherent in mass production, such as genetic drift and intense artificial selection under crowded rearing conditions, may result in the release of sterile male fruit flies that have low sexual competitiveness relative to their fertile, wild counterparts ( Leppla 1989 ; Cayol 2000 ). Such poor field performance may, of course, greatly reduce the cost effectiveness of sterile insect programs.
There exists, therefore, a persistent and pressing need to improve the operating procedures used at fruit fly production and holding facilities in order to enhance the field performance of the released insects. In recent years, considerable research has focused on two potential avenues, both involving the adult stage, for improving the quality of sterile male fruit flies. First, numerous studies ( Ceratitis : see Yuval et al. 2007 for review; Bactrocera : Shelly et al. 2005a ; Anastrepha : Aluja et al. 2001 ) have evaluated the impact of nitrogenous supplements to standard sugar-based diets on male longevity and mating competitiveness. While such supplements often show a positive effect, this result is not universal, and inter-strain and inter-specific differences appear to exist. The second area of research has investigated the effect of chemical treatments - either topical application of juvenile hormone analogs ( Teal et al. 2000 , 2007 ), ingestion of botanical compounds ( Papadopoulos et al. 2001 ; Shelly and Villalobos 2004 ), or exposure to airborne volatiles from such compounds ( Shelly et al. 2007 ) - on the speed of male sexual maturation and/or male mating success. Most of the work regarding the practical application of “chemotherapy” has been limited to two species, C. capitata and the Caribbean fruit fly, Anastrepha suspensa , and the potential usefulness of a chemotherapeutic component in sterile insect programs against other important fruit fly species remains largely uninvestigated.
The use of chemicals, particularly male lures or parapheromones ( Sivinski and Calkins 1986 ; Cunningham 1989 ), to manipulate behavior for management purposes has been well-documented for fruit flies in the genus Bactrocera , most notably B. dorsalis (Hendel) (Diptera: Tephritidae). Long known as a powerful male attractant for certain Bactrocera species ( Howlett 1915 ; Steiner 1952 ), methyl eugenol is widely used to detect incipient infestations of B. dorsalis ( Chambers et al. 1974 ; Jang and Light 1996 ). In addition, when mixed with a toxicant, methyl eugenol was used to eradicate island populations of this species in a procedure termed “male annihilation” ( Steiner et al. 1965 , 1970 ).
Although not frequently used against B. dorsalis , the sterile insect technique may be implemented in those instances where male annihilation alone is ineffective or where environmental concerns preclude widespread use of methyl eugenol, which may attract non-target species and pose a threat to native insect populations. For example, to reduce environmental hazards, Thailand and the International Atomic Energy Agency have implemented an area-wide integrated pest management effort, which involves the release of sterile males to control B. dorsalis (as well as B. correcta ) in mango orchards ( Orankanok et al. 2007 ).
Recent biochemical and behavioral studies indicate that methyl eugenol might be incorporated into such sterile insect programs as a means to increase the mating competiveness of released, sterile males. Methyl eugenol has been shown to function as a precursor for the male sex pheromone in B. dorsalis ( Nishida et al. 1988 , 1997 ) and to increase the attractiveness of the pheromone to females and, correspondingly, male mating success in trials conducted in laboratory cages or individually caged host trees ( Shelly and Dewire 1994 ; Tan and Nishida 1996 ; Shelly and Nishida 2004 ). Like the Mediterranean fruit fly, B. dorsalis appears to exhibit a lek mating system, where males perch on leaf surfaces and emit a pheromone (dispersed by vigorous wing-fanning) to attract receptive females ( Kobayashi et al. 1978 ). Females freely select their mates and, as courtship is absent, the pheromone signal appears critical in affecting male mating success ( Shelly and Kaneshiro 1991 ). Additional studies ( Shelly 1995 ; Shelly et al. 2000) demonstrated that the ingestion of methyl eugenol increased the mating competitiveness of mass-reared, sterile B. dorsalis males (relative to wild males), though these studies were again conducted in small cages in the laboratory. Interestingly, males fed methyl eugenol as larvae did not gain a mating advantage over non-fed males ( Shelly and Nishida 2004 ).
The objective of the present study was to assess the effect of methyl eugenol consumption on the mating performance of sterile B. dorsalis males in more natural conditions. In particular, the level of egg sterility (evidence of successful mating by sterile males) was compared following release of wild flies and either methyl eugenol-fed (treated) sterile males or methyl eugenol-deprived (control) sterile males in large field enclosures. As described below, the enclosures contained multiple host plants and were much larger than the cages placed over single trees. While obviously not a complete substitute for open field tests, the large enclosures provided a more natural setting than the single-tree tents and, just as importantly, allowed for experimental manipulation of over flooding (sterile male: wild male) ratios and for replicated trials at the selected test ratios. | Materials and Methods
Study Insects
Mass-reared B. dorsalis males were from a genetic sexing strain based on a pupal color sexual dimorphism (males = brown; females = white). The strain was developed by McCombs and Saul ( 1995 ) and has been reared at the USDA-ARS laboratory, Honolulu, HI, at levels of 10,000–500,000 pupae per week. Eggs were collected in perforated tubes from large colony cages and placed on a wheat mill feed diet. Approximately 7 d later, the fully developed larvae “popped” into vermiculite, which was sifted 2 d later to collect pupae. To separate the sexes, pupae were passed through a high-speed, photoelectric sorting machine twice, and the male pupae were irradiated at 100 Gy under hypoxia 2 d prior to eclosion with a Gammacell 220 (Nordion Co., Canada) cobalt source.
Following irradiation, flies were held in different sized containers depending on the size of the release. For trials involving lower over flooding ratios (5:1 and 10:1), newly emerged adult males were placed in mesh screen cages (30 cm cubes with a cloth sleeve on one side). For trials involving higher over flooding ratios (30:1 and 60:1), irradiated pupae were placed in paper bags, which, in turn, were placed in plastic adult rearing containers (0.48 × 0.60 × 0.33 m; socalled PARC boxes). Sterile flies were held at 23–27 °C and 60–90% RH, and they received both natural and artificial light with a photoperiod of 12:12 (L:D). Food (a sugar-yeast hydrolysate mixture, 3:1 v:v) was placed in Petri dishes that were placed on the floor of the containers before introduction of the adult (small screen cages) or pupal (PARC boxes) males. In addition, sugar-agar gel and a honey-yeast hydrolysate paste (3:1, v:v) were applied to the top of the screen cages or the screened opening on the top of the storage boxes. For all containers, the sugar-agar gel was replaced every other day, and the food paste was applied daily. At release, the food dishes placed inside the containers invariably contained some residual material, indicating that the flies did not suffer a food shortage.
As described below, 200 wild-like males were released in all trials, which were conducted in two field enclosures concurrently, one involving control males and the other involving treated males. Thus, on any given day, we established equal numbers of containers for control and treated males. For trials involving 5:1 or 10:1 over flooding ratios, 250 sterile males were placed in each of 8 screen cages (4 control, 4 treated) or 16 screen cages (8 control, 8 treated), respectively. Surplus males were also maintained and used to replace dead individuals at the time of release. For trials involving 30:1 or 60:1 over flooding ratios, sterile males were placed in two storage boxes (1 control, 1 treated). For the 30:1 and 60:1 over flooding ratios, 180 and 360 ml of pupae (1 ml ≈ 40 pupae), respectively, were distributed evenly among six bags per storage box. The number of pupae placed in the boxes exceeded the designated over flooding ratio by 20% (≈ 7,200 and 14,400 pupae for 30:1 and 60:1, respectively) to compensate for mortality and developmental abnormalities that prevented flight.
Wild-like flies were from a laboratory colony started with 400–600 adults reared from field-collected guava fruits, Psidium guajava L. (Myrtales: Myrtaceae). The colony was housed in a screen cage (0.6 × 0.6 × 1.2 m) with superabundant food (the sugar-yeast hydrolysate mixture), water, and ripe papayas, Carica papaya L. (Brassicales: Caricaceae) were provided periodically for oviposition. Infested fruits were held over vermiculite for about three weeks, and pupae were then collected by sifting the vermiculite. Adults were separated by sex within 24 h of emergence and were provided the sugar-yeast hydrolysate diet and water. The wild-like flies were held under the same environmental conditions as the sterile males. When tested, the wild-like flies were 3–7 generations removed from the wild.
Exposure to Methyl Eugenol
For all trials, treated sterile males were given access to methyl eugenol 3 d before release, while control sterile males and wild-like males were not supplied with the chemical. Previous work ( Shelly and Dewire 1994 ) showed that consumption of methyl eugenol boosted male mating success over intervals lasting from 1–35 d post-feeding. Methyl eugenol (provided by FarmaTech International, www.farmatech.com ) was applied to cotton wicks resting in aluminum foil-lined Petri dishes (one wick per dish), which were then placed directly in the holding containers. For trials involving the lower over flooding ratios, we applied 0.25 ml of methyl eugenol to a single cotton wick (1 cm diameter, 7.5 cm long), which was then placed in individual screen cages for 1 h. For trials involving the higher over flooding ratios, we applied 1 ml of methyl eugenol to each of three 3 wicks (1 cm diameter; 15 cm long), which were then placed in individual storage boxes for 2 (30:1) or 4 (60:1) h (wicks were introduced and removed through a hole drilled in the side of the storage boxes plugged with a foam stopper). Thus, while the per capita dose of methyl eugenol was lower in the storage boxes than the screen cages, the exposure interval was longer in the storage boxes to increase access to the chemical. Exposure to methyl eugenol was initiated between 0900-1000 hrs and conducted in a room isolated from control males, although prior work ( Shelly and Dewire 1994 ) demonstrated that exposure to the aroma alone (i.e., without ingestion) had no effect on male mating success. When given access to methyl eugenol, the treated males were 6–7 d old (the age at which males of this strain typically attain sexual maturity, TE Shelly, unpublished data).
Methyl Eugenol Consumption and Male Mortality
Steiner ( 1952 ) reported that, given free access to methyl eugenol, B. dorsalis males will “kill themselves with overindulgence.” To determine whether the protocol for pre-release feeding on methyl eugenol influenced male mortality, 30 screen cages (30 cm cubes with cloth sleeve on one side) were set up each containing 20 sterile males (8 d old) and ample food and water. On the same day, methyl eugenol (0.25 ml applied to a cotton wick resting in a Petri dish) was introduced for 1 h to 15 of the cages. Starting the next day, dead males were removed on a daily basis, and after 1 wk, the total number of deaths between cages that did and did not receive methyl eugenol were compared. Cages were held under the environmental conditions described above.
Field Trials
Trials were conducted in four nylon-screen enclosures (16 m long × 6 m wide × 2.5 m high) set up in a guava orchard ( P. guajava ) in Waimanalo, Oahu (elevation 20 m). The tents, which were parallel to one another and separated by 5 m, contained 10–15 guava trees and were covered with shade screen to reduce insolation. As described below, trials lasted 5 d, and treated and control males were tested concurrently (i.e., one treatment per enclosure). Enclosure pairings were the same over the entire study, resulting in a use/nonuse schedule that alternated weekly for each pair of enclosures. Within each pair of enclosures, the type of sterile male released in a given enclosure (i.e. control or treated) was alternated between successive replicates. Trials were conducted between September 2005 and April 2006. Daily minimum and maximum air temperatures were recorded over the entire study at a weather station situated ca. 100 m from the enclosures.
The same schedule was followed for all trials. On day 1, food and water were introduced, and the flies were released. Food (the sugar-yeast hydrolysate mixture) was presented in Petri dishes held in Jackson traps (lacking sticky inserts) suspended 1.5–2.5 m above ground from tree branches at four evenly spaced locations. At each of these sites, water was provided in a covered plastic cup (100 ml volume with an emerging cotton wick) held within a Jackson trap. The wires suspending the resource-laden Jackson traps from branches were coated with Tanglefoot® (Tanglefoot Company, www.tanglefoot.com ) to exclude ants. Food and water were not replaced during a trial. Following the placement of food and water, the flies were released in the center of the enclosures. All releases were performed between 1000 and 1100 hrs. Mating activity in B. dorsalis is restricted to a narrow time window immediately preceding and following sunset ( Roan et al. 1954 , Arakaki et al. 1984 ).
On day 4, 15 Granny Smith apples, Malus domestica (Borkh) (Rosales: Rosaceae), were placed in the enclosures at 1000 hrs for oviposition. Apples were suspended 1.5–2.5 m above ground by piercing the fruit with a nail and connecting the nail to a branch with wire. In addition, to simulate wounds and facilitate oviposition, 10–15 shallow holes were made in each apple using a toothpick. Tanglefoot® was applied to the wire to exclude ants. The apples served as the only available oviposition resource as the guava fruits were removed before the trials. On day 5, the apples were collected, marking the end of the trial (food and water were also removed at this time).
For each trial, the egg hatch of wild-like females mated exclusively to wild-like males in a field cage over a single guava tree adjacent to the large enclosures was also measured. Two hundred individuals of each sex were introduced on day 1, and two apples were introduced on day 2 for a 24 h period.
Upon collection, apples were returned to the laboratory, and eggs were removed using a scalpel and a fine forceps under a dissecting microscope. Eggs were placed on moistened blotter paper in covered Petri dishes and then incubated at 27 °C for 72 h. Hatch was then determined by re-examining the eggs under a dissecting microscope.
Seven replicates were conducted for each over flooding ratio, where a replicate consisted of treated and control males placed concurrently, but separately, in the large enclosures (with associated wild-like flies) and wild-like flies exclusively placed in the single, caged guava tree. At the end of a trial, several Jackson traps baited with food and containing sticky inserts were suspended in the guava trees and left in place until the next trial to remove the remaining, previously released flies.
Fried's Competitiveness Index (C)
For each replicate, Fried's ( 1971 ) competitiveness index (C) was computed to compare the performance of control and treated sterile males vs. wild-like males, where C = (W/S) × [(H w - H c )/(H c - H s )], with W = number of wild-like males released, S = number of sterile males released, Hw = percentage of egg hatch from wild-like females following mating with wild-like males (as determined from the single caged guava tree), Hc = percentage of egg hatch from wild-like females in the test enclosure, and Hs = percentage of egg hatch from wild-like females following mating with sterile males (= 0.2%, DOM, unpublished data).
Daily Fecundity
To estimate the number of females depositing eggs in the apples provided in the field enclosures, daily egg production was estimated in the laboratory. Using wild-like individuals exclusively, mating pairs were collected from Plexiglas cages (30 × 30 × 40 cm) and, the following morning, the mated females were transferred to a screen cage containing food and water but no oviposition substrate. Two days after mating, females were placed singly in screen cages (30 cm cubes) along with an apple (containing puncture wounds as described above) at 0800 hrs and removed at 1800 hrs. Apples were kept in a refrigerator (10 – 12 °C) overnight, and egg counts were made the following morning. Because trials did not span the entire dawn-dusk period (approximately 12 h), the estimates of daily fecundity are likely conservative.
Statistical Analysis
Male mortality in cages that did or did not receive methyl eugenol was compared using a t-test. Values for total egg abundance (normalized using a log 10 transformation), egg sterility (arc sine transformed proportions of unhatched eggs/total eggs), and Fried's Competitiveness Index (arc sine transformed indices) were analyzed using two-way ANOVA with over flooding ratio and male treatment category (treated or control) as the factors. When significant variation was detected, the Tukey multiple comparisons test (critical value q) was used to identify pair wise differences among groups (test statistic q, with df = 48 [ANOVA error df] and k [number of groups] = 8 for all comparisons). Mean values ± 1 SE are presented.
Although daily maximum temperatures varied significantly among the time intervals when the four over flooding ratios were investigated (H = 71.9, df = 3, P < 0.001, Kruskal-Wallis test), temperature was excluded as a factor in the analyses for two reasons. First, the temperature differences, while consistent, were quite small. Among the study periods, the mean daily maximum temperatures ranged only between 25.7 – 29.1 °C. In addition, no significant correlation was found between the mean daily maximum temperature during a replicate and (i) the total number of eggs collected, (ii) the total number of apples containing eggs, or (iii) the proportion of sterile eggs when considering data from treated and control groups separately or collectively within or between over flooding ratios (P > 0.05 in all cases, Spearman rank correlation). | Results
Methyl Eugenol Consumption and Male Mortality
There was no significant difference in the mean number of male deaths recorded for cages that did (2.1 ± 0.4) or did not (2.2 ± 0.4) receive methyl eugenol (t = 0.1, df = 28, P = 0.91).
Total Egg Production
The total number of eggs collected per replicate varied independently of over flooding ratio (F 3, 48 = 1.34, P = 0.27) and male treatment category (F 1, 48 = 1.00, P = 0.32). The interaction term between main effects was also not significant (F 3, 48 = 0.66, P = 0.58). Including all combinations of over flooding ratio and sterile male treatment category, 599–705 total eggs were collected, on average, per replicate ( Fig. 1 ). On average, eggs were found in 8.9 (of a possible 15) apples per replicate (± 0.3, range: 5–11, n = 56).
Daily Fecundity
Egg production was monitored in the laboratory for 41 females over a 10-h period. Approximately 30% (12/41) of the females laid no eggs at all, while the remaining females laid between 7–64 eggs with an average output of 26.2 eggs (± 3.3; n = 29). Based on this average daily output and the average total numbers of eggs collected per replicate, it was estimated that, on average, 23–27 females deposited eggs in the apples during a given replicate.
Egg Sterility and Fried's Competitiveness Index
Levels of egg sterility varied significantly with over flooding ratio (F 3, 48 = 3.8, P = 0.02) and male treatment category (F 1, 48 = 12.8, P < 0.001; Fig. 2 ). The interaction term was not significant (F 3, 48 = 0.7, P = 0.55). The effect of over flooding ratio on egg sterility levels differed between treated and contol males. For treated males, egg sterility levels were fairly consistent across the different over flooding ratios, with average levels varying only between 73%–80%, and correspondingly, there were no significant differences in pair-wise comparisons (P > 0.05 in all cases). In contrast, for control males egg sterility varied markedly with over flooding ratio, increasing from 55% at the 5:1 over flooding ratio to 77% at the 60:1. For control males, significant differences in egg sterility were detected between the 60:1 over flooding ratio and the 5:1 (q = 4.2, P = 0.02) and 10:1 (q = 3.9, P = 0.04) over flooding ratios, respectively. For control males, the average sterility level observed at the 60:1 ratio was higher than that noted at the 30:1 ratio (77% versus 66%, respectively), but this difference was not significant (q = 3.4, P = 0.09).
Consistent with these trends, differences in egg sterility between treated and control males were more pronounced at lower rather than higher over flooding ratios. Egg sterility levels were approximately 33% greater for treated than control males at both the 5:1 over flooding ratio (73% versus 55%, q = 4.6, P = 0.04) and the 10:1 over flooding ratio (75% versus 57%, q = 4.8, P = 0.03). In contrast, the egg sterility values were only slightly higher for treated males at the higher over flooding ratios, and these differences were not statistically significant (30:1: 70% versus 66%, q = 2.4, P = 0.70; 60:1: 80% versus 77%, q = 2.1, P = 0.81).
As with egg sterility levels, values of Fried's Competitiveness Index (C) varied significantly with over flooding ratio (F 3, 48 = 5.5, P = 0.002) and male treatment category (F 1, 48 = 10.1, P = 0.003; Fig. 3 ). The interaction term was not significant (F 3, 48 = 1.3, P = 0.28). Competitiveness values reflected the relationships described above between over flooding ratio and egg sterility ( Fig. 3 ). For treated males, the relatively constant level of egg sterility recorded over the different over flooding ratios resulted in decreasing C values with increasing over flooding ratio: the C value for the 5:1 over flooding ratio was significantly different from C values obtained at 30:1 (q = 5.1, P = 0.02) and 60:1 (q = 6.5, P < 0.001). No significant differences were detected between C values for the 5:1 and 10:1 over flooding ratios (q = 3.8, P = 0.15) or among C values recorded for over flooding ratios ≥ 10:1 (P > 0.05 in all cases). A similar decrease in competitiveness values was observed for control males with increasing over flooding ratios, but there were no significant differences detected among C values for the different over flooding ratios (P > 0.05 in all cases). This result derived from the increased egg sterility noted at the higher over flooding ratios ( Fig. 2 ), which counterbalanced the effect of increasing over flooding ratio in the computation of C values.
Reflecting the convergence in egg sterility levels with increasing over flooding ratio ( Fig. 2 ), competitiveness values for treated sterile and control sterile males differed significantly only at the lowest (5:1) over flooding ratio (q = 6.9, P < 0.001). Competitiveness indices were greater for treated sterile males than control sterile males at all higher over flooding ratios as well, but these differences were not statistically significant (P > 0.05 in all cases). | Discussion
Pre-release feeding on methyl eugenol by sterile males of B. dorsalis resulted in increased levels of egg sterility at all over flooding ratios tested, although significant differences between methyl eugenol-fed and methyl eugenol-deprived sterile males were observed only for the lower ratios. Competitiveness indices computed for treated and control sterile males displayed the same trend. The level of egg sterility achieved by treated males at the lowest over flooding ratio tested (5:1) was similar to that observed for treated or control males at the highest ratios tested (30:1 or 60:1), indicating that pre-release feeding on methyl eugenol could allow reduction in the number of sterile males produced and released in control programs.
Although these data suggest a benefit to pre-release feeding on methyl eugenol, several factors potentially confound this interpretation. First, as control programs may release sterile males on a weekly or bi-weekly basis, these tests (lasting only 5 d) may not have detected any decrease over time in the mating competitiveness of sterile males, due to a possible “wearing off of the methyl eugenol-mediated mating enhancement. As noted above, a laboratory study ( Shelly and Dewire 1994 ) showed increased mating performance for as long as 35 d after consumption of methyl eugenol, but the dose used was extremely high (1.5 ml of methyl eugenol per 40–60 males for 2 h), and it is not known if the doses employed in the current study confer a similar long-lasting benefit. Second, although no adverse effect of methyl eugenol feeding on male survival was found, these trials were conducted in the laboratory, and their applicability to the field is unknown. However, these data were important in revealing that there is no immediate toxic consequence of methyl eugenol ingestion.
In addition, although the large enclosures provided a semi-natural environment, the experiments nonetheless precluded dispersal as an influence on male mating competitiveness. If, for some reason, ingestion of methyl eugenol impairs flight propensity or ability, then treated sterile males may be less likely to locate preferred habitat, including optimal mating (lek) sites. Previous work ( Shelly 1994 ) showed that methyl eugenol-fed males of B. dorsalis , which were subsequently marked and released in the field, were, in fact, less likely to be captured in methyl eugenol-baited traps than were methyl eugenol-deprived males. However, rather than indicating heightened mortality associated with methyl eugneol ingestion, the decreased trap capture apparently reflected decreased responsiveness of once-fed males to methyl eugenol. In other words, assuming dispersal ability is not reduced, pre-release exposure to methyl eugenol may serve to reduce the attraction of sterile male to methyl eugenol-baited traps, allowing concurrent implementation of the sterile insect technique and male annihilation.
As a final consideration, the fact that the wild-like males were not given methyl eugenol in any of the trials implies that the mating competitiveness of treated sterile males was overestimated. In the field, wild males will, of course, seek out sources of methyl eugenol and thereby enhance their mating ability. Thus, denying the wild-like males access to this chemical essentially “handicapped” them relative to treated sterile males. Despite this bias, pre-release exposure to methyl eugenol should prove beneficial, because it eliminates the “need” for sterile males to locate natural sources of the chemical in the environment (thereby eliminating time and energy costs associated with searching) and guarantees that sterile males gain the full benefits derived from methyl eugenol ingestion.
The present results were comparable to those obtained previously for the Mediterranean fruit fly. In that species, laboratory ( Papadopoulos et al. 2006 ) and field-cage trials ( Shelly et al. 2004 ) showed that sterile males exposed to the aroma of ginger root oil ( Zingiber officinale ) competed more successfully against wild males for matings with wild females than non-exposed sterile males. A subsequent experiment ( Shelly et al. 2005b ) using a similar protocol and the same large enclosures described in the present study showed that levels of egg sterility were significantly higher for ginger root oil-exposed sterile males than non-exposed sterile males. As with methyl eugenol, exposure to ginger root oil had its greatest impact at the lowest over flooding ratio tested, and the magnitude of the effect was similar between the two substances. At the 5:1 over flooding ratio, the level of egg sterility achieved by methyl eugenol-fed, sterile males of B. dorsalis was 33% greater than that observed for non-fed, sterile males (73% versus 55%, respectively), and in C. capitata egg sterility associated with ginger root oil-exposed, sterile males was 28% higher than that observed for non-exposed, sterile males (86% versus 67%, respectively). In contrast, at a 60:1 over flooding ratio, the proportional increase in egg sterility observed for treated, sterile males over control, sterile males was only 4% for B. dorsalis (80% versus 77%, respectively) and 7% for C. capitata (96% versus 90%, respectively). Although the trends were similar between species, exposure to ginger root oil had a more pronounced effect than feeding on methyl eugenol: levels of egg sterility and Fried's competitiveness index were significantly higher for treated than control males of C. capitata at all over flooding ratios tested, not only at lower over flooding ratios as observed in B. dorsalis .
Despite this difference, data from both species revealed the same underlying trend: treated sterile males released at low over flooding ratios achieved the same levels of egg sterility as control males released at high over flooding ratios. As noted above, this finding implies that pre-release treatment might increase the cost-effectiveness of the sterile insect technique by reducing the quantity of sterile flies required to achieve programmatic goals. Based on the protocol used for exposing sterile males at the 60:1 over flooding ratio (3 ml of methyl eugenol per 14,400 flies), pre-release feeding would cost approximately $5 per million males (at $22 per liter of methyl eugenol, J. Knapp, personal communication). Assuming production costs for B. dorsalis are similar to those for C. capitata ($100–$200 per million male pupae), methyl eugenol treatment would add only 2–5% to the production cost of the flies. Although data from open field releases are still lacking, the present findings strongly suggest that the increased mating competitiveness of sterile males justifies the small additional cost of implementing pre-release feeding on methyl eugenol. | The sterile insect technique may be implemented to control populations of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), when environmental concerns preclude widespread use of chemical attractants or toxicants. The goal of the present study was to evaluate whether the mating competitiveness of sterile B. dorsalis males could be increased via pre-release feeding on methyl eugenol. Males of the oriental fruit fly are strongly attracted to this plant-borne compound, which they ingest and use in the synthesis of the sex pheromone. Previous studies conducted in the laboratory and small field-cages have shown that males given methyl eugenol produce a more attractive pheromone for females and have a higher mating success rate than males denied methyl eugenol. Here, levels of egg sterility were compared following the release of wild-like flies and either methyl eugenol-fed (treated) or methyl eugenol-deprived (control) sterile males in large field enclosures at four over flooding ratios ranging from 5:1 to 60:1 (sterile: wild-like males). Treated sterile males were fed methyl eugenol for 1–4 h (depending on the over flooding ratio tested) 3 d prior to release. Eggs were dissected from introduced fruits (apples), incubated in the laboratory, and scored for hatch rate. The effect of methyl eugenol was most pronounced at lower over flooding ratios. At the 5:1 and 10:1 over flooding ratios, the level of egg sterility observed for treated, sterile males was significantly greater than that observed for control, sterile males. In addition, the incidence of egg sterility reported for treated sterile males at these lower over flooding ratios was similar to that noted for treated or control sterile males at the 30:1 or 60:1 over flooding ratios. This latter result, in particular, suggests that pre-release feeding on methyl eugenol allows for a reduction in the number of sterile flies that are produced and released, thus increasing the cost-effectiveness of the sterile insect technique.
Keywords | Acknowledgements
We thank Roger Corrales for permission to use the Waimanalo facility and for the weather data, John Knapp of FarmaTech International for supplying methyl eugenol and cost information, and Rick Kurashima for supplying sterile flies. | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Feb 27; 10:8 | oa_package/f3/ff/PMC3014653.tar.gz |
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PMC3014654 | 20575741 | Introduction
In nature, some species live together in the same or in similar niches because they have one or several kinds of similar behaviors, thus one species often conflicts with the other one for food resources. Interspecific competition is defined as a reduction in individual fecundity, survival, or growth as a result of exploitation of resources or by interference with individuals of another species ( Begon et al. 1996 ). A superior species may exclude an inferior one from parts of its fundamental niche or, in extreme cases, drive it to extinction for occupying spatial vertical resources or food resources ( Wissinger 1992 ). Thus, some species often are in an inferior position when competition occurs among various species. The phenomenon is more obvious when food resources are in short supply.
According to the conventional niche theory, the primary determinant of interspecific competition is the overlap and the similarity of the resources used (niche overlap or niche similarity). Two species with highly similar fundamental niches (i.e. the niches potentially occupied in the absence of competitors) will often compete strongly with each other when they first meet ( Duyck et al. 2004 ). This was also the conclusion reached by Shorrocks ( 1991 ), who found that two species of Drosophila coexisted in a spatially divided system, where the inferior species was eliminated in an undivided system.
Recent reviews have shown that interspecific competition is widespread among insects ( Denno et al. 1995 ; Stewart 1996 ; Reitz and Trumble 2002 ). Competition is a universal phenomenon in nature, and in particular, among natural enemies. Natural enemies may conflict for occupying scope and prey, and a superior species of natural enemy may drive an inferior one away, thus limiting the population of the inferior species. Predators may kill their adversaries when they have conflict over spatial resources and food. In general, the adults of parasitoid wasps cannot kill their competitors, but they can drive competitors away for inhabiting advantageous niches and having access to more food ( Bajpai et al. 2005 ). Some larvae of parasitoid wasps can, however, attack the larvae of their adversaries when two species parasitize the same host larvae ( Vinson and Iwantsch 1980 ; Mackauer 1990 ; Tian et al. 2008 ).
In conventional agroecosystems, pest outbreaks are common because these simplified systems are species poor ( Pimentel 1961 ), and agricultural pest suppression has been identified as an important ecosystem service that may be threatened by the loss of natural enemy biodiversity ( Kruess and Tscharntke 1994 ; Wilby and Thomas 2002 ). These ideas suggest that managing for greater natural enemy biodiversity may improve pest suppression ( Cardinale et al. 2003 ; Aquilino et al. 2005 ). Generally, biological control is rarely in conflict with biodiversity in agroecosystems, and thus biological control has been noticed by many researchers ( Geoff and Steve 2000 ). Microplitis prodeniae Rao and Chandry (Hymenoptera: Bracondidae) and Campoletis chlorideae Uchida (Hymenoptera: Ichnumonidae) are both major parasitoid wasps in tobacco fields. In general, both M. prodeniae and C. chlorideae prefer to parasitize the young instar larvae of hosts, and the two parasitoids emerge from the body of the host when the host larvae is in the third instar. Thus they play an important role in the control of the oriental leafworm, Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae) ( Dang and Hung 1999 ; Liu et al. 2004 ; Bajpai et al. 2005 ), and regarded as the two species of candidate biological control agents for controlling the population of S. litura ( Dang and Hung 1999 ; Bajpai et al. 2005 ). The biology and ecology of C. chlorideae has been extensively researched ( Kumar et al. 2000 ; Murugan et al. 2000 ; Pandey et al. 2004 ; Yang et al. 2005 ; Yan and Wang 2006 ; Zhang et al. 2006 ), but a small number of studies have been probed into its biology and ecology since M. prodeniae was recorded as a parasitoid of S. litura larvae. In the search for alternatives to chemical insecticides, the utilization of these two species for protecting crop plants from S. litura has been assigned more importance in the recent decade ( Dang and Hung 1999 ; Bajpai et al. 2005 ). However, most entomologists, ecologists and biologists have chosen only one of the two species as the target of study, and they have not probed into the ecological relationships between them, especially their niches and competition relationships. Therefore, we combined M. prodeniae with C. chlorideae and regard them as one entity. The objective of this study was to understand the ecological relationships of M. prodeniae and C. chlorideae in the field, and the results of this study provide a basis for the utilization of these two species as pest control for S. litura . | Materials and Methods
Study sites
This study was conducted in the tobacco field at the experimental farm of Nanxiong Research Institute of Tobacco, Nanxiong, Guangdong Province. There are 300 ha tobacco, Nicotiana tabacum L. (Solanales: Solanaceae), fields, and S. litura (Fabricious), Helicoverpa assulta Guenée and Helicoverpa armigera Hübner often occur together in tobacco fields in this research station. Several fields, where serious damage was caused by these three species, were taken as the target fields of investigation. The acreage of each of these experimental fields was about 6670 m 2 .
Tobacco variety
N. tabacum 9601 variety was planted on 20 February 2006, and the density was 1.7–1.8 plants / m 2 .
Spatial niches and temporal niches of M. prodeniae and C. chlorideae
Investigations were conducted from 10 May to 3 June in 2006 at the experimental farm of Nanxiong Research Institute of Tobacco. Spatial vertical resources in tobacco plants were divided into three even grades containing upper leaves, mid-leaves and underneath leaves, and temporal resources were divided into five even grades by investigation times (i.e. 10 May, 16 May, 22 May, 28 May and 3 June). Investigations took place once every five days. Second instar larvae of S. litura on the different positions of tobacco plants ( n = 80) were collected by “random sample” every time, and the living larvae from the different positions were taken back to the laboratory. Then they were bred in separate cages, respectively, and fed fresh tobacco leaves every day. The parasitic rates of M. prodeniae and C. chlorideae on S. litura larvae from different positions were recorded when the parasitoid adults emerged from the bodies of S. litura larvae.
Host resources of M. prodeniae and C. chlorideae
Investigations were conducted from 8 May to 19 June in 2006 at the experimental farm of Nanxiong Research Institute of Tobacco. Trophic resources were divided into three grades (i.e. S. litura , H. assulta and H. armigera ). Investigation was carried out once every five days. Second instar larvae from S. litura , H. assulta and H. armigera ( n = 80) were collected by “random sample” every time. The living larvae of these three species were taken back laboratory, and they were bred in different cages. Fresh tobacco leaves were given to them every day, and the parasitic rates of M. prodeniae and C. chlorideae on the larvae from these three species were recorded when the parasitoid adults emerged from the bodies of the larvae of these three species.
Statistical Analyses
Regarding the ecological relationships of M. prodeniae and C. chlorideae , the niches and interspecific competition coefficients of the two species were compared. The ability of M. prodeniae and C. chlorideae to utilize resources was estimated by their niche breadths, by competition occurring between the two species, by niche overlap and niche similarity, and by the competitive degree of the two species to resources based on an interspecific competition coefficient. When the ability of a species to utilize resources is enhanced with the increase of the breadth value of its niche, competition may occur between it and another species when niche similarity proportion between the two species reveals a high value, and competitive degree between them strengthened with an increasing interspecific competition coefficient between the two species. The following formulae are recognized by ecologists, and they are applied for the evaluation of the interrelation between two species.
Niche breadth was calculated by B = 1 / ( s ). In this formula, B is niche breadth of species, S is the number of resource grades and P i is the fraction of all resources that belong to i -th grade resource used by the species ( May 1975 ). Thus, with this formula, P i is the fraction of all spatial resources which belong to upper leaves or mid-leaves or underneath leaves occupied by M. prodeniae or C. chlorideae for the spatial niche calculation, P i is the fraction of all temporal resources which belong to i -th survey date used by M. prodeniae or C. chlorideae for the temporal niche calculation, and P i is the fraction of all host resources which belong to S. litura , H. assulta , or H. armigera parasitized by M. prodeniae or C. chlorideae for the trophic niche calculation.
Niche overlap and the proportion similarity of the niche were measured by ( B i ) and , respectively, where L ij is the nicrie overlap index value and species j is overlapped by species I ; C ij is the proportion similarity of the niches of species i and species j , P ih and P jh are the fractions of total resources which belong to h -th grade resource used by species i and by species j , respectively; and B i is the niche breadth of species i ( Levins 1968 ; Cowll and Futuyma 1971 ). In this survey study, P ih and P jh are the fractions of total spatial resources which belong to h -th grade spatial resource occupied by M. prodeniae and by C. chlorideae , respectively, when the spatial niche overlap was calculated; P ih and P jh are the fractions of total temporal resources which belong to h -th survey date used by M. prodeniae and by C. chlorideae , respectively, when the temporal niche overlap was calculated; and P ih and P jh are the fractions of total host resources which belong to h -th grade host resources parasitized by M. prodeniae and C. chlorideae when the trophic niche overlap was calculated with these two formulas.
Temporal and spatial two-dimensional niche was measured by provided “multidimensional niche models” ( May 1975 ). He suggested that the indices of the multidimensional niche be multiplied by the index of individual niche. Niche breadth value, niche overlap value and the proportion similarity of temporal and spatial two-dimensional niches were multiplied each by its individual counterpoint.
The interspecific competition coefficient was calculated by , where a is the interspecific competition coefficient and where P i and P j are the fractions of all resources which are used by species i and species j , respectively ( May 1975 ). So P i and P j are the fraction of all spatial resources which are used by M. prodeniae and by C. chlorideae , respectively, when interspecific competition coefficient of spatial niche was calculated; and P i and P j are the fraction of all host resources which are parasitized by M. prodeniae and by C. chlorideae , respectively, when interspecific competition coefficient of trophic niche was calculated with this formula. The competition between species intensified if the interspecific competition coefficient increased.
A number of studies reveal that the intensity of competition between two species is not proportional to the niche overlap ( Cowll and Futuyma 1971 ; Zhou et al. 2000 ; Qian et al. 2006 ), and the intensity of competition actually varied inversely with the niche overlap in a few cases ( Qian et al. 2006 ). Two species with highly similar fundamental niches will often compete strongly with each other when they meet ( Duyck et al. 2004 ). So it was assumed that competition between two different species would happen if the niche overlap index, the proportion similarity of niche and the interspecific competition coefficient are all high, or the niche overlap index is low, but the proportion similarity of niche and interspecific competition coefficient are still high. | Results
Temporal niches and spatial niches of M. prodeniae and C. chlorideae
The spatial niche breadth value of C. chlorideae was slightly higher than that of M. prodeniae , and the spatial niche overlap value of C. chlorideae to M. prodeniae was close to that of M. prodeniae to C. chlorideae . The spatial niche proportion similarity between the two species was 0.7855. These results revealed that the active scope of C. chlorideae was more extensive than that of M. prodeniae , and the vertical distributions of the two species on spatial resources were similar. The temporal niche overlap value of C. chlorideae to M. prodeniae was significantly close to that of M. prodeniae to C. chlorideae . The temporal niche proportion similarity between them was 0.9850. The results suggested that M. prodeniae and C. chlorideae may occur together in tobacco fields at the same time. Temporal and spatial two-dimensional niche breadth of C. chlorideae revealed a relatively higher value compared with M. prodeniae , and the two species have a higher value in the two-dimensional niche proportion similarity. The interspecific competition coefficient between the two species was 0.8973, suggesting that severe competition happened between the two species because they each attempt to occupy more advantageous spatial vertical resources than the other ( Table 1 ). In addition, the results of this survey reveal that the coexistence ratios of the two parasitoids on the three different positions on tobacco plants were higher than the individual existence ratios on them ( Figure 1 ), which confirmed the measurement of spatial niche proportion similarity between the two parasitoids in the tobacco fields.
The spatial niche breadth of M. prodeniae decreased with growing of tobacco, except in the case of the last observation (3 June). This was the same as for C. chlorideae , but exceptions were noticed on 3 June and 16 May. In addition, the spatial niche breadths of M. prodeniae were lower than those of C. chlorideae, but an exception was observed on 10 May. The results indicated that the active scopes of the two species in the early growth stages of tobacco were more extensive than those in the later growth stages of tobacco, and the active scope of M. prodeniae was smaller than that of C. chlorideae . The spatial niche overlap values of M. prodeniae were lower than those of C. chlorideae , suggesting that M. prodeniae was always a passive species when the spatial niche overlap occurred between the two species. In general, both the spatial niche proportion similarity and the interspecific competition coefficient between the two species were high during these experiments, revealing that the competition often happened between the two species when they occupied the same spatial resources in different stages. The spatial niche proportion similarity and interspecific competition coefficient between the two species were higher in the early growth stages than in the late growth stages of tobacco. The results show that severe competition between the two species happened in the early growth stages of tobacco ( Table 1 ).
Trophic niche of M. prodeniae and C. chlorideae
The trophic niche breadth of M. prodeniae was only 0.3333, but that of C. chlorideae was 0.8667, suggesting that the host range of M. prodeniae was narrower than that of C. chlorideae ( Table 2 ). At the same time, the results of the survey also revealed that the parasitic rates of M. prodeniae on S. litura were 13.75 % – 48.75 %, and the parasitic rate of M. prodeniae on H. assulta and H. armigera were 0; however, the three lepidopterous host pests could be parasitized by C. chlorideae ( Table 3 ), which is in accordance with the trophic niche breadths of the two species. The trophic niche overlap value of M. prodeniae was lower than that of C. chlorideae , and the results show that M. prodeniae existed in an inferior position when trophic niche overlap occurred between the two species. The trophic niche proportion similarity and the interspecific competition coefficient between the two species were 0.4054 and 0.6537, respectively. The results suggest that competition, to a certain extent, may happen between the two species when they compete for each other's food resources ( Table 2 ).
The trophic niche breadth of C. chlorideae revealed a higher value than M. prodeniae in different stages, and the results indicate that the host range of C. chlorideae was wider than that of M. prodeniae in the tobacco fields. The trophic niche overlap values of C. chlorideae were superior to those of M. prodeniae in different stages, suggesting that C. chlorideae was dominant when the two species competed for food resources. The two species had greater trophic niche proportion similarity and the interspecific competition coefficients on 1 June and 7 June, respectively, indicating that severe competition happened between the two species over food resources during the late growth stages of tobacco compared with the early growth stages of tobacco in the tobacco fields ( Table 2 ). | Discussion
The parasitoid wasps are some of the most useful natural enemies, and many species have been used in biological control ( van Lenteren et al. 1997 ). Both M. prodeniae and C. chlorideae are major parasitoids in the tobacco fields, and they were found to be significantly effective in reducing the natural population of S. litura ( Dang and Hung 1999 ; Liu et al. 2004 ; Bajpai et al. 2005 ).
Those species that live in the same ecological spatial resource often conflict for occupying more food resources, especially when food resources are in short supply ( Wissinger 1992 ; Denno et al. 1995 ; Stewart 1996 ; Reitz and Trumble 2002 ). In order to define the relationships between M. prodeniae and C. chlorideae , and understand whether competition between the two species occurs, niche and competition relationships of the two species were studied. The results showed that when the time and space in which the two species occurred was very similar, severe competition occurred between the two species for spatial resources in different stages, and that M. prodeniae was the passive species when the two competed for spatial resources. Zhou et al. ( 2006 ) considered that the spatial competition between two species was in proportion to the coexistence ratios of the two species on different positions of crop plants, but the intensity of competition may vary inversely with the individual existence ratios of the species. Because the coexistence ratios of the two species of parasitoids were dominant in tobacco plants, spatial competition between them would happen in tobacco fields.
In addition, the host range of C. chlorideae was wider than that of M. prodeniae , and competition would occur between the two species for occupying food resources. In particular, interspecific competition between the two species of parasitoids at the late stages was very severe. The populations of two parasitoids were enhanced with the increasing densities of their hosts ( Zhou et al. 2007 ). The host species, S. litura , maintained a high population ( Zhou et al. 2006 ), and they were concentrated within a certain of the tobacco plant when the yellow leaves were reaped from the bottom up during the late stages in the tobacco fields. As a result, the two parasitic species were also more concentrated during the late stages than those at the early stages of tobacco plant growth. This phenomenon where the population of the parasitoids is enhanced by the increasing densities of their hosts has been confirmed by previous studies ( Cardinale et al. 2003 ; Cronin 2003 ; Garcia-Medel et al. 2007 ). Consequently, these results suggest that the intensity of competition between the two parasitoids will change when the densities and distributions of their hosts vary, and the increasing competition between the two parasitoids can be ascribed to the concentricity of their hosts in tobacco plants.
From these conclusions, it is suggested that the two species often conflict over spatial resources and food resources and that the competitive abilities of C. chlorideae to control spatial and food resources were better than those of M. prodeniae. Thus C. chlorideae may drive M. prodeniae away when it uses the same spatial or food resource. The competitive ability of C. chlorideae may be more greater when compared with M. prodeniae because the body size of a C. chlorideae adult (5.2 – 5.4 mm) is larger than that of an M. prodeniae adult (3.2–3.5 mm). Tian et al. ( 2008 ) reported that first instars of Microplitis mediator could physically attack the larvae of C. chlorideae because when host larvae were parasitized simultaneously by M. mediator and C. chlorideae , the majority of the cocoons produced were of M. mediator. However, whether the larvae of M. prodeniae could be physically attacked by the larvae of C. chlorideae , has yet to be determined.
According to the trophic niche breadths of the two species, the host range of C. chlorideae was wider than that of M. prodeniae. In general, M. prodeniae could only parasitize S. litura larvae, but C. chlorideae could parasitize the larvae of three major lepidopterous pests, S. litura , H. assulta and H. armigera in the tobacco fields ( Kumar et al. 2000 ; Murugan et al. 2000 ; Liu et al. 2004 ; Pandey et al. 2004 ; Yang et al. 2005 ; Yan and Wang 2006 ; Zhang et al. 2006 ). Consequently, the adaptability of C. chlorideae to the environment in tobacco fields was better than that of M. prodeniae , and C. chlorideae retained a higher population compared with M. prodeniae . The results of these experiments suggest that the control of insect pests with C. chlorideae is feasible when S. litura , H. assulta and H. armigera occur together in the fields. | Associate Editor: T. X. Liu was editor of this paper
Both Microplitis prodeniae Rao and Chandry (Hymenoptera: Bracondidae) and Campoletis chlorideae Uchida (Hymenoptera: Ichnumonidae) are major parasitoids of Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae) in tobacco, Nicotiana tabacum L. (Solanales: Solanaceae) at Nanxiong, Guangdong Province, South China. The niches and interspecific competition relationships of the two species were studied. The results show that the competition between the two species for spatial and food resources was very intense, and C. chlorideae was always dominant when the two species compete for spatial and food resources in different periods. Thus C. chlorideae may drive M. prodeniae away when they occupy the same spatial or food resource. The adaptability of C. chlorideae to the environment in the tobacco fields may be greater than that of M. prodeniae , so C. chlorideae can maintain a higher population compared to that of M. prodeniae .
Keywords | Acknowledgements
We would like to thank Nanxiong Tobacco Research Institute of Guangdong for their support and help. This research was funded by the Project of Guangdong Company of Tobacco. We are grateful to Professor Ruiyu He and Professor Foster Ken, South China Agricultural University, for revising the English of this manuscript. | CC BY | no | 2022-01-12 16:13:43 | J Insect Sci. 2010 Mar 2; 10:10 | oa_package/61/5a/PMC3014654.tar.gz |
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PMC3014655 | 20569141 | Introduction
Herbivores can have both direct and indirect negative impacts on all aspects of plant growth, reproduction and productivity ( Whitham et al. 1991 ). Aphids can reduce crop yield through direct consumption, aesthetic damage and virus transmission.
In agricultural systems, the relationships between different trophic levels are disturbed by the effect of agricultural practices such as the use of insecticides and fertilizers that diminish the diversity and structural complexity of the agroecosystem ( Moreau et al. 2006 ). Thus, the structure of herbivore communities and their natural enemies within the same crop may differ according to different agronomic practices. Bottom up and top down forces drive population dynamics ( Walker and Jones 2001 ). In the first case, for example it has been documented that plant nutritional quality influences insect hervivores ( Perrenoud 1990 ) and many of them such phloem feeding aphids and others, are sensitive to changes in macronutrients such as nitrogen or potassium. Myers and Gratton ( 2006 ) found that potassium availability in soil and leaves may play an important role in aphid population dynamics. On the other hand Costamagna and Landis ( 2006 ) found that predators exert top down control on aphid dynamics in soybeans crops.
The strawberry, Fragaria x ananassa Duchesne (Rosales: Rosaceae) is one of the main crops produced in Argentina for export ( Molina et al. 2007 ). Among the herbivore arthropods that feed on strawberry there are several species of aphids such as Aphis gossypii Glover, Aphis fabae Scopoli, Mysus persicae (Sulzer), Macrosiphum euphorbiae Thomas and Chaetosiphon fragaefolii Cockerell (Homoptera: Aphididae), ( Rondon et al. 2005 ). Although aphids are not the main pests on strawberries, they can cause serious problems. The strawberry aphid, C. fragaefolii , can affect yields because it transmits viruses such as the strawberry mild yellow edge virus , strawberry crinkle virus and strawberry mottle virus ( Krczal 1982 ). In Argentina, this aphid has been cited for strawberry crops in the provinces of Buenos Aires and Córdoba ( Cordo et al. 2004 ) and it is known to be a vector for the strawberry mottle virus ( Nome and Yossen 1980 ). In Argentina, the most frequently used method for pest control involves the regular use of insecticides on a calendar basis. However, many strawberry growers are beginning to reduce the use of pesticides and a few producers use no chemicals at all, either due to their cost or in order to obtain premium price for pesticide-free strawberries. As a consequence, different agronomic practices are currently used in strawberry fields.
The goals of the present study were: (1) to record the seasonal abundance of the aphid complex in strawberries under different input of fungicides and pesticides, (2) to obtain information about the life history of C. fragaefolii and (3) to estimate its demographic parameters. | MATERIALS AND METHODS
Field sampling
Aphid populations were monitored on commercial strawberry fields in Los Hornos and Colonia Urquiza, two localities near La Plata city (province of Buenos Aires) from April to December 2006. Strawberry cultivars used by farmers were Festival, Camarosa and Aromas. Crops under three different agrochemical treatments were surveyed twice a month. Fertilization was similar in all fields, N, P, K in agronomic standard doses for the region: N = 1Kg/ha, P = 0.5 kg /ha and K = 1.7 Kg/ha.. Eleven strawberry crops located in different fields were grouped according to different fungicide and insecticide input.
Four crops of 5000 m 2 (0.9 m rows by 50 m) fields received a high input of chemicals (HIC) under calendar spray weekly to prevent mites, thrips and aphids. Insecticides used included: New Mectin abamectin at 0.6 lt/ha (AgriMarketing www.agrimarketing.com.ar ), methomyl at 250 g/ha (Lannate, Dupont, www.dupont.com ), methamidophos at 1.5 lt/ha, (Metamidofos 60 SINER S.A, www.asaprove.com.ar ), and dicofol + tetradifon at 1–2 lt/ha (Tetranyl, Triavet S.A, www.triavet.com.ar ). The fungicide used was benomyl at 50 gr/ha (Benosem 50 PM, Sembrado S.A, www.asaprove.com.ar ).
In five other 10000 m 2 (0.90 m rows by 50 m) fields, only fungicides (OF) were used to control fungi and earthworms: benomyl at 50 gr/ha (Benosem 50 PM, Sembrado S.A, www.asaprove.com.ar ).
In two 5000 m 2 (0.90 m by 50 m) fields, little insecticide (LI) was used. Abamectin at 0.6 lt/ha (New Mectin) was used once to diminish aphid density.
A total of 237 aphid samples were collected. Systematic sampling was performed by collecting one leaflet at 5–10 m intervals in each row (approximately 100 leaflets). Leaflets, 6 and 7 cm in diameter, were selected at random from mature leaves in the middle level of the plant. The number of leaflets per sample ranged between 90 and 115, depending on the crop size. Individuals of all the aphid species found were counted under stero-microscope and the number of mummies/leaflet was recorded.
When the abundance of C. fragaefolii began to increase the samples were placed in Petri dishes lined with filter paper moistened in distilled water and maintained at 25°C, 70% RH with 14:10 L:D to search for signs of natural enemies. Samples were observed every four days to record the development of any fungi or parasitoids that could be present. Infected aphids were preserved in 70% ethanol for further identification.
The effect of different crop management practices on aphid abundance was assessed by profile analysis following von Ende ( 2001 ). The first three sample dates were excluded from the analysis due to the number of samples in which no aphids were present. Because not all the samples from each field were collected on the same dates, they were pooled by 4-week periods.
Life history traits and demographic parameters
The C. fragaefolii colony used in the experiments was started using specimens from Colonia Urquiza. They were maintained on strawberry plants under controlled conditions at 21°C, 60–70 RH and 14:10 L:D. Thirty-six viviparous females of C. fragaefolii were randomly selected from the colony and individually transferred to the abaxial side of a strawberry leaflet from Centro de Estudios Parasitológicos y de Vectores stock plants. Plants of Festival, Camarosa and Aromas cultivars were transplanted to 2 liter plastic pots with soil plus humus (50:50) outdoors under natural seasonal photoperiod at CEPAVE. The plants grew vigorously without fertilizers or insecticides being used. Each leaflet was placed in a Petri dish. The petioles were covered with moistened cotton and the leaflets were replaced every four days with a fresh mature leaflet. After 24 hours, the female and all of her offspring were removed except for one recently born nymph. These thirty-six nymphs formed a cohort. Nymphal development was observed once a day, and the presence of exuviae was used to determine molting. When the aphids reached the adult stage, the number of progeny produced per adult and adult mortality were recorded daily. Reproductive and post-reproductive periods and longevity were recorded. The following demographic parameters were calculated: net reproductive rate (R 0 ), intrinsic rate of increase (r m , = ln R 0 / T), reproductive value (V x ), and generation time (T) ( Begon et al. 1986 ). The mean and standard error of R 0 , r m and T were estimated using the jackknife method ( Krebs 1999 ; Caswell 1989 ). Estimations were done using Microsoft Excel. | RESULTS AND DISCUSSION
Aphid abundance was significantly affected by agricultural management practices (Wilks 1 Lambda: 0.000001, Rao's R: 524.80, df1: 12, df2: 6, p < 0.000001).
In LI the strawberry aphid, C. fragaefolii , was the dominant species. Two peaks in population were observed, the first in autumn and the second in spring, and population density gradually decreased towards the end of the sampling period ( Figure 1a ). Considering the average density throughout the whole sampling period, the mean number of aphids per leaflet was 1.34 ± 1.80 and the maximum density was of 14.84 ± 2.12 aphids /leaflet at the end of October. It should be noted that these estimates are relative densities, as aphids were sampled only from mature strawberry leaves.
C. fragaefolii usually infects young leaves. Rondon et al 2005 found that nymphs of C. fragaefolii were more frequently found on leaves than on the developing buds but apterous adults predominated on the buds. In most plant species young leaves are more heavily attacked by insects than mature leaves ( Slansky and Rodriguez 1987 ). One reason may be because they are less tough and easier to suck, chew and digest. Another factor that may contribute to high herbivory on young leaves is their greater nutritional value. Young leaves typically have to two to four times the nitrogen content of mature leaves ( Mattson and Scriber 1987 , Coley and Aide 1991 ). Diet with higher nitrogen can increase herbivore fitness ( Mattson and Scriber 1987 ). A diet with higher nitrogen can increase herbivore fitness ( Mattson and Scriber 1987 ).
Between July and August aphid densities were very low. The insecticide application to LI crops was made in October, so these low densities could be due to conditions of the temperate climate with a frost period ranging from mid-May until mid-September.
During the spring, C. fragaefolii coexisted only with A. gossypii in LI, although at very low densities, never being over 6% of the total aphids per sampling date. During September and October the fungus Entomophthora planchoniana Cornu (Zygomycetes: Entomophthorales) was found to be present. In samples that came from the field, the mortality due to fungi was between 10.3 % in mid September and 4.5 % in late October when no fungicides were applied. Application of fungicides is known to reduce entomopathogeniic fungi ( Lagnaoui and Radcliffe 1998 , Kouassi et al. 2003 )
A. gossypii was parasitized by Aphidius sp. and Praon sp., in percentages that fluctuated between 18% (20/9/06) and 10% (26/10/06) in all three field types (HIC, OF and LI), but no parasitism was observed on C. fragaefolii. This finding coincides with the report of Rondon and Cantliffe ( 2004 ) who suggested that the capitate hair constitutes a barrier that prevents parasitism. The ladybird beetle, Cycloneda sanguinea , and immature syrphids were the most conspicuous predators observed in these crops.
Figures 1b and 1c show the abundance of all aphids in OF and HIC, respectively. In HIC and OF, A. gossypii , M. euphorbiae and M. persicae were present, but C. fragaefolii was not recorded. Agricultural practices did not significantly affect aphid density (Wilks' Lambda: 0.034, Rao's R: 9.31, df 1 : 6, df 2 : 2, p < 0.1). Maximum density in OF was 0.35 aphids /leaflet (late November), and mummies of parasitic wasps, Aphidius sp and Praon sp. were found in 28.4 % of samples. Predators were scarce in these fields, but Crysopa lanata (Neuroptera: Chrysopidae) and immature stages of syrphids were observed but not quantified.
In HIC aphid density was lower than in OF and mummies were observed only twice. Maximum density was 0.012 aphids /leaflet (late August). No predators were observed in these crops.
Life traits and demographic parameters
The developmental times of immature and adult stages of C. fragaefolii are summarized in Table 1 . Reproduction began 24 to 36 h after the last molt. Twenty-three out of 32 nymphs reached adulthood after completing four nymphal instars. However, 12.5 % of the total aphids went through a fifth instar that lasted 2.25 ± 0.95 days. Diaz and Ferere ( 2005 ) and La Rossa et al. ( 2000 ) also reported an extra nymphal instar for the lettuce aphid, Nasonovia ribisnigri . The Russian wheat aphid, Diuraphis noxia , also exhibited a variable number of instars (four to six) during the course of its development at different temperatures ( Michels and Behle 1988 ; Nowierski et al. 1995 ).
The reproductive period represented 51 % of the entire life cycle, leading to stage overlap. Survival ( Figure 2 ) recorded at 11 and 20 days was 31.5 and 8.2 days, respectively, which was higher than the values recorded by Krczal ( 1982 ). This parameter decreased slowly and remained relatively constant (l x = 0.63) during the oviposition period ( Figure 2 ). After that it decreased from day 23 until death of all the individuals. The mean number of nymphs/ female/day was 2.4 ± 0.3.
Strawberry cultivar type influences growth capacity of C. fragaefolii . For example, females produced an average of 3.7 ± 0.8 nymphs on Festival, 4.3 ± 1.8 nymphs on Carmine and 2.1 ± 1.1 nymphs on Diamond ( Rondon and Cantliffe 2004 ). This information is interesting because it suggests that cultivars could be selected to diminish the growth potential of aphids.
Demographic parameters of C. fragaefolii were as follows: the net reproductive rate Ro= 14.55 ± 0.096 nymph/female, generation time T= 16.91 ± 0.035 days and the intrinsic rate of increase r m = 0.158 ± (0.004) nymph/female/day. Although the r m value was calculated at optimum temperature for this species, it is quite low when compared to that of other species such as A. gossypii (r m = 0.33 to 27° C, Razmjou et al 2006 ), with which it coexists. In the present paper, demographic parameters were estimated from aphids living on mature leaves, thus they could be underestimations because young leaves might be a better source of nutrients, such as nitrogen. However, Rondón et al. ( 2005 ) showed that C. fragaefolii infest the whole plant, not just young leaves.
The highest reproductive value of mature age ( Figure 3 ) was reached between days 14 and 17; after that offspring production decreased abruptly.
Although the aphids are not the main pests in strawberry crops, C. fragaefolii can be a serious problem because it can transmit several virus diseases of strawberry. According to Mellow and Frazier ( 1970 ) it can acquire viruses 24 hours after being born. After a latency period of 10 to 20 days, the infected aphid can transmit viruses for up to two weeks. C. fragaefolii was not detected during 2007 and this sporadic behavior is particularly dangerous because the aphid may not be detected until it is too late for effective control.
During the course of this study, damage caused by the strawberry mottle virus and signs of damage by the strawberry crinkle virus were observed (C. Cédola, personal observation). No further information is available about this species in Argentina. Accordingly, these new findings about life traits and mortality factors are useful to design appropriate control strategies for these aphids. This contributes to set the basis for effective pest management. | RESULTS AND DISCUSSION
Aphid abundance was significantly affected by agricultural management practices (Wilks 1 Lambda: 0.000001, Rao's R: 524.80, df1: 12, df2: 6, p < 0.000001).
In LI the strawberry aphid, C. fragaefolii , was the dominant species. Two peaks in population were observed, the first in autumn and the second in spring, and population density gradually decreased towards the end of the sampling period ( Figure 1a ). Considering the average density throughout the whole sampling period, the mean number of aphids per leaflet was 1.34 ± 1.80 and the maximum density was of 14.84 ± 2.12 aphids /leaflet at the end of October. It should be noted that these estimates are relative densities, as aphids were sampled only from mature strawberry leaves.
C. fragaefolii usually infects young leaves. Rondon et al 2005 found that nymphs of C. fragaefolii were more frequently found on leaves than on the developing buds but apterous adults predominated on the buds. In most plant species young leaves are more heavily attacked by insects than mature leaves ( Slansky and Rodriguez 1987 ). One reason may be because they are less tough and easier to suck, chew and digest. Another factor that may contribute to high herbivory on young leaves is their greater nutritional value. Young leaves typically have to two to four times the nitrogen content of mature leaves ( Mattson and Scriber 1987 , Coley and Aide 1991 ). Diet with higher nitrogen can increase herbivore fitness ( Mattson and Scriber 1987 ). A diet with higher nitrogen can increase herbivore fitness ( Mattson and Scriber 1987 ).
Between July and August aphid densities were very low. The insecticide application to LI crops was made in October, so these low densities could be due to conditions of the temperate climate with a frost period ranging from mid-May until mid-September.
During the spring, C. fragaefolii coexisted only with A. gossypii in LI, although at very low densities, never being over 6% of the total aphids per sampling date. During September and October the fungus Entomophthora planchoniana Cornu (Zygomycetes: Entomophthorales) was found to be present. In samples that came from the field, the mortality due to fungi was between 10.3 % in mid September and 4.5 % in late October when no fungicides were applied. Application of fungicides is known to reduce entomopathogeniic fungi ( Lagnaoui and Radcliffe 1998 , Kouassi et al. 2003 )
A. gossypii was parasitized by Aphidius sp. and Praon sp., in percentages that fluctuated between 18% (20/9/06) and 10% (26/10/06) in all three field types (HIC, OF and LI), but no parasitism was observed on C. fragaefolii. This finding coincides with the report of Rondon and Cantliffe ( 2004 ) who suggested that the capitate hair constitutes a barrier that prevents parasitism. The ladybird beetle, Cycloneda sanguinea , and immature syrphids were the most conspicuous predators observed in these crops.
Figures 1b and 1c show the abundance of all aphids in OF and HIC, respectively. In HIC and OF, A. gossypii , M. euphorbiae and M. persicae were present, but C. fragaefolii was not recorded. Agricultural practices did not significantly affect aphid density (Wilks' Lambda: 0.034, Rao's R: 9.31, df 1 : 6, df 2 : 2, p < 0.1). Maximum density in OF was 0.35 aphids /leaflet (late November), and mummies of parasitic wasps, Aphidius sp and Praon sp. were found in 28.4 % of samples. Predators were scarce in these fields, but Crysopa lanata (Neuroptera: Chrysopidae) and immature stages of syrphids were observed but not quantified.
In HIC aphid density was lower than in OF and mummies were observed only twice. Maximum density was 0.012 aphids /leaflet (late August). No predators were observed in these crops.
Life traits and demographic parameters
The developmental times of immature and adult stages of C. fragaefolii are summarized in Table 1 . Reproduction began 24 to 36 h after the last molt. Twenty-three out of 32 nymphs reached adulthood after completing four nymphal instars. However, 12.5 % of the total aphids went through a fifth instar that lasted 2.25 ± 0.95 days. Diaz and Ferere ( 2005 ) and La Rossa et al. ( 2000 ) also reported an extra nymphal instar for the lettuce aphid, Nasonovia ribisnigri . The Russian wheat aphid, Diuraphis noxia , also exhibited a variable number of instars (four to six) during the course of its development at different temperatures ( Michels and Behle 1988 ; Nowierski et al. 1995 ).
The reproductive period represented 51 % of the entire life cycle, leading to stage overlap. Survival ( Figure 2 ) recorded at 11 and 20 days was 31.5 and 8.2 days, respectively, which was higher than the values recorded by Krczal ( 1982 ). This parameter decreased slowly and remained relatively constant (l x = 0.63) during the oviposition period ( Figure 2 ). After that it decreased from day 23 until death of all the individuals. The mean number of nymphs/ female/day was 2.4 ± 0.3.
Strawberry cultivar type influences growth capacity of C. fragaefolii . For example, females produced an average of 3.7 ± 0.8 nymphs on Festival, 4.3 ± 1.8 nymphs on Carmine and 2.1 ± 1.1 nymphs on Diamond ( Rondon and Cantliffe 2004 ). This information is interesting because it suggests that cultivars could be selected to diminish the growth potential of aphids.
Demographic parameters of C. fragaefolii were as follows: the net reproductive rate Ro= 14.55 ± 0.096 nymph/female, generation time T= 16.91 ± 0.035 days and the intrinsic rate of increase r m = 0.158 ± (0.004) nymph/female/day. Although the r m value was calculated at optimum temperature for this species, it is quite low when compared to that of other species such as A. gossypii (r m = 0.33 to 27° C, Razmjou et al 2006 ), with which it coexists. In the present paper, demographic parameters were estimated from aphids living on mature leaves, thus they could be underestimations because young leaves might be a better source of nutrients, such as nitrogen. However, Rondón et al. ( 2005 ) showed that C. fragaefolii infest the whole plant, not just young leaves.
The highest reproductive value of mature age ( Figure 3 ) was reached between days 14 and 17; after that offspring production decreased abruptly.
Although the aphids are not the main pests in strawberry crops, C. fragaefolii can be a serious problem because it can transmit several virus diseases of strawberry. According to Mellow and Frazier ( 1970 ) it can acquire viruses 24 hours after being born. After a latency period of 10 to 20 days, the infected aphid can transmit viruses for up to two weeks. C. fragaefolii was not detected during 2007 and this sporadic behavior is particularly dangerous because the aphid may not be detected until it is too late for effective control.
During the course of this study, damage caused by the strawberry mottle virus and signs of damage by the strawberry crinkle virus were observed (C. Cédola, personal observation). No further information is available about this species in Argentina. Accordingly, these new findings about life traits and mortality factors are useful to design appropriate control strategies for these aphids. This contributes to set the basis for effective pest management. | Associate Editor: Eileen Cullen was editor of this paper
Seasonal abundance of the strawberry aphid complex under different agronomic practices in the outskirts of La Plata, Argentina was studied on strawberry, Fragaria x ananassa Duchesne (Rosales: Rosaceae). Aphid densities were low in strawberry fields in which insecticides and fungicides were used. In addition to Aphis gossypii , Aphis fabae , Mysus persicae and Macrosiphum euphorbiae , the aphid, Chaetosiphon fragaefolii (Cockerell) (Homoptera: Aphididae), was recorded for the first time in this horticultural area. Life history and some demographic parameters were calculated for C. fragaefolii . The mean duration of nymphal stages was 10.44 days, the oviposition period was 11.8 days, and the mean number of nymph/female/day was 2.4 ± 0.3. Demographic parameters analyzed included the net reproductive rate R o = 14.55 ± 0.096 nymph/female, generation time T=16.91 ± 0.035 days, and the intrinsic rate of increase r m = 0.158 ± (0.004). No parasites were found associated with C. fragaefolli . The pathogenic fungus, Entomophthora planchoniana Cornu (Zygomycetes: Entomophthorales) was the main mortality factor. Although aphids are not the main pests in strawberry fields, C. fragaefolii can be a serious problem because it can transmit several virus diseases of strawberry. Greater knowledge of life history traits and mortality factors of this species is needed in order to design appropriate control strategies.
Key words | ACKNOWLEDGEMENTS
We thank Rubén La Rossa for the determination of C. fragaefolii , Ana Clara Scorcetti for the identification of fungal pathogens, M. Roggiero, F. Gugole, F. Cingolani and N. Cluigt for their assistance in the field and laboratory, and two anonymous referees for their comments and suggestions. | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Mar 2; 10:9 | oa_package/83/0e/PMC3014655.tar.gz |
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PMC3014657 | 20575742 | Introduction
The yellow dung fly Scathophaga stercoraria (L.) (Diptera: Scathophagidae) is a widespread and locally abundant fly associated with the dung of large mammals. The species was first named by Linnaeus as Musca stercoraria in 1758. In 1800, Meigen renamed the genus as Scopeuma , and in 1803 he reclassified the species, together with several relatives, into a new family Scathophagidae . For unknown reasons, from 1805 onwards, Fabricius used the genus spelling Scatophaga for several of the related species (but not S. stercoraria , which remained Scopeuma for a while). There has been confusion about the spelling of the whole family group, but the spelling was officially settled to become Scathophaga in all the standard catalogues (see Gorodkov 1986 ).
In the early part of the last century, the predatory S. stercoraria first attracted interest as a possible bio-control agent of pest flies affecting livestock ( Cotterell 1920 ). During the past 40 years, this fly has been the subject of numerous studies on mating behavior and sperm competition ( Parker 1970a , b , c , 1978 ), post-copulatory sexual selection and sexual conflict ( Ward 2000 ; Hosken et al. 2001 ), reproductive physiology ( Hosken and Ward 1999 ; Reim et al. 2006 ), foraging ( Blanckenhorn and Viele 1999 ), life history evolution ( Blanckenhorn 1998a , b ; Teuschl et al. 2007 ), thermal biology ( Blanckenhorn and Llaurens 2005 ), developmental stability and fluctuating asymmetry ( Strong and James 1992 ; Swaddle 1997 ; Hosken et al. 2000 ; Webb et al. 2007 , Floate and Coghlin 2010 ), phylogenetics ( Bernasconi et al. 2001 ), quantitative genetics ( Blanckenhorn 2002 ), and population genetics ( Kraushaar et al. 2002 ). S. stercoraria also have been used to test for non-target effects of chemical residues in dung of livestock treated with veterinary pharmaceuticals (e.g. Sommer et al. 1992 ; Strong and James 1992 ; Floate 1998 , 2007 ; Webb et al. 2007 , Floate and Coghlin 2010 ). In this latter context, the international community has approved S. stercoraria as a standard test species to evaluate the toxicity of drug residues in livestock dung ( OECD 2008 ). As a result, tests using S. stercoraria will become a requirement for the registration of new veterinary compounds (e.g., Römbke et al. 2009 ).
No papers specifically written to provide information on the rearing of S. stercoraria in laboratory culture exist in the literature. Such information may facilitate future studies on the insect's biology and ecology, but is particularly needed for studies in response to the new regulatory requirements for novel livestock medical products. Here a general description of the biology, life history, ecology, and behavior of S. stercoraria is provided, including control and reference test data relevant for the assessment of toxicological tests. Subsequently, laboratory rearing and handling methods plus some associated field methods are described. In this review, some original information is included on pupal survival and standard life history traits relevant for the conductance of ecotoxicological applications, which due to availability primarily reflect the situation of Swiss populations. Blanckenhorn ( 2009 ) more completely covers the S. stercoraria literature.
General biology, distribution, phenology, behavior and life history
Adult S. stercoraria flies are between 7 and 13 mm long. The males are hairy and yellow to orange in color, whereas the females are much less hairy, greenish and more cryptic. Atypically for insects, the males are considerably larger than females ( Kraushaar and Blanckenhorn 2002 ), and unlike most Scathophagids, the sexes differ quite starkly in color ( Figure 1 ). The species inhabits temperate regions of the entire northern hemisphere ( Stone et al. 1965 ; Gorodkov 1986 ), favoring cooler climates at high altitudes such as the Swiss Alps ( Blanckenhorn 1997 ) and high latitudes up to Iceland or even Spitzbergen ( Sigurjónsdóttir and Snorrason 1995 ). Its distribution appears limited by hot temperatures toward the south ( Hammer 1941 ; Ward and Simmons 1990 ; Blanckenhorn 1998a , Blanckenhorn et al. 2001 ), where the flies occur only at higher elevations, such as the Pyrenees and Sierra Nevada in Spain or the Sierras of Mexico ( Stone et al. 1965 ). S. stercoraria is also reported from South Africa, although this is probably a closely related sister species, S. soror ( Werner et al. 2006 ). It is not reported from anywhere else in the southern hemisphere. In North-Central Europe, S. stercoraria is one of the most abundant and widespread insect species associated with cow dung, although some similar scathophagid species may co-occur on cattle pastures, depending on location, but usually at much lower densities (e.g. S. furcata in North America and S. inquinata, S. suilla , and S. lutaria in Europe). The species' distribution pattern is likely influenced by human agricultural practices. While S. stercoraria is considered a cow-dung specialist, it can successfully breed on dung of other large mammals such as sheep ( Hirschberger and Degro 1996 ), horse, deer, or wild boar (Blanckenhorn et al., unpublished data).
S. stercoraria larvae are coprophagous, feeding on the dung of large mammals, which they thereby help to decompose along with many other species of competing earthworms, beetles and flies ( Hammer 1941 ; Holter 1979 ). Adult S. stercoraria , in contrast, are sit-and-wait predators of small insects, but they also imbibe nectar and fresh dung as energy sources ( Cotterell 1920 ; Hammer 1941 ; Gibbons 1980a , b ; Sasaki 1984 ). Adult flies are nutritionally anautogenous (or capital breeders) requiring protein and lipids from prey to become sexually mature producers of eggs and sperm ( Foster 1967 ).
S. stercoraria phenology depends strongly on local climate. In Central Europe, fly populations often exhibit a sharp population decline in summer ( Hammer 1941 ; Parker 1970; Gibbons 1987 ; Blanckenhorn 1997 ; Jann et al. 2000 ; Blanckenhorn et al. 2001 ), subdividing the year into a spring (March — June) and an autumn (September — November) flight season. The summer decline is mediated by temperatures above 25° C, which tend to kill larvae, pupae and adults. Adult flies avoid this by moving into cooler, forested microhabitats and entering some sort of physiological quiescence ( Gibbons 1987 ; Ward and Simmons 1990 ; Blanckenhorn 1998a ; Blanckenhorn et al. 2001 ). In contrast, the flies are present only in summer in the highlands of the Swiss Alps (1500–2800 m) ( Blanckenhorn 1997 ) and in northern European countries such as Iceland and Finland ( Sigurjónsdóttir and Snorrason 1995 ; Otronen 1996 ). Accordingly, the number of generations per year varies with latitude and altitude, with two to three (overlapping) generations per year in lowland Switzerland ( Jann et al. 2000 ), and one or two generations per year in the Swiss highlands and Northern Europe. The flies diapause over winter in the pupal stage.
S. stercoraria females spend most of their time foraging for prey and nectar in the vegetation surrounding the pasture and only come to the dung to oviposit. Males also need to forage in the surroundings but spend most of their time waiting on or around fresh dung pats to mate with incoming females ( Parker 1970b ). During copulation, which typically lasts about 20–50 minutes (though there is considerable variation), and during the ensuing oviposition, the male guards the female against competitors so his sperm are not displaced before oviposition ( Parker 1978 ; Simmons 2001 ). Competition among males for females is typically intense, as the operational sex ratio at the dung is highly male biased; a single dung pat may host as many as 400 males ( Jann et al. 2000 ). After copulation, the female lays about 30–90 eggs into the dung and then leaves the pat for further foraging in the vegetation. The male waits for other females at the same pat or switches to another, fresher pat, as pats lose their attractiveness to this fresh dung specialist within 1–2 h of being deposited ( Parker 1970a ).
Larvae hatch from the eggs within 1–2 days, depending on temperature, and immediately enter the dung to avoid desiccation or drowning. Thereafter, they regularly surface for oxygen. Other sources of egg, larval and pupal mortality in the dung community include numerous egg and larval predators such as staphylinid beetles and hymenopteran parasitoids ( Hammer 1941 ; Sowig et al. 1997 ). Larvae undergo three molts and, at 20° C, grow exponentially and rapidly during the first five days of development; thereafter they require an additional five days to empty their guts and prepare for pupation, during which time no additional body mass is accumulated ( Blanckenhorn 1999 ; Teuschl et al. 2007 ).
Individuals pupate in the encrusted parts of the dung or in the ground under or near the dung pat. Pupal development takes an additional 10 days at 20° C. In the case of direct (i.e. non-diapause) development, adult flies emerge after a total pre-adult (egg + larval + pupal) development time between 17 days (at 25° C) and 80 days (10° C and below; Table 1 ). On average, the smaller females emerge a few days earlier.
Collecting wild S. stercoraria in the field
S. stercoraria can be easily collected while mating on a pasture at the appropriate time in the season (see above). Overcast conditions (no rain), mild weather (not too hot) and certain times of day (the last two hours before sunset) usually work best. A sweep-net can be used, although copulating pairs (as well as single males) are more efficiently caught by carefully lowering a long (e.g. 50 ml) glass or plastic vial over the insects while they copulate on or near the dung and then plugging it with a permeable paper or foam stopper. Live single flies or pairs then can be brought back to the lab in these vials, to which a bit of sugar and/or moist paper or cotton is best added, preferably in a dark box under mild refrigeration. Alternatively, freshly laid eggs can be scooped with a small spatula directly into a plastic vessel containing fresh dung and plugged with a stopper, wherein the larvae can subsequently hatch and develop in the laboratory. Emerging adults then can be collected as described in the egg processing section below. If the flies are not needed alive for procedures such as molecular analyses, individuals can be transferred directly into pure ethanol or can be frozen in the lab and subsequently stored in ethanol.
Laboratory holding conditions
Adult S. stercoraria are cannibalistic in the absence of alternate prey, as they require prey to produce eggs and sperm ( Foster 1967 ). Cannibalism can be prevented by keeping flies individually in glass or plastic culture bottles (100 ml volume, for example) capped with paper or foam stoppers. Stoppers should fit the bottles snugly to prevent phoretic and parasitic mites from moving between bottles. Each bottle should be equipped with a water source (e.g. gauze or cotton submerged in water in a small dish), plus sugar in a separate dish; diluted sugar water is also suitable. Rearing conditions not higher than 24° C with humidity of 50–60% and a light period of 12–14 h are recommended.
Ideal prey include flies smaller than dung flies. Mass-reared Drosophila spp. or Musca domestica work well, both of which are commercially available and cultured in many laboratories. Start freshly emerged S. stercoraria with ad libitum amounts of ca. 50 Drosophila melanogaster per week (or equivalents thereof) and re-feed when all prey are eaten. Otherwise, feed every week or even two weeks, making sure that the water does not dry up. After attaining sexual maturity ( Table 1 ), males will often stop eating prey while females will require new prey to replace each clutch of eggs laid. Ten Drosophila melanogaster per week will limit reproductive output ( Jann and Ward 1999 ). Holding bottles tend to become filthy and moldy after about one month, when they should be replaced. All bottles, stoppers and containers for food and water need to be disposed of, or carefully cleaned, to avoid mite infestations within the lab culture. For material that is not easily washed (such as paper stoppers), autoclaving in an oven at 120° C is adequate to prevent infestations.
Holding flies individually in bottles is time-consuming and requires considerable laboratory space. When doing so, subsequent generations can be produced by mating a number (for example, n = 30) of randomly chosen pairs and raising their offspring, typically from the first-laid (partial) clutch, in disposable containers as described below and keeping a subset of the emerging adults to be further propagated. As an alternative, S. stercoraria can be held in groups in large, well-aerated cages. S. stercoraria colonies have been maintained for several years in plexiglass cages (60 cm × 60 cm × 60 cm), each housing several hundred flies allowed to feed ad libitum on suitable prey with access to water and sugar. Lower densities of about five flies per litre (= 10 cm × 10 cm × 10 cm) can be used to optimize production, with the sexes initially separated. Otherwise, males will harass non-receptive females and ultimately delay reproduction.
For ease of colony maintenance when flies are housed in large cages, dung pats (ca. 1 litre) on Styrofoam plates with an underlying layer (ca. 1 cm thick) of sand and vermiculite (1:1) can be placed into the cage with the adult flies. This can be started approximately 2–3 weeks after the first emergence of adults to allow time for sexual maturation and mating. After 2–3 days of exposure to ovipositing females, the dung pat is removed and replaced with a fresh one, if more eggs are needed. The Styrofoam plates with the dung containing eggs and larvae are subsequently held for 2–3 weeks in a plastic tub for larval development and pupation. The plate is elevated above the floor of the tub by placing it on a Petri dish. The tub is then almost fully covered with a sheet of plastic to reduce dehydration of the dung. However, one corner of the tub should be left uncovered. Otherwise, the heat generated by larval metabolism will cause the tub to overheat and reduce larval survival. Larvae will typically reduce the dung pat to a granular consistency, which is readily sieved to remove pupae. Larvae also will crawl off the plate to pupate underneath. If the plate is not elevated, there is insufficient space for pupation, in which case pupae are often deformed and adult emergence is low. Each dung pat will produce from a few dozen to several hundred adult flies. Pupae removed from tubs can be placed in shallow Petri dishes in clean cages for emergence of the adults.
In general, when handling S. stercoraria adults, keep in mind that they are positively photo-tactic, and, therefore, they are best moved from one into another container, or kept in a particular bottle, by orienting the opening toward or away from the light, respectively. Thus it is best to work at a window or facing a strong lamp (at night) with overhead lighting switched off.
Adult maturation and reproductive period
Newly emerged S. stercoraria females take about 2 weeks (10–16 d) and males about 1 week (5–8 d) to become sexually mature when held individually and given an excess of sugar, water and prey ( Table 1 ; Gibbons 1980a ; Blanckenhorn and Henseler 2005 ). Although females will mate after about 6 d, they usually require at least 10 d to produce a batch of eggs. The abdomens of fully gravid females look swollen and whitish when viewed from the side or below. Inter-clutch intervals are between 3–7 days in the laboratory, depending on nutrition and temperature (but probably are considerably longer in the field; cf. Gibbons 1987 ). After first producing sperm, males can and will mate at least 5 times, indeed more or less continuously if their sperm stores are refilled by having continuous access to prey. Under (benign) laboratory conditions, flies live, on average, for 1–2 months, up to a maximum of several months, during which females can produce 10 clutches or more. Females store sperm from each copulation, which should last to fertilize at least 4 clutches, so repeated mating is not strictly necessary, although a recent mating will increase the likelihood of oviposition when females are offered dung. Also, gravid females may dump fertilized or unfertilized eggs into the holding bottle if not given an opportunity to lay.
Laboratory matings
When males and females are sexually mature, matings of individually-held flies are best staged in a 50 or 100 ml vial containing a smear of dung on wet filter or blotting paper. Males will also mate without dung, but the presence of dung is believed to stimulate copulation, although this has not been tested rigorously. To reflect the prior arrival of males at the dung in nature, the male is typically placed in the vial first, and then female is added. Copulations can begin almost instantly, or there can be a delay of a few minutes up to one hour. Some pairs may never mate, in which case the male should be exchanged after 10 to 15 min. For experimental matings, which last between 20 and 50 min, the following information can be easily recorded: (1) the time the female was added, (2) the time copulation started, and (3) the time copulation ended. These give measures of ‘latency’ to copulate (2 minus 1) and copulation duration (3 minus 2). After copulation ends (i.e. the male and female abdomens have become detached), males may guard (i.e. stand over the female without genital contact) while the female deposits her eggs into the dung, which takes about 20 min. However, some males may not guard the females in the vials used for laboratory copulations, and they will dismount and then likely copulate again with the same female (presumably not recognizing her identity); thus males should be removed after copulation to avoid further harassment. When re-mating non-virgin, gravid females, there is a risk that she will start ovipositing into the dung before the new male copulates. This risk can be reduced by only providing dung after the focal copulation terminates.
Egg processing
S. stercoraria eggs are white and banana-shaped, similar to, but larger than, the eggs of Drosophila melanogaster ( Figure 2A, B ; Table 1 ). Females will stick the eggs lengthwise (vertically) into the dung singly or in groups, so that only the upper pole will be exposed to the air, the rest being submerged. The upper pole features a yellowish flap (plastron), through which the larva will eventually exit, flanked by two little lateral respiratory horns ( Figure 2B ; Arthur et al. 2008 ).
Individual females will typically lay entire clutches of eggs (30–90 eggs, depending on body size; Table 1 ; Blanckenhorn 2009 ), although they sometimes only deposit a partial clutch. After oviposition into the dung portion on filter paper, the female can be transferred to another vial and the filter paper with the eggs removed to be counted in situ under a binocular microscope. Using curved, smooth forceps, as many eggs as necessary can then be transferred face up (in groups, with a bit of dung) into a disposable plastic container with excess fresh dung (i.e. > 2 g per individual, Amano 1983 ) for propagation of the next generation, to be capped with a paper or foam stopper or paper towel fastened with rubber bands. This should be done quickly (within several hours) after egg deposition to avoid excessive drying of the dung surface, which can make the extraction of intact eggs difficult. The respiratory horns should not be submerged below the dung surface. Also, the containers should be sufficiently large (e.g. 100 ml) and only about half full of dung, leaving some space for emerged adults.
To measure the size of eggs, and to easily score larval hatching success (e.g. when conducting eco-toxicological tests), single eggs can be randomly removed from the clutch using a small, wetted paint brush, to be placed horizontally on a piece of wet filter paper, brushing off most of the excess dung. Length and width (as egg height and width are roughly the same) of 3–5 randomly chosen eggs of a given clutch are typically measured using an ocular eyepiece or by taking digital photos and then measuring with image analysis software (e.g. ImageJ, http://rsb.info.nih.gov/ij ). The volume of eggs can be estimated using the formula for an ellipsoid:
After measuring, the wet filter paper with the eggs is placed onto the (wet) dung in a disposable plastic container as described above. After about 24 h (at room temperature) the larvae will have hatched and crawled into the dung, and deflated egg cases are easily distinguished from the remaining intact (infertile) eggs. Egg development time, i.e. the time from egg deposition to larval hatching, can thus be scored by checking for hatched larvae at regular intervals (e.g. hourly).
When eggs are transferred into a refrigerator at 2–5° C immediately after laying, larval hatching can be delayed for 2–3 d. This is necessary, for example, when eggs are to be sent to a laboratory for immediate ecotoxicological testing. Thus, individual clutches in the dung portion on a round filter paper can be placed in Petri dishes of corresponding size, stacked, and sent via overnight express mail in a styrofoam box containing padding and an ice pack for cooling.
Collecting emerged flies and scoring development time, emergence success, and body size
As for any ectothermic organism, egg-to-adult development time (as well as body size) is highly temperature dependent ( Blanckenhorn 1997 ; Table 1 ). It can take as little as 17 d (above 23° C), or about 21 d at 20° C, 35 d at 15° C, and ca. 75 d at 10° C, although at the latter, cooler temperatures some flies will go into winter diapause as pupae ( Blanckenhorn 1998b ). About half of the development period is spent as a larva, the other half as a pupa ( Blanckenhorn 1999 ; Teuschl et al. 2007 ). Emergence occurs over several days for a whole clutch (family) reared together in the same dung container, the smaller females emerging first. Egg-to-adult development time is obtained by checking containers for emerging adults on a daily basis. Emergence success (= egg-to-adult survival) is simply the proportion of adults emerged relative to the number of eggs entered or larvae hatched (to be written on the container). Mean emergence success is 60–80% at optimal conditions in the lab ( Table 1 ). Somewhat higher survival can be obtained when raising eggs individually in small dung containers, thus avoiding larval competition, but this is laborious.
When checking emergence, adults can be collected daily from the larval family dung containers and immediately frozen for later measurement of body size, or alternatively transferred into a holding bottle for further propagation, as described above. In principle, any morphological structure can be used as a measure of body size, as most body structures covary positively with one another. However structures composed of individual exoskeletal elements (e.g., a segment of a leg) are most consistent because they do not change size with body condition or female gravid state. Hind tibia length (cut off the leg at the femur) is traditionally used in this species ( Parker 1978 ; Simmons 2001 ), but teneral fresh (wet) weight or dry weight (after 24 h of drying at 60° C) of freshly emerged adults is also good ( Table 1 ). When measuring live animals, wet weight or head width (= widest extension of head including eyes) are best and should be obtained from individuals anaesthesized with CO2 while measuring.
Alternatively, when simply interested in collecting emerged adults without scoring, the small (e.g. 100 ml) plastic dung container with the developing offspring can be placed open into a larger (e.g. 1.5 liter plastic) container, to be capped with a paper towel lid held with rubber bands. If some sugar and water is added, the flies will survive for a couple of days as a group and hopefully will not eat each other before being processed further.
Collecting larvae or pupae for further processing
Newly hatched larvae can be collected with a wet paint brush if eggs are processed via the filter-paper technique described above, provided they received no dung; otherwise, it is cumbersome, but possible, to dig out larvae from the dung. Alternatively, single eggs (e.g. for molecular analysis of single larvae) can be transferred directly into wells of a 96-well plate, sealed with sealing foil, and the whole plate can be frozen at -80° C when larvae have hatched.
Because S. stercoraria spend about 10 d in the hardy pupal stage, pupae are easy and inexpensive to send using regular mail, in contrast to eggs (as explained above) or adults, which can be sent singly or in groups in plastic vials equipped with water-drenched gauze and some sugar. To collect pupae, eggs are placed in sufficient dung directly onto dry sand in a larger plastic container. The larvae will hatch, feed on the dung, and eventually crawl down into the sand to pupate. After 8–12 d, they can be removed by sieving. Pupae can be mailed in a sturdy shallow Petri dish with a bit of sand. Still, they should not be exposed to temperatures exceeding 25° C.
Blanckenhorn ( 1998b ) showed that larvae from Swiss populations facing low temperature (10° C) and photoperiod combinations (indicating the advent of winter) will enter winter diapause as pupae for up to 5 months, after which adults will emerge at survival rates comparable to direct development. In general, there may be occasions when it is desirable to store at cool temperatures pupae generated from adults held at room temperature, until needed for colony expansion or experimental studies. To test the effect of cold storage on survival (novel data presented in the Results section), 15 replicates of fly pupae ( n = 10 pupae per replicate), aged 24–48 h, were stored at 4, 10, 12, or 15° C for periods of 1, 2, 3, 4, 5, or 6 weeks. Upon completion of the storage period, pupae were removed and held at 20° C for an additional week. Numbers of emergent flies were recorded. Puparia not producing flies were dissected to determine whether pupae were dead or in a state of arrested development. As a control, 20 replicates of pupae were held continuously at 20° C. Pupae used for different combinations of storage time and temperature, and for the control replicates, were from the same generational cohort. Flies occasionally emerged while in storage at temperatures of 10, 12 and 15° C. This is not surprising, as individuals held at temperatures exceeding 12° C are unlikely to enter pupal diapause ( Blanckenhorn 1998b ), so adults are expected to emerge from pupae after roughly half the development time listed in Table 1 for that particular temperature. Hence, the experiment was repeated using storage temperatures of-3, 0 and 3° C, with 15 (rather than 20) replicates of pupae held at 20° C for use as controls. No flies emerged during storage at these lower temperatures. The results presented below indicate that larvae developing at warm temperatures will not be properly prepared physiologically for pupal diapause, and thus keeping the resulting pupae at cold temperatures for extended periods of time will inevitably result in significant mortality ( Figure 3 ). Longer storage of non-diapausing pupae in the fridge is therefore not feasible.
Processing cow dung for experimentation
Cow dung can be collected in large quantities from the pasture in the field, mixed thoroughly in larger vats or buckets in the laboratory (to reduce variation in dung quality), and frozen in portions of suitable size for extended periods of time to be used later, marking the date on the pot lid or zip-lock bag with a waterproof pen. Any parasites or other organisms in the dung can be killed by freezing the dung first at -20° C and thereafter at -80° C for a minimum of 1 week. (Freezing at merely -20° C might not kill some parasites, and storing fresh dung at -80° C without an intermediate -20° C step causes most freezers to trip their alarms because the samples do not cool quickly enough.) A ca. 5 cm gap should be left at the top of the dung container to allow for expansion as the dung freezes, or the container may crack. When needed, dung can be thawed overnight and, if necessary, mixed with some warm water to obtain a looser consistency. If needed more quickly, dung containers can be submerged in hot water or defrosted in a microwave oven.
Dung quality (i.e. water, nutrient, parasite or medication content, etc.) has great impact on larval development and ultimately the fitness of the individual fly. Unfortunately, dung quality usually cannot be controlled effectively over longer periods of time. For example, cows fed on dried hay over the winter produce dung of a lower food quality than those fed on summer grass. Therefore, the same, homogenized and thoroughly mixed dung batch should be used for any particular experiment. Because insecticidal residues from veterinary drugs may be excreted in dung of treated livestock for at least 3 months after application (e.g. Floate 1998 ; Floate et al. 2008 ), dung is best collected from animals that have not been treated with parasiticides in the previous six months.
When testing the effect of pharmaceuticals on S. stercoraria according to the OECD guideline ( 2008 ), the dung should have been frozen for at least 1 week (preferably longer). Dung samples should be taken to determine its moisture and pH. The test substance in question has to be mixed thoroughly into the dung, usually at various, roughly logarithmicly-scaled concentrations. If chemicals volatile solvent (typically acetone or ethanol) and mixed thoroughly for ca. 10 minutes. The two mandatory control treatments are inoculated with a known amount of solvent (solvent control) and with an equivalent amount of water only (untreated control). When a solvent carrier is used, it must be allowed to fully evaporate for at least 4 hours at room temperature before the test organisms are added. All test concentrations must be given on a dry weight basis in order to ensure comparability of the results from different studies. | Results
As a guide, means and ranges for all relevant, standard life history traits stemming from previously published sources are reported here, referring to primarily one Swiss population (Fehraltorf near Zurich). These data, stemming from lab and field experiments, are summarized in Table 1 , which includes the original references.
Cold storage of pupae
For the experiment in which pupae were stored at temperatures of 4, 10, 12 and 15° C, survival (= fly emergence) of control pupae was 83.5% ± 2.5 (SD; Figure 3A ). Using linear regression, no effect of weeks in storage was detected for the survival of pupae held at temperatures of 10° C ( F 1, 108 = 0.01; p = 0.911) or 15° C ( F 1, 108 = 0.53; p = 0.466). A weak, though overall positive, effect of storage ( F 1, 108 = 3.99; p = 0.048) was detected for pupae held at 12° C. This can be attributed to the unexpectedly low survival of pupae removed from storage after one week; survival of pupae held for 6 weeks at 12°C (85.3% ± 3.8) did not differ from that of control pupae. However, at 4° C ( F 1, 108 = 17.76; p < 0.001) survival declined to 50.7% ± 7.1 after 6 weeks in storage. Dissection of puparia from which flies did not emerge revealed pupae to be dead and desiccated rather than in a state of arrested development.
For the experiment in which pupae were stored at temperatures of -3, 0 and 3° C, survival of control pupae was 57.3% ± 37.1 ( Figure 3B ). At all three temperatures survival declined with time to 12.0% ± 2.9 after storage for 6 weeks at -3 and 0° C, and to 2.0% ± 1.3 at 3° C ( F 1, 73 = 46.0 to 128.6; p < 0.001). | Conclusions
Baseline information and summary data are provided here on the natural history, field and laboratory handling of S. stercoraria for the benefit of the international scientific community, as well as for government regulators or testing agencies that use this fly for eco-toxicological testing. Based on the availability of data and experience with this fly, the information primarily reflects the situation of Swiss dung fly populations. It is clear that variation in the life history of this fly will be considerable given its wide distribution on at least three continents (North America, Asia, Europe) in climates ranging from sub-arctic to Mediterranean. Systematic comparisons of geographic, latitudinal and altitudinal populations on a worldwide scale are consequently necessary and underway. | Editor: Allen Cohen was editor of this paper.
The yellow dung fly Scathophaga stercoraria (L.) (Diptera: Scathophagidae) is a widespread and locally abundant fly associated with the dung of large mammals, especially farm animals. This species has recently become a standard test organism for evaluating toxic effects of veterinary pharmaceuticals in livestock dung. In this context, a review of its natural history and a general description of the field and laboratory rearing methods of this species are provided here to benefit the scientific community as well as government regulators and applicants of eco-toxicological studies. For guidance, means and ranges are included for all relevant standard life history traits stemming from previously published data on Swiss populations.
Keywords | Acknowledgements
We thank all regulating agencies, laboratories and companies involved over the years in initiating and participating in the Dung Organism Toxicity Test Standardization (DOTTS) effort and group, an advisory group of the Society of Environmental Toxicology and Chemistry (SETAC), which ultimately prompted us to write this rearing note. | CC BY | no | 2022-01-12 16:13:43 | J Insect Sci. 2010 Mar 2; 10:11 | oa_package/f6/d9/PMC3014657.tar.gz |
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PMC3014658 | 20575743 | Introduction
The evolution and diversification of carabid beetles has been proposed to be tightly linked to changes in delivery, composition and potency of their defensive secretions ( Erwin 1981 ; Will et al. 2000 ). Although the use of defensive allomones produced from the pygidial glands of adephagan beetles is well documented in terms of the various admixtures of compounds released ( Dazzini-Valcurone and Pavan 1980 ; Dettner 1985 ; Will et al. 2000 ) and mechanisms of delivery of those compounds ( Forsyth 1972 ; Moore 1979 ; Rossini et al. 1997 ; Eisner and Aneshansley 1982 ), there are no studies that measure the quantity released in a typical spray event.
Pygidial gland secretions are employed defensively, and in this context the actual amount released is important for several reasons, including that semiochemical concentration is a determinant in what behaviors are elicited. For example, knowledge of spray quantities may help clarify whether carabid defensive gland compounds, when applied to a given competitor or predator will tend to trigger escape responses, alarm behavior, death, or some alternative. The amount held in reserve after initial and successive chemical emissions, critical to further defense, may also be ecologically important. Thus, knowledge of how carabid beetles apportion defensive compounds will help establish the ecological context within which defensive chemistry can be best understood. This study is the first to measure the quantity of defensive gland secretions released defensively by carabid beetles.
In a study by Rossini et al. ( 1997 ) on Galerita lecontei several individual beetles were held in the laboratory for 3.5 months to ensure gland fullness and then sampled to determine the quantity of formic acid held in the pygidial glands. They determined that the formic acid content was approximately 80% of the estimated total gland contents, or about 4.56 ± 0.72 mg per beetle. They found that G. lecontei on average would spray 6.5 times before depletion and reasoned that a typical discharge of 0.7 mg of formic acid (∼0.8 mg total discharge) would be the average minimal quantity ejected. However, this average was based on a sample of only six individuals and a series of spray events from beetles with anywhere from replete to nearly empty reservoirs. Therefore these data cannot address changes in quantity over the series of spray events and is not conclusive regarding the amount ejected in a usual spray event.
Like G. lecontei , the beetles in this study, Platynus brunneomarginatus (Mannerheim) (Coleoptera: Carabidae: Platynini), P. ovipennis (Mannerheim) (Platynini) and Calathus ruficollis Dejean (Sphodrini), produce formic acid, a common compound in carabid beetles ( Dazzini-Valcurone and Pavan 1980 ; Will et al. 2000 ) and other arthropods ( Blum 1981 ). Formic acid is mixed with hydrocarbons in these taxa, creating an admixture known to be a potent toxin ( Löfqvist 1977 ) and an alarm pheromone in ants ( Löfqvist 1976 ). Hydrocarbons and other lipophilic compounds have frequently been associated with formic acid as surfactants ( Eisner et al. 1961 ; Rossini et al. 1997 ; Schildknecht et al., 1968a ) that increase the effectiveness of the primary irritant and are themselves potentially defensive ( Peschke and Eisner 1987 ). When agitated, carabid beetles emit these defensive chemicals from their pygidial glands by oozing them onto the body ( Moore 1979 ), crepitation of fluids directed via a flange of Coanda ( Eisner and Aneshansley 1982 ), pulsatile crepitation ( Aneshansley et al., 1969 ) or as is the case for all formic acid spraying beetles, like those in our study, by spraying an aimed mist or droplets ( Moore 1979 ; Rossini et al. 1997 ). | Materials and Methods
Terms
Terminology for morphological structures follows Forsyth ( 1972 ). Herein a spray event refers to a single spray episode from a beetle, induced by a pinch on a meso- or meta-leg.
The beetles
To determine the average quantity sprayed, specimens of two species of Platynini were used, P. brunneomarginatus (n = 31; 18 females and 13 males) and P. ovipennis (n = 17; 8 females and 9 males), and one species of Sphodrini, C. ruflcollis (n = 9; 4 females and 5 males). To determine the relative quantities of formic acid 18, eight and 30 individuals were used for P. brunneomarginatus , P. ovipennis and C. ruficollis respectively. Specimens were collected from October to December from four sites in California: 1. Concord, Contra Costa Co., 2. Berkeley, Alameda Co., 3. Big Creek, Monterey Co., and 4. Mt. Tamalpais, Marin Co. Beetles were transported to laboratory facilities at the University of California, Berkeley, and kept individually on damp sphagnum in a plastic, 5 cm diameter Petri dishes with snap-on covers. They were housed in a growth chamber with a day-length and temperature routine that simulated local conditions. Beetles were fed commercial dog food. To reduce the artifact of laboratory conditions beetles were collected with minimal handling to prevent premature release of defensive chemicals and processed within 10 days of collection.
Dissection and gland preparation
Various internal parasites are known to attack adult carabid beetles ( Thiele, 1977 ) and breeding conditions may have physiological and behavioral consequences that could impact this study. Therefore, breeding conditions and the macro-parasite load of individual beetles was determined by dissection after sampling. Individual beetles were killed by briefly freezing them. Abdomens were dissected under distilled water using a stereo-microscope. Testicular and accessory glands or ovary development was noted. Females with developed ova were further dissected and their spermatheca checked for sperm using a compound microscope. For individuals forced to spray to exhaustion, fluid volume in the gland reservoirs was measured and recorded.
For the determination of the relative amounts of formic acid, specimens were briefly frozen, their abdomens removed and pygidial glands excised. Each specimen's pair of gland reservoirs was retained if equivalent in size. One gland reservoir of each same-sized pair was sealed in a capillary tube for the quantitative analysis of formic acid. The other reservoir of the pair was placed on a microscopic slide and excess external water removed. For the accurate measurement of weight, multiple reservoirs of approximately the same size were accumulated on a single slide and the pooled series was used for weighing ( Table 1 ). After weighing the full reservoirs, they were crushed and all fluid was removed by wicking and evaporation at room temperature. The weight of the remaining glandular tissue (cuticle and muscle) was measured. The weight difference between the full and empty glands provides the weight of the fluid in the glands ( Table 1 ). The average for the series was used as an approximation of the individual weights.
Size measurements
As a standard measure of beetle body size, the area of the left elytron was used. Elytron length, from humeral angle to apex, and width along its widest point (approximate midpoint), were measured with a stereo-microscope equipped with an ocular reticule. In some studies the weight of an individual insect is reported, (e.g., Löfqvist 1977 ; Rossini et al. 1997 ), however, because weight varies depending on recent feeding, breeding condition, hydration, amount of stored fat body and quantity of stored defensive chemicals, weight is not a reliable metric for scaling size-related change in secretion output. The fixed size of the hard cuticle and relationship of elytra, which cover the abdomen to the capacity of the abdominal cavity, make elytron area (greatest length × greatest width) the preferred metric.
Spray weight measurements
To obtain spray weight measurements, each beetle was affixed with an apparatus consisting of a piece of wire bent on one end into a hook, and on the other into a loop embedded in cheese wax, which was warmed and affixed to the beetle. To prevent defensive chemical discharge while the apparatus was being affixed and to promote wax adhesion, each beetle was placed in a -17 °C freezer for 4 min. The wire/wax apparatus was held over a flame to partially melt the wax, and was then attached to the beetle's elytra just after the beetle was removed from the freezer. The beetle was then suspended from a metal frame for 4 min to allow for a return to room temperature and normal locomotory behavior. Weights were taken using a Cahn Instruments C-31 microbalance, set at the 250 mg weight range. To simulate the humidity conditions of the damp sphagnum, a wet paper towel was kept in the chamber to maintain a high moisture level. Weights were considered reliable if a fluctuation of 0.002 mg or less was observed over 10 s. After an initial weight was taken, the beetle was held by the attached wire over filter paper soaked in an acid-indicating phenolphthalein-KOH solution. Typically the right meta-leg was pinched using forceps mounted with super glue onto a clothespin. A standardized pinch force was produced by allowing the clothespin spring to press the forceps closed onto the beetle's leg. The presence of defensive chemical secretion was indicated by smell and color change on the filter paper. The beetle was immediately re-weighed, and the difference between the second and first weight values was taken to be the mass of defensive chemicals secreted. Beetles were then returned to their respective Petri dishes to be re-tested the following day.
To induce beetles to spray to exhaustion, the procedure was as above, except beetles were pinched and re-weighed until no color change was seen on the reactive paper and no odor was detected. Re-weighing in the absence of color change or odor resulted in weights comparable to those obtained for negative controls (n=3), which were weighed, allowed to remain undisturbed for 30 s, and then reweighed.
Of the 31 individuals of P. brunneomarginatus a spray reaction was successfully induced in 23 for the first round (day one) and 29 in the second round (day two). Twenty individuals were successfully weighed in both rounds. Of the 17 individuals of P. ovipennis used, spray was induced in 16 for the first round and 17 in the second round. Of the nine individuals of C. ruficollis used, the spray reaction was induced in seven in the first round and nine in the second round
Seven (three female, four male) P. brunneomarginatus and three (two female, one male) P. ovipennis were induced to spray until exhaustion.
Estimating amount contained in reservoirs at the time of a spray event
In the spray to exhaustion study the approximate quantity contained in the reservoirs was ascertained at the time of each spray event by summing weight differences from all spray events and then sequentially subtracting previous emission quantities ( Table 2 ).
Analytical chemistry methods
For GC-MS analysis, all reservoirs of the series in a capillary tube were carefully transferred into a vial (1 ml) filled with about 200 μl of dichloromethane. For a quantitative transfer, the empty capillary tube was thoroughly rinsed with dichloromethane and the rinsed fluid was carefully transferred into the vial containing the reservoirs. The reservoirs were crushed with a needle and the internal standard, 2 ml of pivalic acid solution (100 ppm) of dichloromethane was added to the crushed glands.
Chemicals . Formic acid and pivalic acid were purchased from Aldrich Chemical Company, www.sigmaaldrich.com . Potassium hydrogen phthalate and sodium hydroxide were purchased from Fischer Scientific Company, www.fishersci.com .
Instrumental . For the quantitative analysis of formic acid, one microliter aliquot of each sample was injected into a Shimadzu-QP5050 GC-MS ( www.shimadzu.com ) installed with a ZB-FFAP coated capillary column (30 m × 0.25 mm × 0.25 mm, Phenomenex, www.phenomenex.com ). The oven temperature was initially held at 40 °C for 3 min, raised 6 °C/min to 250 °C, and held for 10 min. Helium was used as the carrier gas at a flow of 1.6 ml/min, and a split ratio of 12:1 on the Shimadzu instrument. The injections were made in splitless mode by an auto injector (Shimadzu, AOC-20i) on the Shimadzu-QP5050 GC-MS. Injection was repeated three times for each sample. Mass spectra were obtained under 70-eV electronionization with selected ion monitoring (m/z 41, 44, 45, 46, 57, 102) mode.
Calibration curve . About 0.1 M NaOH solution was prepared and standardized with potassium hydrogen phthalate (110 °C for 2 h). The percentage of commercial formic acid was determined to be 98.58%) (w/w) by titration with the standardized NaOH solution. An internal standard, pivalic acid solution (100 ppm in dichloromethane), was accurately prepared. Stock solution (2500 ppm) of formic acid in dichloromethane was prepared and used to make 50, 100, 200, 300, 400, 500, and 600 ppm of standard solutions of formic acid. Each standard solution of formic acid (1 ml) and pivalic acid solution (1 ml, 100 ppm) was combined, and one microliter aliquot of the mixture was injected to a GC-MS. From the gas chromatogram, a calibration curve was plotted using the chromatographic area of formic acid/chromatographic area of pivalic acid versus the amount of formic acid/amount of pivalic acid, and the relative response factor was calculated
Statistical methods
The two tailed t-test was used to determine if the two rounds of spray weights were significantly different, or were possible artifacts of our handling method, and if spray weights differed significantly between males and females of a species. Pearson correlation analysis was used to look for within- and between-species correlation for individual body size and quantity sprayed. Regression analysis was used to examine the relationship between amount contained in the pygidial gland reservoirs and amount sprayed. The final spray event — the spray that voids all remaining fluid in the reservoirs — was not included in this analysis as this spray event would always equal 100% of remaining fluid. | Results
Dissections
All individuals in this study were found to be free of macro-parasites. All individuals forced to spray to exhaustion were found to have empty gland reservoirs. Within species breeding condition was found to be comparable in individuals of the same sex. All P. brunneomarginatus males had a minute, undeveloped testis and scarcely any fluid in the accessory glands while females showed no sign of developing ova. All P. ovipennis males had a well-developed testis and significant quantities of fluid in the accessory glands (male P. brunneomarginatus and P. ovipennis have only one testis ( Will et al., 2005 )). Of the eight P. ovipennis females seven contained between seven and sixteen well developed eggs. No sperm was found in the spermathecae of any female P. ovipennis . All C. ruficollis males had well-developed testes and significant quantities of fluid in the accessory glands. Females had between two and four well developed eggs. No sperm was found in the spermathecae of any female C. ruficollis .
Nine to eleven different compounds were identified from analyses of whole-gland contents of the three species in this study ( Table 3 ). Undecane, formic acid and doceyl acetate were the major compounds for C. ruficollis. Undecane, tridecanone, pentadecanone and formic acid are the major compounds present in P. brunneomarginatus and P. ovipennis.
Spray events
Difference in sequential weighing of negative controls was ∼ 0.01 mg. No significant difference was found between averages in round 1 and round 2 of spray weights within species ( P. brunneomarginatus (P = 0.49), P. ovipennis (P = 0.97) and C. ruficollis (P = 0.45)). Within species weights were pooled from both rounds of spraying to give the overall average for each species.
No statistical difference was found between males and females for spray weights within species ( P. brunneomarginatus (P = 0.55), P. ovipennis (P = 0.14) and C. ruficollis (P = 0.78)). Within species weights were pooled for both sexes.
Within species there was no correlation between size of an individual and the spray quantity emitted ( Figure 1A ). The Pearson correlation was calculated for spray quantity relative to elytral area for P. brunneomarginatus (r2 < 0.001), P. ovipennis (r2 < 0.032) and C. ruficollis (r2 < 0.001). For these three species average spray quantities were positively correlated to the average individual size for each species (r2 = 0.991, Figure 1B ).
On average individuals of P. brunneomarginatus emit 0.313 ± 0.172 mg of pygidial secretions per spray event (median 0.318 mg; ∼ 0.370 μ 1); individuals of P. ovipennis emit 0.337 ± 0.229 mg (median 0.261mg; ∼ 0.398 μ1); and individuals of C. ruficollis emit 0.197 ± 0.117 mg (median 0.142 mg; ∼ 0.233 μl).
In the spray to exhaustion study individuals of P. brunneomarginatus initially contained between 0.206 – 2.048 mg of secretion in their pygidial glands ( Figure 2A ) and P. ovipennis individuals ranged from 0.589 – 1.645 mg ( Figure 3A ). In P. brunneomarginatus three of seven individuals sprayed more in the second spray event, as did two of three P. ovipennis. Subsequent sprays tended to decrease in weight ( Figure 2B , 3B ).
Based on regression analysis, the amount emitted per spray event was positively correlated with the amount held in the beetle's reservoir at the time of spraying (r2 = 0.85, Figure 4 ). For P. brunneomarginatus the amount emitted in the first and second spray events was positively correlated with the amount held in the beetle's reservoir at the time of spraying (spray-1: r2 = 0.91, Figure 5A ; spray-2: r2 = 0.97, Figure 5B ). However, the third spray event was found to have a weak positive correlation (r2 = 0.24, Figure 5C ). Regression analysis of only those spray events where individuals of P. brunneomarginatus had reserves at or greater than the average spray quantity for the species (≥ 0.31 mg) show a strong positive correlation (r2 = 0.82, Figure 6A ), while spray events where beetles had reserves less than the average spray quantity (excluding final spray events) showed only a weak positive correlation (r2 = 0.41, Figure 6B ).
Relative quantities of formic acid
On average the amount of formic acid contained in a single gland reservoir of P. brunneomarginatus is 34.2%, P. ovipennis 73.5% and C. ruficollis 34.2% ( Table 4 ). | Discussion
Average spray events
Conspecific individuals showed no relationship between spray output and sex or body-size. There was no behavioral or physiological reason to expect a difference between the sexes of conspecifics. This is consistent with the expectation that males and females would encounter similar predators, requiring a similar defensive response. The average spray output did vary between species and was related to the average body size of the individuals sampled for that species. However, there was interspecific overlap ( Figure 1A ). Increases in spray distance as well as output relative to body size are expected as reservoir lumen area, quantity of muscle and abdomen size are known to be positively correlated ( Forsyth, 1972 ). A relationship of size to output was not found within species, perhaps because the within species size-variation was quite small relative to the variation in secretion output. This is also possibly an artifact of sampling from a narrow part of species' ranges and a small sample of very similar individuals. Given the strong correlation for mean spray quantity and body size between species, it is likely that if the full range of sizes for each species was sampled a similar positive correlation may emerge. This does limit the extent to which we can suggest these results will be able to predict quantities of spray released by other species. However, it is likely that formic acid producing beetles of similar size and breeding condition will produce similar quantities of defensive chemicals.
Spray quantity and lethality
The average individual spray event quantities in our study are well below the smallest quantity tested and found to be lethal to ants by ( Löfqvist, 1977 ). In that study the smallest quantity of formic acid or hydrocarbons used was 0.5 μl (∼ 0.57 mg). The largest average single spray quantity we found was in individuals of P. ovipennis , which produce less than two-thirds the minimal amount used by Löfqvist. Löfqvist ( 1977 ) remarked that the quantities of formic acid used were probably similar to what one or a few ants could produce, but the hydrocarbons were perhaps as much as “10 times the total gland content in one ant.” However, Löfqvist considered that many ants may respond to a single threat with many individuals simultaneously spraying toxins. Carabid beetles are not known to be social beyond brooding and mate guarding behaviors ( Anderson, 1984 ), and cooperative defense between individuals would not be expected. A single individual, however, can spray multiple times and does, typically, contain more than enough formic acid and hydrocarbon compounds to kill an individual ant. In the spray to exhaustion tests, five of the ten individuals emitted quantities equal to or up to about three times the 0.57 mg used by Lofqvist ( 1977 ) ( Table 2 ). However, it seems unlikely that lethal toxicity is used or required against ants. An irritating or incapacitating effect and general disruption by eliciting an alarm signal is probably sufficient action. Variation in the ratio of the amount of defensive chemical emitted (Q) to the response threshold of the target (K) (Q/K of Bossert and Wilson ( 1963 ), see below) would be expected to yield different results ranging from death at a high Q/K to a more information rich message at lower Q/K.
Alarm behavior
Several ant species have been shown to exhibit alarm behavior when exposed to formic acid and various hydrocarbons ( Hölldobler and Wilson 1990 ; Löfqvist 1976 ) and the interaction of carabids is thought to be both ecologically and evolutionarily significant (e.g. Darlington 1971 ; Hawes et al. 2002 ; Reznikova and Dorosheva 2004 ; Liebherr and Krushelnycky 2006). Carabid formic acid-based chemical secretions would likely elicit a similar alarm effect in ants. Because of the frequent similarity of the compounds secreted this has even been considered a case of chemical mimicry ( Blum 1980 ).
These data can be used to calculate a number of properties of a typical release and speculate on how this may affect ants. Three assumptions must be made to do this: 1) the diffusion constant (D) of formic acid in the sprayed mix is nearly the same as for formic acid alone (0.127cm2/sec ( Gibson et al. 1997 )), 2) the threshold value for an intense alarm behavior response to formic acid found for Formica rufa (K = 7×1015 molecules cm-3, Löfqvist 1976 ) is typical for ants, and 3) the formulae for diffusion from an instantaneous emission in still air can be applied as a reasonable model for an alarm semiochemical ( Bossert and Wilson 1963 ). The maximum radius of the threshold sphere is then R max = 0.527(Q/K) 1/3 ( Bossert and Wilson 1963 , formula 1.4) where R is the radius, Q is the number of molecules released at the point of origin, and K is the behavioral threshold density. The time it takes to reach this maximum is TR max = (0.0464/D)(Q/K) 2/3 ( Bossert and Wilson, 1963 , formula 1.5), where D is the diffusion constant of formic acid. Based on this calculation, the average emission quantities found for single spray events for each of the three species in our study, and the relative quantity of formic found for these species in this study, it would be expected that an individual beetle would create a maximum of a 5–8 cm diameter hemisphere of threshold density for formic acid in 9–22 seconds ( Table 5 ). Natural conditions in the field would be more complex than a simple hemispherical region of effect, but emissions based on this model do appear reasonable for beetles that would be expected to interact with ants and that are of a size-class where ants are a significant threat. The size of the region within the threshold density is sufficient to elicit behavioral responses from ants well away from contact with the beetle. It also persists long enough to allow a beetle to escape during the chemically induced confusion. Ants in immediate proximity to a beetle at the moment of spray release would of course be subject to a greater formic acid concentration than appears at R max .
Spray to exhaustion
None of the species we sampled delivered a uniform quantity over a series of sequential spray events. The averages for spray quantities we found are much more accurate than the methods used to estimate spray output by Rossini et al. ( 1997 ) for G. lecontei. If their methods were used for our spray to exhaustion data, P. brunneomarginatus estimates would range from 0.056 to 0.290 mg for individuals and would have a 0.17 mg average over all events, while P. ovipennis , would be 0.15–0.27 mg, with an average of 0.22 mg overall. These are both well below our estimates for a single spray because the Rossini et al. ( 1997 ) method calculates the average including the terminal spray event (whatever remains in the reservoir) and other events with low reservoir volumes (less than average spray quantity available). The turgid condition of the gland reservoirs achieved in the laboratory after 3.5 months by G. lecontei in the Rossini et al. ( 1997 ) study is probably rarely found in nature where the glandular secretion might regularly be used. Therefore the average spray quantities found in our study probably represent a more realistic measure.
There are a number of conceivable ways that beetles could apportion their gland contents through sequential spray events, e.g., the quantities could have been random, constant for all events, increasing or decreasing. Beetles in this study exhibited a trend of decreasing emission quantities in sequential spray events ( Figure 2 – 3 ). The quantity of fluid in the gland reservoir is apparently an important factor of, or at least highly correlated with, the quantity of fluid sprayed ( Figure 2 – 4 ). One might be tempted to propose that beetles are actively assessing their defensive chemical reserve and ejecting a minimal amount that allows them to administer multiple sprays. To some extent this is possible; however this trend can be explained mechanistically without invoking a feed-back mechanism in the beetle.
Action of the glands
The gland reservoirs are roughly ovoid in form, and have three layers of muscle, an outer layer running longitudinally and attached to the base of the efferent duct, an oblique middle layer attached to the dorsal lobe and an inner layer that attaches to the base of the collecting canal ( Forsyth 1972 ). Given that any muscle has a maximum potential contraction length, if each muscle layer was recruited sequentially its contraction would be acting on a sequentially smaller volume as the reservoir is emptied, resulting in mechanically declining emissions. This can be roughly modeled by considering the gland to be an ovoid whose volume can be calculated as two elliptical paraboloids, base to base. The only way beetles could maintain or increase emission quantity with this system would be to exert an ever increasing amount of muscular effort. The increase in spray quantity shown between spray event 1 and event 2 in more than half the beetles ( Figure 2B , 3B ) is possibly due to a greater muscular effort or a bilateral rather than unilateral spray response. The methods we used cannot distinguish between use of both or only one of the contralateral pair.
The emission-reservoir volume relationship holds only when the reservoir volume is at or above the average spray weight quantity ( Figure 6 ), as is normally the case during the first two sequential spray events ( Figure 5 ). There is only a weak positive correlation between the reservoir content and spray weight once the reservoir content is below the average emission quantity. Reservoirs at this point are presumed to be nearly empty and the inner cuticle variously in-folded ( Forsyth 1972 ), resulting in a very small inner volume.
Chemical constituents
The admix of lipophilic and hydrophilic compounds present in the gland reservoirs of the three species in this study are consistent with what is known for other members of these genera ( P. assimilus, P. magnus, P. protensus , C. fucipes , C. melanocephalus ( Kanehisa and Kawazu 1985 ; Kanehisa and Murase 1977 ; Schildknecht 1970 ; Schildknecht et al. 1964 ; Schildknecht et al. 1968a ; Schildknecht et al. 1968b )). However, these earlier studies only identified three or four major components in each species. We found a greater than twofold difference in the relative quantity of formic acid between P. ovipennis (73.5%) and the other two species (34%). This is notable, but not obviously explained by differences among these species. Platynus ovipennis is larger, more heavily sclerotized, flightless (as is C. ruficollis ) and restricted to the wetter forests of the west coast of North America. Calathus ruficollis and P. brunneomarginatus are both very widespread species in the western United States. Both are found in relatively drier habitats. Calathus ruficollis is common in open grassy areas, while P. brunneomarginatus is generally near a source of water, e.g. permanent and vernal streams or springs. Too little is known of the specific ecological interactions, diet and behaviors of these beetles to know if any of these aspects are correlated with formic acid content.
Limitations
A number of limitations of this study are important to point out. Sampling was specifically designed to limit the amount of within species variation due to physiological differences such as breeding condition and age. Samples were all collected from a very small number of sites in California and most of each species from a single site. The sample was of a comparable set of individuals in terms of probable age and physiological condition. This could mean the sample used is not necessarily representative of variation across the distribution of these beetles, and it may not reflect the potential effects of seasonal changes.
Our study also used a standard amount of stimulation to elicit the spray response. This means the full set of spray events are comparable measurements, probably representing the minimal stimulation needed to cause spray release. However, it does not address the question of possible variation in spray quantities that may be emitted under different levels of attack severity.
Our study establishes average quantities and variation of defensive chemicals in three carabid species. This finding provides a basis for further experimentation, using realistic quantities in bioassays, regarding the effect of the chemical constituents of carabid defensive compounds on ants and perhaps other likely predators, e.g. spiders, scorpions and other beetles. | This study is the first to measure the quantity of pygidial gland secretions released defensively by carabid beetles (Coleoptera: Carabidae) and to accurately measure the relative quantity of formic acid contained in their pygidial gland reservoirs and spray emissions. Individuals of three typical formic acid producing species were induced to repeatedly spray, ultimately exhausting their chemical compound reserves. Beetles were subjected to faux attacks using forceps and weighed before and after each ejection of chemicals. Platynus brunneomarginatus (Mannerheim) (Platynini), P. ovipennis (Mannerheim) (Platynini) and Calathus ruficollis Dejean (Sphodrini), sprayed average quantities with standard error of 0.313 ± 0.172 mg, 0.337 ± 0.230 mg, and 0.197 ± 0.117 mg per spray event, respectively. The quantity an individual beetle released when induced to spray tended to decrease with each subsequent spray event. The quantity emitted in a single spray was correlated to the quantity held in the reservoirs at the time of spraying for beetles whose reserves are greater than the average amount emitted in a spray event. For beetles with a quantity less than the average amount sprayed in reserve there was no significant correlation. For beetles comparable in terms of size, physiological condition and gland reservoir fullness, the shape of the gland reservoirs and musculature determined that a similar effort at each spray event would mechanically meter out the release so that a greater amount was emitted when more was available in the reservoir. The average percentage of formic acid was established for these species as 34.2%, 73.5% and 34.1% for for P. brunneomarginatus, P. ovipennis and C. ruficollis , respectively. The average quantities of formic acid released by individuals of these species was less than two-thirds the amount shown to be lethal to ants in previously published experiments. However, the total quantity from multiple spray events from a single individual could aggregate to quantities at or above the lethal level, and lesser quantities are known to act as ant alarm pheromones. Using a model, one directed spray of the formic acid and hydrocarbon mix could spread to an area of 5–8 cm diameter and persisted for 9–22 seconds at a threshold level known to induce alarm behaviors in ants. These results show that carabid defensive secretions may act as a potent and relatively prolonged defense against ants or similar predators even at a sub-lethal dose.
Keywords | Acknowledgments
We thank Robert Dudley, University of California, Berkeley, for very helpful discussion and information regarding beetle pygidial glands. This research was funded (in part) by the United States Department of Agriculture, Cooperative State Research, Education, and Extension Service in partnership with the California State Agricultural Experiment Station. | CC BY | no | 2022-01-12 16:13:44 | J Insect Sci. 2010 Mar 2; 10:12 | oa_package/08/da/PMC3014658.tar.gz |
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PMC3014659 | 20575744 | Introduction
The study of insect-plant interactions is currently one of the most actively investigated areas in chemical ecology, partly owing to its interesting perspectives for the development of new biopesticides with plant origins. These interactions involve numerous secondary plant metabolites that may interfere with behaviour, growth and/or development of insects. Phytoecdysteroids are secondary metabolites produced by many plants. They represent analogues of insect steroid hormones (ecdysteroids) that control insect growth, development, and reproduction ( Koolman 1989 ; Lafont 1997 ). It has been suggested that they form part of the plant's defences against phytophagous insects ( Lafont 1997 ; Adler and Grebenok 1999 ) and soil nematodes ( Soriano et al. 2004 ).
Several studies have investigated the effects of phytoecdysteroids on phytophagous insects. The exogenous application of phytoecdysteroids to a number of species resulted in marked growth and developmental disruption, e.g. in Spodoptera frugiperda ( Kubo et al. 1983 ), Bombyx mort ( Tanaka and Takeda 1993a , 1993b ), Lobesia botrana ( Mondy et al. 1997 ), Inachis io and Aglais urticae ( Blackford and Dinan 1997a ), and Bradysia impatiens ( Schmelz et al. 2002 ). This disruption involves a number of effects including inhibition of growth, induction of supernumerary larval instars, death without moulting, and death during or after induced moulting. However, certain insect species remain unaffected by dietary phytoecdysteroids. This is the case for Heliothis virescens ( Kubo et al. 1987 ), Heliothis armigera ( Robinson et al. 1987 ), Spodoptera littoralis ( Blackford et al. 1996 ), and Lacanobia oleracea ( Blackford and Dinan 1997b ). These species have developed effective detoxification mechanisms against ingested phytoecdysteroids.
Due to the obvious differences in the susceptibility of lepidopterans to ingested phytoecdysteroids, it is of interest to determine whether other lepidopteran pests, such as Plodia interpunctella Hübner (Lepidoptera: Pyralidae), can tolerate ingested phytoecdysteroids and of interest to analyze their relative susceptibility to different molecules of this family.
The Indian meal moth, P. interpunctella, is a world-wide insect pest of stored-products and processed food commodities ( Mohandass et al. 2007 ). In Morocco, this insect is a major problem during processing and storage of dried fruit such as dates ( Azelmat et al. 2005 ).
In this work, the effects of four phytoecdysteroids on the development of P. interpunctella were studied. The phytoedysteroids used were 20-hydroxyecdysone (20E), polypodine B (PolB), ponasterone A (PonA), and makisterone A (MakA). These were used in preference to other phytoecdysteroids because they are among the most common phytoecdysteroids present in plants ( Dinan 2001 ). In addition, the minimal structural differences between these molecules were assessed to determine their effects on toxicity toward P. interpunctella. | Materials and Methods
Insect rearing
P. interpunctella were collected as larvae infesting dates from the Errachidia province in the southeast region of Morocco. The larvae were reared under standard conditions at 28 ± 2° C with a relative humidity of 70 ± 5 % and a photoperiod of 16:8 L:D. Insects were placed in 0.25 L glass containers half-full of wheat flour as a medium. Emerging adults were removed and allowed to mate in new 0.25 L glass containers. Eggs were allowed to develop in their oviposition sites.
Under these conditions, P. interpunctella displays 5 larval instars and the life-cycle lasts 36 to 44 days.
Phytoecdysteroid extraction and purification
Phytoecdysteroids were isolated from two plants known for their high phytoecdysteroid content. The phytoecdysteroids 20E and MakA were purified from Ajuga iva Schreber (Lamiales: Lamiaceae), and 20E and PolB were purified from Silene nutans L. (Caryophyllales: Caryophyllaceae). PonA was prepared chemically from 20E ( Dinan 1985 ). The purity of all molecules was checked by HPLC and was superior to 95%. The structures of the 4 analogues are shown in Figure 1. Plants for extraction and purification of phytoecdysteroids were collected from different sites. A. iva was collected around the Tangier region (Morocco), and S. nutans was collected in the Pradelles region (Haute-Loire, France).
Classical procedures based on liquid-liquid partitions and different types of chromatography (low-pressure column chromatography and HPLC) were used for phytoecdysteroid extraction and purification (e.g. Báthori 1998 ).
Treatments
Each phytoecdysteroid was dissolved in 5% (v/v) methanol in distilled water. A volume of 5 ml was incorporated into 5 g wheat flour at 200 ppm. For control larvae, 5 ml of 5% methanol in distilled water were added to wheat flour. The solvent was evaporated from the diet at 35° C in an oven over a period of 48 hours. To verify that the effect was due to phytoecdysteroid ingestion and not to a deterrency effect, another experimental group (starved) was generated by transferring larvae into Petri dishes with no food. The choice of a concentration of 200 ppm was based on previous results that have been obtained with different concentrations of 20E. In fact, 200 ppm 20E corresponded to its EC50 on P. interpunctella larvae. The use of other phytoecdysteroids was to compare their effects to those of 20E and possibly to identify more efficient molecules.
Post-embryonic development
Fourth instar larvae were starved for 24 h prior to use to induce a higher feeding rate. Then, 10 larvae were placed in a Petri dish in the presence of either 5 g treated or 5 g control diet. Observations, including larval weight, mortality, cannibalism, pupation and adult emergence, were made every second day over 30 days. Mortality was identified by brown colouration with no observable movements. Cannibalism was noted by the disappearance of larval bodies from the Petri dishes. Larvae were maintained under standard conditions throughout the experiment. Five replicates were performed for treated, starved, and control larvae.
Statistical analysis
To determine the statistical significance of treatment effect, results were analysed by ANOVA Statistica Software using the Tukey HSD test ( Statistica 1997 ). A significance level of 0.05 was applied to all statistical tests. | Results
Weight
The results of weight change in treated, starved, and control larvae are shown in Figure 2. Phytoecdysteroid ingestion had a negative effect on the evolution of larval weight. During the first days, a slight decrease of weight of treated insects was recorded for each compound, except for PolB, where there was a slight increase in weight. This effect increased with time. Thus, 10 days after treatment started, weight loss was 33.4%, 34.1%, and 40.3% for larvae fed 20E, PonA, and MakA, respectively. For larvae fed PolB, the weight increased during the first four days before registering a decrease from the sixth day forward. Overall, the weight loss was only 6.3%. For starved larvae, the weight reduction was more pronounced (43%). In contrast, control larvae gained 28.6% in weight. Statistical analysis showed that the treatment with 20E, PonA, or MakA, as well as starvation, had a very highly significant, negative effect on larval weight evolution (p < 0.001). For PolB, the effect was not significant when compared with control larvae. The effect of starvation was significantly more pronounced when compared between the 20E and PolB groups (p < 0.001).
Mortality
Phytoecdysteroid ingestion also caused high larval mortality (Figure 3). The mortality started two days after the beginning of treatment for 20E, MakA, and PolB, and from the fourth day for PonA. It increased throughout the observation period. After eight days, it was 17.5%, 36.7%, 44%, and 76% for 20E, PolB, PonA, and MakA, respectively. Then, 22 days after the beginning of the experiment, it was 46.7%, 52.5%, 64%, and 84% for PolB, 20E, PonA and MakA, respectively. In starved larvae, mortality appeared after six days of treatment, and it was 60%) on day twelve. For controls, the larval mortality did not exceed 11% during the whole experiment. ANOVA analysis showed that for all treatments and for starvation there was a very highly significant larval mortality as compared with controls (p < 0.001). For starvation compared to treated groups, the significant difference was noted compared to MakA and PolB (p < 0.001).
Cannibalism
The presence of phytoecdysteroids in the larval diet provoked high levels of cannibalistic behaviour between larvae (Figure 4). We noted the disappearance of larval bodies in the batches treated with 20E, PonA and MakA from the second day, with a rate that varied between 4 and 6%. This percentage increased with time. It reached 10%, 16% and 26% for 20E, MakA and PonA, respectively. For PolB, cannibalism appeared only six days after the treatment began and it reached its maximum on day ten at 10%. For starved larvae, the cannibalism was more pronounced, being 40% after just six days. The rate of cannibalism in control larvae did not surpass 7%. Statistical analysis showed that the treatment with PonA, as well as starvation, had a very highly significant effect on cannibalism (P < 0.001) and a highly significant effect was observed for MakA (P < 0.01). The effect of starvation was highly more pronounced across with all phytoecdysteroid treatments (P < 0.001).
Pupation
It seems clear that pupation was affected by phytoecdysteroid ingestion (Figure 5). In larvae fed MakA, pupation was completely inhibited, as in starved insects (p < 0.001). However, for other molecules, precocious pupation appeared by two days after for larvae fed PonA or PolB and four days in the case of 20E as compared with control group. The maximum pupation percentage was 43.4%, 35%, and 8% for PolB, 20E, and PonA, respectively (p < 0.001). Pupation of control larvae began on day 8 and reached 79% over 28 d.
Adult emergence
From the pupae, the percentages of adults emerging were very different between experimentally treated larvae (Figure 6). Moreover, as for pupation, this developmental parameter was significantly affected by the presence of phytoecdysteroids in the larval diet (p < 0.001). For insects fed phytoecdysteroids, an acceleration of adult emergence by two and four days for PonA and PolB, respectively, was noted when compared with controls. In contrast, in the case of 20E, a delay of two days was recorded. At the end of the experiment (after 30 days), the percentage of adult emergence was 15%, 50%, and 54% for 20E, PonA, and PolB, respectively. For untreated larvae, it occurred over sixteen days and reached 72% by day 28. | Discussion
A number of studies have investigated insect susceptibility to dietary phytoecdysteroids. Most of the species examined have been lepidoptera, due to the pre-eminence of this order among phytophagous pests. Some species are very tolerant, such as H. virescens ( Kubo et al. 1987 ), H. armigera ( Robinson et al. 1987 ), and S. littoralis ( Blackford et al. 1996 ). Other species, Cynthia cardui and Tyria jacobaeae, for example, are semitolerant ( Blackford and Dinan 1997a ), while others are highly susceptible, such as S. frugiperda, Pectinophora gossypiella ( Kubo et al. 1981 , 1983 ), Acrolepiopsis assectella ( Arnault and Sláma 1986 ), A. urticae, and I. io ( Blackford and Dinan 1997a ).
The data presented in this report clearly show the susceptibility of P. interpunctella to phytoecdysteroid ingestion. The toxicity of phytoecdysteroids is associated with a decrease in larval weight, induced cannibalism, increased mortality, together with disruption of development. Such effects have been observed in P. interpunctella reared on other pure allelochemicals with insecticidal activity, such as harmaline ( Rharrabe et al. 2007a ) and azadirachtine ( Rharrabe et al. 2008 ). The addition of phytoecdysteroids to the diet of P. interpunctella resulted in an array of metamorphic disorders. These effects can be divided into three categories: (i) precocious pupation and adult emergence, (ii) delay in adult emergence (in the case of 20E), and (iii) reduction in pupation and adult emergence.
These larval growth and developmental defects could result from the cytotoxicity of phytoecdysteroids on the larvae's midgut. In fact, 20E ingestion caused, in a dose-dependent manner, a severe cytotoxicity of the midgut epithelial cells of P. interpunctella larvae ( Rharrabe et al. 2009 ). Tanaka and Yukuhiro ( 1999 ) also found that, in B. mori larvae fed 20E, the morphology of midgut epithelial cells was disrupted.
Exposure of P. interpunctella larvae to phytoecdysteroids induced significant cannibalism. Cannibalistic behaviour is a common phenomenon in arthropods, including insects ( Polis 1988 ). Cannibalism is commonly increased under stressful conditions, such as high population density or scarce food, when cannibals obtain a nutritional benefit ( Via 1999 ). In Helicoverpa zea larvae reared on Bacillus thuringiensis transgenic corn, the cannibalism was enhanced, and this diet appeared to represent a particularly stressful environment for this -insect ( Chilcutt 2006 ). For P. interpunctella, incorporation of harmaline into the larval diet provoked high levels of cannibalistic behaviour ( Rharrabe et al. 2007a ).
Starved P. interpunctella larvae displayed a marked disruption of development with total prevention of pupation, extensive cannibalism, and high mortality. However, the intensity of these perturbations differed from those caused by phytoecdysteroid ingestion. For example, the weight loss was more pronounced in starved larvae than in larvae fed phytoecdysteroids. On the other hand, mortality in starved larvae began four days later than in larvae treated with phytoecdysteroids, and it reached a maximum just after six days.
The effects of phytoecdysteroids on P. interpunctella depend on the analogue used and the developmental parameters observed. These results show that even the small structural differences between these four phytoecdysteroids significantly affect their toxicity toward P. interpunctella. Similar observations were recorded in other species. For example, in B. mori, the fate of the larvae depended on the ecdysteroid analogue; the addition of ecdysone caused supernumerary moults ( Tanaka and Takeda 1993a ), while 20E provoked synchronised moults at low doses and death at high doses ( Tanaka and Takeda 1993b ). This difference in sensitivity to ingested phytoecdysteroids might have different explanations, for example: (i) differences of the target site(s) of each analogue in larvae, (ii) different rates of uptake of each analogue by larvae, or (iii) difference in the ecdysteroids detoxification rates/pathways. Insects have developed diverse detoxification mechanisms toward ingested ecdysteroids, and in P. interpunctella larvae, these include oxidation at C3 and conjugation with fatty acids of the 22-OH group ( Rharrabe et al. 2007b ). Among the tested molecules, MakA showed the highest toxicity toward P. interpunctella larvae. This could be explained by the extra methyl group in position C-24 that may make 22-acylation less efficient and therefore may increase its half-life. This explanation, however, requires direct experimental evidence.
These results in P. interpunctella (a polyphagous species) do not fully fit with the hypothesis of Blackford and Dinan ( 1997a ), which proposed that the physiological and the behavioural sensitivity of insects to phytoecdysteroids is correlated to their feeding habits (monophagous = sensitive, oligophagous = semi-tolerant, and polyphagous = tolerant). The reported susceptibility of P. interpunctella may be correlated with the fact that it feeds on food commodities that are phytoecdysteroid-negative. At the other extreme, truly polyphagous species, such as S. littoralis and L. oleracea, feed on richly ecdysteroid-containing plants without exhibiting any abnormal effects, due to the presence of a very efficient detoxification pathway. These species have such a wide host-plant range that there is a very high probability that their diet will include phytoecdysteroids (in Blackford and Dinan 1997a ).
In summary, this work establishes the potent growth inhibitory effects of phytoecdysteroids on P. interpunctella. They caused significant disturbance to growth and development, confirming that phytoecdysteroids can be a valuable plant defence against insect pests. It would be of interest to check the activity of some other phytoecdysteroids on P. interpunctella and to perform similar experiments on other species in order to determine which compounds are the most efficient in every case. This could provide a reason for the presence of complex ecdysteroid cocktails in plants ( Bâthori et al. 1999 ).
Together, these results and the literature data indicate that phytoecdysteroids play a role as defensive substances against phytophagous insects. But, is it feasible to use phytoecdysteroids for crop protection against insect pests? To answer this question, much work is necessary in the laboratory and also in the field. For the moment, certain strategies could be foreseen. First, the use of such compounds by treating with the extracts of plants rich in phytoecdysteroids is difficult to conceive, except in confined spaces, like the conservation of stored-products in stock. Second, it is possible to imagine cultivated plants protecting themselves against insect pests by stimulating them to produce phytoecdysteroids. Indeed, the taxonomical distribution of plants producing ecdysteroids suggests that the genes necessary for their production are widespread in the plant kingdom ( Dinan 2001 ).
The use of phytoecdysteroids to fight against insect pests is not an alternative for the methods used currently, but they represent interesting molecules that could have an important part in integrated pest management strategies for cultures and stored products. | Using pure phytoecdysteroids isolated from Ajuga iva (L.) Schreber (Lamiales: Lamiaceae) and Silene nutans L. (Caryophyllales: Caryophyllaceae), plants known for their high ecdysteroid content, a study was carried out on the effects of ingestion of four different phytoecdysteroids (20-hydroxyecdysone, polypodine B, ponasterone A and makisterone A) on the growth and development of the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae) larvae when added at a concentration of 200 ppm in their diet. The experiments clearly showed the susceptibility of P. interpunctella to phytoecdysteroid ingestion. The toxicity of phytoecdysteroids manifested itself by a decrease in larval weight, induction of cannibalism and an increase of mortality, together with disruption of development. The severity of the phytoecdysteroid effect on P. interpunctella depended on the structure of the molecule. The results demonstrate that the minimal structural differences existing between these four phytoecdysteroids significantly affected their toxicity toward P. interpunctella. Makisterone A was the most toxic of the four compounds towards P. interpunctella larvae. In conclusion, phytoecdysteroids ingestion evokes disruptive growth effects on P. interpunctella. This work supports a role for phytoecdysteroids in plant defence against phytophagous insects.
Keywords | Acknowledgements
This work was supported by the program “CEEM- Pôle d'Excellence Régional-AUF” (FST. Tangier, Morocco). The authors are grateful to Dr. Laurence Dinan (University Paris 6, France) for critical reading of the manuscript.
Abbreviation
20-hydroxyecdysone,
polypodine B,
ponasterone A,
makisterone A | CC BY | no | 2022-01-12 16:13:44 | J Insect Sci. 2010 Mar 2; 10:13 | oa_package/d5/4c/PMC3014659.tar.gz |
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PMC3014660 | 20569122 | Introduction
Army ants are notorious for their raids of ant colonies of other species ( Schneirla 1971 ; Mirenda et al. 1980 ; Gotwald 1995 ). During raids, army ant trails are often very direct, with a strong column trail at the base and network of trails at the swarm front ( Rettenmeyer 1963 ; Burton and Franks 1985 ). The front line of these trails can span a width of 5m to 20m, depending on the army ant species ( Schneirla 1971 ; Gotwald 1995 ). Captured prey is immediately dismembered while prey that gets away is usually so weakened that it becomes susceptible to parasitism or secondary predation ( Gotwald 1995 ). Some army ants are generalists while others, such as species in the genus Neivamyrmex, are specialists, preying primarily on ant larvae, pupae, and/or adult workers ( Gotwald 1995 ). Thus, there is strong selective pressure on prey to evolve effective defensive strategies against army ant raids.
When under attack by army ants, some social wasps such as Protopolybia exigua and Angiopolybia pallens exhibit vibrational alarm calls that eventually lead to synchronized nest evacuation ( Chadab 1979 ; Chadab-Crepet and Rettenmeyer 1982 ). Other social wasps build nests on long, thin pedicels that are laced with ant-repellent chemicals or build nests that are completely enveloped, except for a small nest entrance where guards are present ( Jeanne 1970 , 1975 ). There are also instances where social wasps construct nests in trees that are occupied by ants that aggressively protect the tree from invading ants ( Chadab-Crepet and Rettenmeyer 1982 ). Other social insects, such as termites, employ highly specialized soldier workers with large, biting mandibles or head nozzles that spray a glue-like substance in response to ant raids ( Wilson 1971 ). However, under more intense raids by army ants, Macrotermes termites construct makeshift chambers that help protect the termite king and queen from attacks ( Darlington 1985 ).
In the desert grasslands of the southwestern United States, army ants particularly favor Pheidole ants because they are highly abundant in these habitats and because most species have a relatively small body size and/or lack specific defensive strategies for army ants ( Mirenda et al. 1980 ). In contrast, army ants tend to be deterred from invading Pogonomyrmex, Myrmecocystus, and Forelius ant colonies because of the large worker body size, physical strength, and/or defensive secretions of these species ( Mirenda et al. 1980 ). Other ants such as Stenamma expolitum and S. alas build elevated nest entrances to minimize detection by army ants; build adjacent, normally unoccupied chambers for hiding after evacuations; and close off entrances with a single round pebble ( Longino 2005 ). Army ant raids on Camponotus festinatus and Novomessor ( Aphaenogaster ) albisetosus often lead to nest evacuation ( LaMon and Topoff 1981 ; McDonald and Topoff 1986 ). In contrast, Atta leaf-cutting ants have a minor worker caste and multiple major worker size castes ( Fowler 1983 ). One of the defensive strategies Atta uses against raiding army ants is to deploy separate teams of large major workers (primary combatants) and smaller workers (assistant combatants) to counter-attack army ants outside their nest ( Powell and Clark 2004 ). The use of large major workers as the primary defenders shows the importance of having an extra caste size. Occasionally, Atta leaf-cutting ants will also use soil and organic debris to plug their entrances in response to army ant attacks ( Powell and Clark 2004 ).
The genus Pheidole is characterized by its dimorphic worker caste system consisting of minor workers and major workers ( Wilson 2003 ). Pheidole major workers, in general, have a disproportionately greater head size than minor workers (MH Huang and DE Wheeler, in prep). The worker caste system of P. obtusospinosa Pergande (Hymenoptera: Formicidae) is unusual in this genus because head size varies considerably within the major worker caste. Super majors of P. obtusospinosa have a greater absolute head size than smaller majors, but both have a similar head size to body size ratio (MH Huang and DE Wheeler, in prep). P. obtusospinosa and six other New World Pheidole species with a similar worker caste system are all found primarily in U.S. southwestern deserts and Mexico ( Wilson 2003 ). Interestingly, army ants in the genus Neivamyrmex have an overlapping geographical distribution ( Gotwald 1995 ) and are frequently found within the vicinity of various Pheidole species (MH Huang, personal observation). Interactions between army ants and Pheidole species with polymorphic major workers have not been previously documented.
Here, the head size distribution of workers of P. obtusospinosa is characterized to clearly define the size ranges that represent the different worker sub-castes. The difference in head morphology of different worker castes often correlates with the ability to perform various tasks. For example, smaller workers of some ant species are more efficient at feeding brood while larger workers are more effective defenders ( Hölldobler and Wilson 1990 ). After defining the different worker castes of P. obtusospinosa, field observations are reported of a successful, multi-phase nest defense strategy used by these ants against the army ant, Neivamyrmex texanus. In this system, only workers with the largest head size are involved in head-blocking at the nest entrance while workers of all sizes participate in aggressive combat outside the nest. | Methods and Materials
Worker colony demographics
A total of five P. obtusospinosa colonies were reared in the lab from founding queens collected in Tucson, Arizona in mid-July 2004. All colonies were kept in constant darkness, humidity, and temperature (30°C). They were sampled for major workers once in either March or April 2005, well after worker size distributions had stabilized [∼8 to 9 months after colony founding (Huang and Wheeler, unpublished)]. For each colony, all major workers were isolated into a large Petri dish, and a sample of that subpopulation was taken by randomly placing a smaller Petri dish upside-down into the larger Petri dish. All majors lying within the small Petri dish were collected and measured. The number of majors collected for each colony ranged from 76 to 111 individuals. Minor workers from each colony were sampled on different dates from major workers; three colonies were sampled for minors in February 2005 while two colonies were sampled in both February and May 2005. During each sampling date, 15 to 16 minors were randomly collected directly from each colony. A total of 446 majors and 111 minors were measured for the five colonies sampled. Head width measurements were made for both minor and major workers by using a microscope reticle. Head width was obtained by measuring the distance between the two most widely separated points on the two sides of the head, as seen from the frontal view. A cluster analysis was performed on the worker size distribution (with the assumption that there were two modes) to determine where the cutoff of the large and small major worker ranges were. The statistical package JMP 5.1 was used.
Field observations
Observations were made between 1430 and 1530 hours Mountain Standard Time on July 2, 2006 in an oak, sycamore, and juniper forest in Gardner Canyon in Tucson, Arizona (31°42.56′N and 110°42.58′W; Elevation: 1618 meters). The observed P. obtusospinosa colony nested at the base of a living oak tree and had a triangular-shaped nest entrance (base: ∼ 12mm height: ∼ 9mm) partially bordered by hard tree bark and the softer soil ground surface. The head sizes of the Pheidole major workers involved in headblocking were estimated using measurements of army ant specimens collected in the vicinity of the head-blocking event for calibration of the photographs taken. The army ant specimens were measured using a microscope reticle. The thorax length of the army ants collected ranged from 1.6 mm to 1.85 mm (mean = 1.72 mm, S.D. = 0.096, n = 11). Army ant thorax length (i.e., anterior margin of pronotum down to the beginning of the first petiole) was used because this body dimension varies the least between individuals and because it was the most visible in the photographs. The estimated size of the P. obtusospinosa nest entrance in the photographs was also used to confirm the head sizes of Pheidole major workers performing head-blocking. The nest entrance size was roughly measured using the dimensions of my fingernails. The average thorax length of the army ant specimens and the estimated dimensions of the nest entrance were used together to create the scale bar (3 mm) at the bottom right corner of the photographs shown in Figure 2 . This scale bar was ultimately used to estimate the head sizes of the Pheidole majors. | Results
Worker colony demographics
Minor workers of P. obtusospinosa have an extremely narrow size range (head width = 0.5mm to 0.7mm) and are discretely separated from major workers ( Figure 1a ). The major worker head width distribution was bimodal and ranged from 1.1 mm to 2.4 mm with smaller major workers present in colonies approximately three times as frequently as larger major workers. Results of the cluster analysis (assuming two modes) suggested that small majors range from 1.1 mm to 1.7 mm in head width while larger majors range from 1.7 mm to 2.4 mm. Figure lb shows that there is no major change in overall head shape when comparing small and larger majors, despite an increase in absolute head size. Here, the larger majors are referred to as super majors.
Field observations
At the field site, a strong column of army ants ( N. texanus ) was sighted running across a dirt path toward a P. obtusospinosa colony at the base of a large oak tree. Initially, the army ants focused their attacks on P. obtusospinosa majors of all sizes outside the nest entrance. Groups of 4–6 army ants attacked the P. obtusospinosa majors by biting and stinging them. The P. obtusospinosa majors bit back with their thick, crushing mandibles. Most of the attempts by individual majors at defending themselves were futile because they were outnumbered. Meanwhile, groups of P. obtusospinosa minor workers tried to assist majors by stretching out the legs of individual army ants to hold them down.
As the army ants attacked the P. obtusospinosa major workers outside the nest, the super majors guarding the entrance retreated into the nest and formed a blockade using their enlarged heads ( Figure 2a ); these super majors had head widths between 2 mm and 3 mm. The heads were packed tightly together with little space between them. Super majors forming the blockade remained motionless despite continuous biting and stinging attempts by army ants. Minor workers and small majors played no role in implementing the head blockade.
After failing to penetrate the nest entrance of the P. obtusospinosa colony, some of the army ants turned away and swarmed around the base of the oak tree, possibly trying to find another entrance into the colony. As the number of army ants at the nest entrance dwindled, the P. obtusospinosa super majors broke their head-blockade formation ( Figure 2b ) and stormed out of the nest. One group of P. obtusospinosa majors (large and small) attacked the army ants circling around the tree base from behind, while another group attacked the front line of the incoming army ant reinforcements by heading straight into the army ant foraging trail, occasionally dragging their abdomens on the ground. These actions of the second group of P. obtusospinosa majors resulted in the disorientation of army ant reinforcements at the front end of the trail.
The major sign of army ant disorientation was their change from moving in an initially straight path to moving in various, random, directions.
Without reinforcements, the group of army ants circling around the tree trunk was left behind. As this isolated group of army ants returned their attention toward the original nest entrance, both groups of P. obtusospinosa majors retreated into the nest, and the super majors resumed their head-blockade formation at the entrance ( Figure 2a ). Even after the P. obtusospinosa majors had abandoned the front line of the army ant trail, army ant reinforcements continued to show signs of disorientation. After 30 to 45 min of switching at least three times between the defensive head-blockade formation and the dual offensive attacks, the P. obtusospinosa colony drove away the raiding army ants. | Discussion
This study shows that P. obtusospinosa used a multi-phase defensive strategy against invading army ants that is distinct from strategies implemented by other ants. Similar to Atta leaf-cutting ants, P. obtusospinosa has multiple major worker castes in addition to the minor worker caste ( Figure 1a, b ). The Pheidole super majors in this study played a critical role in defending the nest because they blocked nest entrances with their large heads and assisted in combating the army ants outside of the nest. In contrast, large major workers of Atta leaf-cutting ants have never been shown to use head-blocking ( Powell and Clark 2004 ). Unlike P. obtusospinosa, the majority of Pheidole species are dimorphic, only having a minor worker caste and a single major worker caste with a narrow size range ( Wilson 2003 ). Without majors with extra large heads, these species may not use blockade formation as a defensive strategy. For example, P. desertorum and P. hyatti (both have dimorphic worker caste systems) immediately evacuate their nest in response to army ant attacks ( Droual and Topoff 1981 ; Droual 1983 ).
Ants such as Colobopsis nipponicus (Szabó-Patay 1928, as cited in Wilson 1971 ; Hasegawa 1993 ) and Cephalotes (= Zacryptocerus = Cryptocerus ) ( De Andrade and Baroni Urbani 1999 ) also use major workers for blocking nest entrances. Blocking of a nest entrance with the body is also known as phragmosis. In the case of C. nipponicus and Cephalotes, however, the majors have extreme modifications in head morphology for phragmosis, such as a disc-shaped or truncated head. Also, neither C. nipponicus nor Cephalotes exhibit aggressive combat outside of the nest.
Head-blocking has been suggested as a defense mechanism in other ants, such as C. nipponicus and Cephalotes, but this study is the first account of head-blocking in an ant species with super majors that have a noticeably enlarged head that is neither discshaped nor truncated. Therefore, extreme head modifications may not be necessary for implementing head-blocking. Having a more generalized head shape may allow P. obtusospinosa super majors to perform other tasks efficiently. Such additional tasks potentially include processing large food items, transporting large objects, and dismantling large enemies. The ability of P. obtusospinosa super majors to perform these additional tasks needs to be further investigated. In the case of Cephalotes, the disc-shaped heads of major workers are so morphologically specialized for head-blocking that their mandibles are reduced in size ( De Andrade and Baroni Urbani 1999 ). As a result, they are less competent at predation, processing intact prey, and transporting large items ( Wilson 1976a ; Cole 1980 ). In addition, Powell ( 2008 ) has shown that different Cephalotes species have an increasingly specialized head shape as both the size of the nest entrance and the number of workers involved in head-blocking decreases. If this trend is consistent in other ant genera, P. obtusospinosa super majors may maintain a relatively non-specialized head shape given the relatively large size of the nest entrance and the numerous super majors involved in head-blocking, as observed in this study.
Head-blocking, however, may be a more consistently effective strategy for ants such as Cephalotes because they nest in dried hardwood, with the nest entrance completely surrounded by a hard substrate ( Creighton and Nutting 1965 ; De Andrade and Baroni Urbani 1999 ). On the other hand, P. obtusospinosa has nest entrances that are surrounded by both a hard substrate such as a boulder or wood and a softer substrate such as variably loose soil. As a result, reliance on head-blocking alone in P. obtusospinosa may not always be effective because intruders may eventually dig through the soil. This may explain why the P. obtusospinosa colony observed in this study exhibited a multi-phase strategy consisting of both defensive and offensive tactics.
The behavioral specialization of P. obtusospinosa super majors observed here is consistent with the predictions stemming from the findings of Pie and Traniello ( 2007 ). They predict, by comparing allometric measurements of workers across various Pheidole species, that major workers are more behaviorally specialized than minor workers since there is a partial dissociation in head morphology between the two subcastes. Although they only examined minors and majors, observations in the present study suggest that this trend may be extended to comparisons between small major workers and super majors in species with polymorphic majors. Here, only the major workers with head widths in the largest size range ( Figure 1 ) are involved in the specialized task of headblocking.
One aspect of the multi-phase strategy used by P. obtusospinosa that needs further study is how their major workers were able to cause disorientation of army ant reinforcements at the front line of the army ant trail. Since Pheidole majors were seen dragging their abdomens on the ground surface in the vicinity of the army ant trail, Pheidole majors may have altered the trail by physical or chemical manipulation. Work by Couzin and Franks ( 2003 ) has shown that the initial trailfollowing stage of the army ant Eciton burchellii is disordered. However, these army ant foragers are eventually able to collectively decide on a common raid direction by assessing the relative position of nest mates along the swarm trail. Assuming that Neivamyrmex army ants have similar microdynamic properties in trail following, manipulations of the front line of the army ant trail by P. obtusospinosa majors may have disrupted the interactions between individual army ant foragers, thus contributing to the overall disorientation of the army ants observed here.
Since the observations here were only based on one invasion event on one colony, behavioral experiments with more field or lab colonies of P. obtusospinosa are needed to determine how frequently head-blocking is implemented by P. obtusospinosa against army ants. In addition, the stimuli involved in coordinating the initiation and termination of head-blocking by super majors must be further examined; this defensive phase may only be implemented when the army ant raid is very intense and direct. Pheidole dentata, for example, can go through a sequence of up to three defensive phases against invading fire ants depending on the intensity of the invasion ( Wilson 1976b ). Nevertheless, this study shows strong evidence that head-blocking by super majors at the nest entrance in combination with aggressive combat outside the nest can be an effective defensive strategy, at least for the one P. obtusospinosa colony observed. This strategy was successful even though P. obtusospinosa super majors do not have extremely modified head morphology specialized for head-blocking. Although evidence is yet to be provided, it is possible that this multi-phase defensive strategy can be used effectively by other P. obtusospinosa colonies, as well as other Pheidole species with polymorphic majors, for several reasons. First, there is strong selective pressure from army ant species that have a high preference for them as prey ( Mirenda et al. 1980 ). Second, the only seven described Pheidole species with super majors have a geographical distribution that completely overlaps with various army ant species ( Gotwald 1995 ; Wilson 2003 ). This coexistence further increases the selective pressure on Pheidole ants to evolve defensive strategies against army ant raids since the likelihood and frequency of raids most likely increases with the number of predator-prey encounters. If the above is proven to be true, the observations in this study could help partially explain why Pheidole species with polymorphic major workers have evolved convergently in multiple occasions ( Moreau 2008 ). | Assotiate Editor: Robert Jeanne was editor of this paper
Army ants are well known for their destructive raids of other ant colonies. Some known defensive strategies include nest evacuation, modification of nest architecture, blockade of nest entrances using rocks or debris, and direct combat outside the nest. Since army ants highly prefer Pheidole ants as prey in desert habitats, there may be strong selective pressure on Pheidole to evolve defensive strategies to better survive raids. In the case of P. obtusospinosa Pergande (Hymenoptera: Formicidae), the worker caste system includes super majors in addition to smaller majors and minor workers. Interestingly, P. obtusospinosa and the six other New World Pheidole species described to have polymorphic major workers are all found in the desert southwest and adjacent regions of Mexico, all co-occurring with various species of Neivamyrmex army ants. Pheidole obtusospinosa used a multi-phase defensive strategy against army ant raids that involved their largest major workers. During army ant attacks, these super majors were involved in blocking the nest entrance with their enlarged heads. This is the first description of defensive head-blocking by an ant species that lacks highly modified head morphology, such as a truncated or disc-shaped head. P. obtusospinosa super majors switched effectively between passive headblocking at the nest entrance and aggressive combat outside the nest. If this multi-phase strategy is found to be used by other Pheidole species with polymorphic majors in future studies, it is possible that selective pressure by army ant raids may have been partially responsible for the convergent evolution of this extra worker caste.
Key words | Acknowledgements
I am eternally grateful to Alex Yelich and Jennifer Patton for accompanying me on this one of many exciting excursions and taking all the amazingly vivid photographs. Thanks to D.E. Wheeler and T. Cao, M.J. Couvillon, A. Dornhaus, N. Duong, G. Holt, J. Jandt, E. Jones, and M. Lanan for feedback on the manuscript. Also, thanks to G. Snelling and R. Snelling for the identification of army ant specimens. | CC BY | no | 2022-01-12 16:13:43 | J Insect Sci. 2010 Feb 18; 10:1 | oa_package/29/24/PMC3014660.tar.gz |
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PMC3014661 | 20569123 | Introduction
During recent years an extensive survey on the foraging behavior of the most damaging subterranean termites of Khuzestan province (Iran) has been undertaken with a view to the development of appropriate strategies for control. Four genera namely Microcerotermes, Amitermes (Termitidae), Anacanthotermes (Hodotermitidae) and Pssamotermes (Rhinotermitidae) were the main genera collected in Iran. Current management of subterranean termites in Iran mainly involves the application of a soil insecticide to reduce/isolate their foraging populations ( Habibpour 2006 ). Organochlorine insecticides are still used in Iran to control subterranean termites. These compounds have undesirable environmental effects.
Microcerotermes diversus (Silvestri) (Isoptera: Termitidae) is an extremely destructive structural wood pest, and is considered to be the major species with a wide distribution in Iran, Iraq and Oman ( Edwards and Mill 1986 ). M. diversus was identified as the major pest of date palms ( Phoenix dactylifera L.) in Iran, Iraq and Saudi Arabia ( Logan and El Bakri 1990 ). Its nest is very complicated, diffuse and cryptic. The developmental pathway of the genus Microcerotermes has, as in all Termitidae, an irreversible bifurcation at the first molt, separating the nymphal/alate line from the worker / soldier line. Small workers are male, large workers are female, and soldiers develop from them. Workers are capable of developing into presoldiers at the first instar ( Roisin 1990 ). This species tends to form secondary nests containing both reproductives and brood. In locations where the ground water is high, secondary nests are usually built in above-ground sites such as tree trunks and wall voids.
Soil treatments with organochlorines, organophosphates and carbamates do not persist for long in this environment and proved ineffective against aerial colonies. In other parts of the world insecticidal baits have been shown to be an effective alternative to conventional soil insecticides for remedial termite control ( Su 1991 ). Bait systems can eliminate entire colonies of subterranean termites ( Grace et al. 1996 ; Su and Scheffrahn 1996 ). Methods for the control of termites including chemical control, baiting system and wood protection, have hardly been investigated scientifically in Iran.
Cellulose baits impregnated with insect growth regulators can be used for termite control ( Su et al. 1985 ; Jones 1984 , 1989 ; Jones 1984 , 1989 ; Haverty et al. 1989 ; Su and Scheffrahn 1993 ; Su 1994 ). Many insect growth regulators induce development of superfluous presoldiers and soldiers, cause morphological abnormalities (intercastes), defaunation of cellulose digesting microbes, and exert various levels of acute and delayed toxicity. However, termite responses to insect growth regulators are not uniform ( Jones and Lenz 1996 ). Most laboratory and field experiments with insect growth regulators so far have focused on the lower termites (Rhinotermitidae) and higher termites have been studied only rarely. For example, the efficacy of insect growth regulators to control the genus Microcerotermes spp. was studied in the field by Faragalla et al. ( 1985 ) and laboratory efficacy of flufenoxuron 10 DC on mortality of Microtermes obesi (Isoptera: Termitidae) in soils was evaluated by Ahmed and Farhan ( 2006 ) in Pakistan, but their results were inconclusive.
The objective of this study was to determine the general effects of the chitin synthesis inhibitor Flurox (flufenoxuron) on M. diversus including mortality and molting inhibition and differences in response between castes. | Materials and Methods
In Khuzestan province, in the period of 2006– 2007, termites (workers, nymphal 3 rd to 5 th instars and soldiers) were collected from a field colony of M. diversus in the campus of Shahid-Chamran University (without exposure to pesticides) using a collection trap unit as described by Sornnuwat et al. ( 1996 ) with some modifications. The unit was made of PVC pipe with a roll of toilet paper and a beech wood ( Fagus orientalis ) stake in it, buried in the soil. The chitin synthesis inhibitor tested was Flurox (common name: flufenoxuron), provided by Janssen Pharmaceutica, Belgium.
Laboratory no choice trial
Flurox was dissolved in methanol (Merck, www.merck.com ). Four concentrations (100, 500, 1000 and 5000 ppm) were prepared. Whatman No. 1 filter papers (9.0 cm in diameter) were uniformly wetted with 1 ml of each concentration or with 1 ml of methanol as a control. After overnight air drying each filter paper was then placed in a Petri dish (10 cm in diameter) and 1 ml of distilled water added to moisten the paper. One hundred workers, 30 nymphs and 2 soldiers were introduced into each dish. The soldiers were added to reflect natural caste ratios and prevent molting of workers to soldiers. Four replicates were prepared for each concentration. Experimental units (Petri dishes) were kept in constant darkness at 28 ± 1° C and 90 ± 5% relative humidity in an incubator. The number of dead workers and dead nymphs (caused by molting inhibition or direct toxicity) were counted daily for each treatment for 28 days after treatment. Dead workers were counted and removed daily from the test unit ( Remmen and Su 2005 ). Data were normalized by and then subjected to a one-way ANOVA (SPSS 16). Significant means were separated by the Tukey multiple range test ( P < 0.05), however, untransformed means are reported. In tables mortality was corrected by Abbott's formula.
Laboratory choice trial
Three concentrations of Flurox (500, 1000 and 5000 ppm) were prepared as above. Two filter papers (4.25 cm in diameter) were placed in each Petri dish (10 cm in diameter), and they were treated with 0.5 ml of one of the concentrations of Flurox or methanol alone as a control, and allowed to air dry. There was a space (1 cm) between the papers. Other conditions were similar to that of the no-choice tests. Analysis was the same as above. | Results
Laboratory no-choice trial
The mortality of workers and nymphs in all control groups was always lower than 4%. All concentrations of Flurox greater than 100 ppm elicited 100% mortality of the workers ( Table 1 ). Significant differences were observed among the concentrations throughout the experimental period ( Table 1 ). In all concentrations (with the exception of the control groups) nymphs died due to direct toxicity or during metamorphosis when nymphs showed symptoms of molting failure after exposure to Flurox, Figure 1 . The largest number of dead nymphs was observed at concentrations of 1000 and 5000 ppm ( Table 1 ).
Laboratory choice trial
In choice tests, Flurox treatment at 500 and 1000 ppm worker mortality varied from 32.5%, to 100% ( Table 2 ). The percentages of nymph mortality due to direct toxicity or during metamorphosis (molting failure) varied from 53%, to 100% ( Table 2 ). No difference in feeding behavior was noted at a concentration of 5000 ppm; they did not appear to be repelled by the presence of Flurox. Flurox was found to have relatively low (< 21%) contact toxicity, so the toxic effect was mainly due to consumption ( Table 3 ). The nymphs directly fed on the filter paper disks as they died faster without workers present (compare Tables 2 and 4 ). | Discussion
The development of alternative strategies to control multigenera/multispecies termite infestations that avoid pesticide accumulation in soil is an important goal. One of the commonly used strategies of integrated termite management is the use of baiting systems. Several novel slow-acting pesticides are being screened for the control of subterranean termites ( Hrdy et al. 2004 ; Rojas et al. 2004 ; Kubota et al. 2006 ; Yeoh and Lee 2006 ) because use of slow-acting insecticides in baiting systems can be distributed within, and kill, the entire colony. This is the first report of using Flurox as oral bait toxicant to control the genus Microcerotermes.
Concentrations of Flurox did not produce high mortality in M. diversus workers and nymphs at the first week, although this mortality was higher in no choice treatments. The results clearly show that Flurox is a slow acting toxicant, which is an important characteristic for use in baits. This compound could be distributed among termites in the field colonies when applied in bait stations. This agrees with other studies that showed delayed toxicity for chitin synthesis inhibitors ( Su and Scheffrahn 1993 ; Lenz et al. 1996 ; Kubota et al. 2006 ).
Flurox treatments against M. diversus in no-choice and choice feeding tests produced similar results at concentration of 5000 ppm. Flurox is a potent chitin synthesis inhibitor to control wood pests. Flurox has been found to be extremely active against wood borers, when applied in preventive or curative way ( Valcke and Pallaske 1995 ). During the reevaluation of molt-inhibitors it was found that Flurox could be an appropriate replacement for the pyrethroids in wood preservation ( Pallaske 1997 ).
Control of termites with a baiting method would be successful if the foraging behavior is properly understood. Laboratory evaluations, using groups of older workers and soldiers, can fail to accurately predict the impact of the insect growth regulators at the level of greatest concern, the colony ( Lenz et al. 1996 ). Usually, in M. diversus winged nymphal instars 3–5 were observed in feeding sites. They were found in the galleries leading to different food sources, including cellulose baits and monitoring stakes near the colonies in the summer ( Habibpour 2006 ). To simulate the conditions of field treatment, the nymphal instars were used in laboratory trials.
The results presented here show that concentrations of 1000 and 5000 ppm of Flurox have the potential to be used in termite baiting. The results suggest that the caste system and foraging behavior may be the most important factors affecting the success of termite control with baiting. We have an ongoing field program aiming to develop effective, cheap bait formulations with locally occurring bait toxicants and bait matrices for the control of termites in Iran. | Associate Editor: James Ottea was editor of this paper
Microcerotermes diversus (Silvestri) (Isoptera: Termitidae) is the most economically destructive termite in structures in southwest Iran. One sustainable control strategy that usually helps to reduce subterranean termite damage in buildings, is the use of insect growth regualtors in a suitable bait matrix that are safe to the user and the environment. In the laboratory assays described here, the delayed toxicity of Flurox, a chitin synthesis inhibitor, to M. diversus was evaluated under force-feeding and choice trials. Flurox induced worker and nymph mortality and incomplete ecdysis in nymphs of M. diversus under no-choice and two-choice feeding tests. These adverse effects may cause disruption of the caste balance in M. diversus , leading to the collapse of the colony. These assays determined concentrations of Flurox that can be used in bait formulations.
Key words | Acknowledgments
I would like to thank Dr Michael Lenz, CSIRO Entomology, Australia, for their valuable comments. Many special thanks to Drs. Alex Valcke and Dany Bylemans, Janssen Pharmaceutica, Belgium, for providing the samples of Flurox. This research was supported by Department of Plant Protection, College of Agriculture, Shahid-Chamran University, Ahwaz, Iran. | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Feb 18; 10:2 | oa_package/51/2b/PMC3014661.tar.gz |
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PMC3014662 | 20569124 | Introduction
A common way of organizing work in insect societies is through the division of labor in which individuals consistently perform a subset of tasks for periods ranging from a few days to their whole lives. At the colony level, the simultaneous performance of different tasks by different groups of specialized individuals is assumed more efficient than if tasks were performed sequentially by unspecialized πindividuals ( Oster and Wilson 1978 ; Jeanne 1986 ; Gordon 1989 ; Robinson 1992 ; Bourke and Franks 1995 ).
Task specialization is known to be influenced by the age of workers, their morphology, their genetic background and their individual experience in bees and in ants (for bees, see Robinson and Page 1988 ; Seeley 1995; Dreller and Page 1999 ; Huang and Robinson 1999 ; Fewell and Bertram 2002 ; Hurd et al. 2007 ; and for ants, see Gordon 1989 , 1996 ; Hölldobler and Wilson 1990 ; Theraulaz et al. 1998 ; Beshers et al. 1999 ; Tripet and Nonacs 2004 ; Heredia and Detrain 2005 ; Seid and Traniello 2006 ; Ravary et al. 2007 ). Several studies have shown that division of labor between workers is a dynamic phenomenon: the species-specific behavioral profile of each caste can be altered by day-to-day or seasonal changes in the colony composition due to predation, competitive pressure or changes in environmental resources ( Davidson 1978 ; Sorensen et al. 1984 ; Johnston and Wilson 1985 ; Wheeler 1991 ; Aarab and Jaisson 1992 ; Schmid-Hempel 1992 ; Tschinkel 1993 ; Gordon 1996 ; Passera et al. 1996 ). In order to maintain colony organization at a high efficiency level even after large perturbations in its caste ratio, one colony should ideally be able to fill-in for missing individuals. Over a long time-scale, a colony can increase the production of new adults belonging to the depleted caste through an adaptive demography response ( Calabi and Traniello 1989 ; Passera 1977 ; Passera et al. 1996 ). Over a shorter time-scale, a more flexible response can be achieved by the behavioral plasticity of individuals that perform tasks considered atypical for their caste ( Wilson 1980 , 1983 , 1984 , 1986 ; Detrain and Pasteels 1991 ; Cassill and Tschinkel 1999 ) and/or change their activity rate ( Wilson 1984 ; Brown and Traniello 1998 ).
Regarding dimorphic ant species, physical castes are expected to differ in their behavioral capabilities. From an ergonomic perspective ( Oster and Wilson 1978 ) it is assumed that the numerically dominant minor caste consists in “generalists,” workers that take care of all tasks necessary to colony development. Contrastingly, majors are “specialists” that perform only a subset of the minors' behavioral repertoire ( Wilson 1976a b , 1984 ; Calabi and Traniello 1989 ; Brown and Traniello 1998 ). Species of the ant genus Pheidole typically possess such dimorphic workers: minor workers perform most tasks within the nest and forage while majors, with their disproportionately large head, are specialized for colony defense, seed milling and food storage ( Wilson 2003 ). With its worldwide distribution and with its diversity (more than 900 described species, see Bolton 1995 ; Wilson 2003 ), Pheidole has become a key genus for investigating the adaptive nature of caste morphology among species living under different ecological conditions. Furthermore, the diversity of Pheidole has provided a framework to investigate questions concerning the evolutionary ecology of morphological variation as well as the interplay between morphological and behavioral specialization ( Pie and Traniello 2006 ; Moreau 2008 ).
Several authors have suggested that the breadth of the behavioral repertoire of minors has been a stabilizing factor, buffering the need for morphological specialization in specific tasks such as brood care and nest construction ( Bolton 1995 ; Pie and Traniello 2006 ; Moreau 2008 ). Conversely, the distinct head morphology of majors appears related to their behavioral specialization for a limited number of tasks. In conditions of highly disturbed caste ratio, majors may enlarge their repertoire size and/or increase their performance rate of some behaviors to compensate for a depletion of the minor caste ( Wilson 1984 , 1986 ; Calabi and Traniello 1989 ; Brown and Traniello 1998 ; Burkhardt 1998 ). However, this behavioral flexibility of majors needs to be examined by separating possible sampling size effects ( Sempo and Detrain 2004 ) from actual changes in their behavioral capacities.
The close relationship between caste morphology and behavioral specialization in the Pheidole genus has been highlighted on several species of the New World, which is assumed to be the cradle of this “hyperdiverse” genus ( Moreau 2008 ). The Old World species, Pheidole pallidula (Nylander) (Hymenoptera: Formicidae), offers a contrasting picture of division of labor. With a degree of morphological specialization similar to New World species, P. pallidula majors show an unexpectedly large behavioral repertoire: they are involved in defense ( Detrain and Pasteels 1992 ) as well as foraging ( Detrain and Pasteels 1991 ; Detrain and Deneubourg 1997 ), food storage ( Lachaud et al. 1992 ) and brood care ( Sempo and Detrain 2004 ). With such an extended behavioral repertoire of both minor and major castes, whether and how does social regulation occur in P. pallidula colonies? Would majors further enlarge their behavioral repertoire following deep changes in the colony's caste ratio? If their behavioral repertoire remains unchanged, would majors change their activity rate or their engagement in social behaviors within the nest? | Methods
Collection and rearing of colonies
P. pallidula colonies were collected on rocky calcareous in Gonfaron (France). Colonies were composed of one queen and 4,500 to 5,500 workers. Eggs, nymphs and larvae belonging to all three instars of P. pallidula were present in the colony. The worker caste consisted of 10 to 12% majors, which was a percentage close to that reported in the field by Passera ( 1977 ).
In the laboratory, colonies were housed in moistened nest tubes (length: 16 cm; diameter: 1.5 cm) placed in plastic trays (length: 30 cm; width: 20 cm; height: 4 cm). The plastic trays' walls were coated with Fluon to prevent ants from escaping. In the foraging area, ants had permanent access to water and brown sugar solution (concentration: 1M), and they received one dead cockroach ( Periplaneta americana ) per week. Colonies were maintained in the dark at 28°±1°C which is a temperature that maximizes the production of major workers among larvae ( Passera 1974 ).
Definition of behavioral acts
Inner-nest behaviors possibly performed by P. pallidula workers were listed based on preliminary observations ( Sempo and Detrain 2004 ) and data reported for other Pheidole species ( Wilson 1976a , 1984 ; Calabi et al. 1983 ; Patel 1990 ; Brown and Traniello 1998 ).
Queen care was excluded as a possible behavior because all tested colonies were queenless. There were 38 remaining behaviors included in the P. pallidula repertoire. These behaviors were classified in the following main categories: egg care, care of first and second instars larvae, care of third instar larvae, care of pupae, cleaning the nest, self-grooming, allogrooming, antennal contacts and trophallaxies. Ants were arbitrarily considered inactive if they did not move or engage in any tasks for at least 5 seconds.
Worker behavior in colonies with modified caste ratio
Experimental colonies were reared in nests (4 cm × 4 cm × 0.2 cm) dug in a plaster of Paris layer poured in a polyethylene box (15 cm × 10 cm × 7 cm). A red glass plate covered the top of the nest to create the darkness necessary for the ant settlement while allowing observation of the brood and of the ants' behavior within the nest.
From 16 mother colonies, 56 queenless colonies were made consisting of 10, 25, 50, 60, 75 or 90% of majors. For each of the tested caste ratios, excepting 10 and 90% of majors, four genetically distinct colonies were used. For extreme caste ratios (10 and 90%) a higher number of colonies (n=20) were required: these were made from 16 distinct mother colonies with four of them being used twice. The use of 16 additional colonies containing 10% and 90% implies that for these caste ratios only, 4 pairs of colonies come from the same 4 mother colonies.
Each of the colonies were made by picking up ants from all parts within the mother nest as well as from its foraging area. After allowing workers to acclimate to their experimental nest for 24 hours, brood was deposited in front of the nest entrance allowing workers to retrieve it into the nest chamber. The amount of brood introduced in each experimental nest was arbitrarily fixed to 15 eggs, 15 larvae of first and second instars, 15 third instar larvae, 13 minor pupae and 2 major pupae.
The proportion of majors among workers staying within the nest was significantly correlated to their proportion among the 100 ants introduced in the setup (Spearman rank correlation: rs = 0.92, n = 260, P < 0.0001). Since the fraction of majors introduced in the setup is highly representative of the fraction of majors actually present within the nest, it is used as the X-axis unit for all figures presented hereafter.
The first set of behavioral observations took place two days after the introduction of brood. This time allowed workers to organize brood spatially and to re-allocate tasks within the new experimental set-up. For each colony, five sessions of behavioral observations were carried out at 9a.m., 12p.m., 3p.m., 6p.m., and 9p.m. respectively. Each observation session consisted of scanning the entire colony and recording the behaviors performed by every minor and major workers present within the nest. The behavioral repertoire of each nest was determined by pooling results from these five observation sessions. The nest was covered with a grid network (4cm × 4cm) composed of 16 numbered quadrates (1cm × 1cm) to systematize observations during the scanning.
Four different colonies were used for each tested caste ratio (25%, 50%, 60% or 75% majors). For each of the five observation sessions, the behavior of all individuals in the nest was recorded once through a systematic scanning of all quadrates. Consequently, the proportion of observed individuals belonging to one caste was directly related to their proportion within the colony. Knowing the influence of the sampling size on the estimated behavioral repertoire size ( Sempo and Detrain 2004 ), the number of observations made on the two castes was balanced for the two extreme conditions of caste ratio (10% majors and 90% majors). Therefore, more scanning of the minority caste (i.e. majors in 10 % majors colonies and minors in 90% majors colonies) was performed by observing 20 colonies instead of 4.
To investigate social regulation in colonies with modified caste ratio, how the major caste adjusted its relative investment in social behaviors including brood care, allogrooming, trophallaxies, and social interactions with adults to compensate for the minors' depletion was determined. The “non-social behaviors” category included all other activities such as self-grooming, feeding on an insect, nest maintenance and movement inside the nest. In social hymenoptera, colony size is one factor influencing division of labor with workers being less specialized in small nests than in mature, large colonies ( Bourke and Franks 1995 ; Karsai and Wenzel 1998 ). To check for such a colony size effect for a normal caste ratio (90%) minors) on P. pallidula repertoire size, the frequency distribution of minors' behavior in the experimental colonies (100 ants) was compared to that observed in large, mature nests (around 5,000 ants, from data of Table 1 in Sempo and Detrain 2004 ).
Statistical analysis
Means are expressed with standard deviations. Probability values are given for two-tailed tests and the null hypothesis was rejected at p < 0.05. Survival curves were drawn to account for the evolution of the behavioral repertoire size of majors with sampling size. The statistical difference between two survival curves was determined by comparing the slope and the elevation of the regression lines obtained after curve linearization through a natural logarithmic data transformation ( Zar 1999 ). The activity rates between castes were compared using a paired t -test. A Spearman rank test was used to test the correlation between the percentage of active individuals and the fraction of majors in the colony. The influence of the colony caste ratio on the performance rate of castes was tested using a Kruskal-Wallis test. The influence of colony caste ratio on the total number of social behaviors performed by both castes was determined by using a one-way ANOVA Test followed by a Tukey-Kramer Multiple Comparisons Test. | Results
Social regulation through extended repertoire of majors
It is known that the number of observations made on one caste may modify the observer's perception of its behavioral repertoire ( Fagen and Goldman 1977 ; Sempo and Detrain 2004 ). In these experiments, the sampling size performed on one caste is directly related to its proportion within the nest chamber. Therefore, this estimate of majors' repertoire size is expected to vary with their ratio in the colony. In particular, when majors are few, their repertoire (the number of different behaviors observed) could be underestimated due to rarely occurring behaviors missing. To account for such a possible bias, a rarefaction curve was drawn out from the data set in which majors were the most frequently observed (i.e. colonies with 90% majors). Such a curve predicts how a decrease in sampling size alters an estimate of majors' repertoire. This mathematical tool helps to determine, in colonies with a modified caste ratio, whether changes in majors' repertoire size are due either to changes in sampling size or to genuine changes of their behavioral profile. For each tested colony composition (10, 25, 50, 60, 75 and 90% of majors; Fig. 1 ), the observed repertoire size of majors fit the value predicted from the rarefaction curve for the same sampling size. The apparent increase of majors' repertoire as a function of their ratio in the colony is thus mainly explained by an increased sampling instead of an enlargement of their repertoire. This statement is confirmed by the wide repertoire displayed by majors even when they account for only 10% of the nest population. Indeed, there is a similarity between the rarefaction curves that were drawn out from the 10% majors and from the 90% majors experiments. After a linearization of these two rarefaction curves using natural logarithmic data transformation, the slopes and elevations of the resulting regression lines were not statistically different ( Fig. 1 ; test of equality of slopes: df = 115, t = 0.3; p<0.05; test of equality of elevations: df = 116, t = 0.6, p<0.05). The low level of behavioral specialization of majors is not due to a bias related to the small size of experimental nests (100 ants). Indeed, there is a wide overlap with the behavior expressed by majors that live in large, mature colonies of around 5,000 ants ( Table 1 ). Even in these large colonies, the division of labor does not rest on a highly specialized worker caste performing strictly compartmentalized tasks.
Even though the size of the majors' repertoire is large, the type of acts performed by the majors may differ from those expressed by the minors. Therefore, the behaviors expressed by majors was compared to those expressed by minors in colonies where they, the majors or minors, are the most numerous and thus the more likely to cope with everyday tasks (i.e. the 90% majors and 90% minors colonies, respectively) ( Table 1 ). Regarding social interactions between adults, majors antennated nestmates twice more frequently than minors ( Table 1 : 0.406 versus 0.243 for antennations done by majors or by minors, respectively). On the other hand, the two castes displayed, at similar frequencies, allogrooming (0.083 for minors vs. 0.078 for majors), regurgitation (0.036 for minors vs. 0.034 for majors) and aggression (0.003 for minors vs. 0.001 for majors). Most differences in frequency values are related to between-caste interactions since the likelihood of one caste interacting with another will depend on the numerical abundance of its partner. For instance, the frequency of allogrooming is higher towards a minor (0.039) in a 90% minor colony than in a 90% major nest (0.004). Moreover, one cannot detect any preferential interactions between individuals of the same caste. An example is that the percentages of allogrooming directed towards a worker of the same caste (For minors: 83%; for majors: 89.1%) is always close to the proportion of this caste in the colony (90% in the two experimental conditions, Table 1 ).
P. pallidula castes also differ in the ways they care for brood both qualitatively and quantitatively. Although most brood-care tasks are performed by majors, they were never seen performing the following 4 brood-care behaviors: lick the eggs; feed first, second and third instar larvae; or carry and roll pupa. Likewise, in the minor caste these behaviors have a very low occurrence (relative frequency < 1%), even though they are important for brood development. Castes, then, differ mainly in a quantitative way since minors are twice more often involved in brood care than majors (relative frequency for minors: 0.205; for majors: 0.104, Table 1 ).
Social regulation through increased activity rate
As shown above, the depletion of minors in a colony did not induce qualitative changes in the behavioral repertoire of majors.
Nevertheless, social regulation can still occur through quantitative changes of the behavioral profile, by altering either the global activity level of majors or their performance through quantitative changes of the rate of a subset of behaviors. In our study, the activity level of ants within the nest differed between castes. In small, normal colonies (10% majors), minors were involved in more inner-nest tasks than majors were. The activity rate, the percentage of active individuals among all workers of one caste, reached 70.1% ± 10.8 for minors but only 50.8% ± 17.3 for majors ( Fig. 2 ; mean ± SD, n = 20; paired t-test: t19 = 7.5, p0.0001). For all of the tested caste ratios, minors continued to display a higher activity rate than majors ( Fig. 2 ). The activity level of minors even increased slightly following their depletion in modified colonies. A significant correlation was observed between the percentage of active minors and the fraction of majors in the colony ( Fig. 2 ; Spearman rank correlation: r = 0.37, n = 56, p < 0.01).
Majors did not significantly change their average activity rate regardless of the composition of the nest population ( Fig. 2 ; Spearman rank correlation: r = -0.23, n = 56, p = 0.09). Indeed, majors always kept a low activity rate ranging between 40% and 50% ( Fig. 2 ). This suggests that P. pallidula majors are not a stand-by caste, since they did not shift from a resting to an active status in order to compensate for the depletion of minor workers.
Social regulation through increased performance of social behaviors
Since the caste ratio did not influence the size of the behavioral repertoire or the global activity level expressed by minors and majors, social regulation could be restricted to a subset of behaviors that matter for social cohesion and ergonomic efficiency. Therefore, 28 social behaviors were considered out of the P. pallidula repertoire, including all brood care behaviors and all contacts between adults (antennal, allogrooming, trophallaxies, etc.).
For each caste and for each colony composition, the relative frequency of these social behaviors was calculated. The comparison of these frequencies obtained for the different colony compositions highlights the relative influence of the caste ratio on social behaviors. For minors, no significant changes were observed in the frequency of social behaviors as the proportion of majors increased in the nest ( Fig. 3 ; Kruskal-Wallis test: KW = 6.96, n = 52, p > 0.05). Thus, minors did not compensate for their large depletion in the colony by performing more social behaviors.
Likewise, changes in the colony caste ratio did not significantly alter the performance rate of social behaviors by majors ( Fig. 3 ; Kruskal-Wallis test: KW = 6.14, n = 52, p > 0.05). Consequently, no conclusion can be drawn on a possible compensation of minors' depletion by majors shifting from idle state to higher implication level in social behaviors.
Finally, at the colony level, the total social investment of both castes could be influenced by a changed caste ratio. A significant decrease in the total number of social behaviors was observed (ANOVA Test: p <0.0001; Tukey-Kramer Multiple Comparisons Test: p < 0.05 only for comparison between colonies composed of 10% versus 25%, 50%, 60%, 75% or 90% majors) ( Fig. 4 ). As soon as the colony differed from its natural composition (10% majors), the average number of social behaviors observed per scanning was lower and stood between 9 and 17.8 behaviors for colonies composed of 25% majors or more ( Fig. 4 ). The colony was thus able to keep social behaviors at a relatively high level of performance, even after a large depletion of minors. | Discussion
It is commonly admitted that, in the dimorphic Pheidole ant genus, minors carry out nearly all colony tasks while majors are specialized and display only a small part (13% to 19%) of the minors' behavioral repertoire ( Wilson 1984 ; Brown and Traniello 1998 ).
Besides, it was reported that majors can act as an emergency stand-by caste by enlarging their repertoire in order to compensate for minors depletion due to demographic changes or predation ( Wilson 1984 ). However, the role of majors in social regulation should be reconsidered by paying attention to possible bias due to sampling size. For instance, the largest majors' repertoire ( Wilson 1984 ) was reported for colonies composed of only majors which also implies the largest number of observations (more than 500 majors observed). As this sampling size was up to 20 times higher than on colonies with “natural” caste ratio, the extended repertoire of majors can simply result from the observation of less frequent behaviors. Here it is shown that, in P. pallidula , changes in the size of majors' repertoire with colony caste ratio can be explained simply by sampling size effects without invoking qualitative changes in majors behavioral profile (see also Jaisson et al. 1988 ; Sempo and Detrain 2004 ). Whatever the caste ratio, the majors' repertoire is strikingly similar to that of minors. Even though they are morphologically specialized with their hypertrophied head and powerful mandibles, P. pallidula majors perform all tasks excepting some brood care behaviors and are nearly as totipotent as the minors.
Besides changes in the behavioral repertoire size, social regulation can also take place through an increase in the number of active individuals and/or through an enhancement of individual activity rate. Indeed, inactive individuals constitute a reserve workforce that can assume new tasks depending on colony needs. Those inactive individuals that have no fixed role belong mainly to a transition group intermediate in behavior and age between nurses and foragers (e.g. Lasius niger ( Lenoir and Ataya 1983 ) and Cataglyphis cursor ( Retana and Cerda 1991 )). In polymorphic ant species, differences in the activity level between workers are usually related to body size, with minors being more active than majors as shown in Pheidole guilelmimuelleri and P. pubiventris ( Wilson 1984 ), P. morrisi ( Patel 1990 ), Atta sexdens ( Wilson 1980 ), Erebomyrma nevermanni ( Wilson 1986 ), Megaponera foetens ( Villet 1990 ) or Solenopsis invicta ( Mirenda and Vinson 1981 ; Sorensen et al. 1984 ).
Likewise, in the dimorphic P. pallidula ant species, inactive individuals are nearly twice more frequent among majors than among minors. Nevertheless, whatever the colony caste ratio, the activity level of P. pallidula majors is rather high and quite stable in comparison with other Pheidole species (see Wilson 1984 ; Brown and Traniello 1998 ). For instance, the ratio of the majors to minors activity rate remains stable at around 0.65 in P. pallidula , while it varies between 0.05 and 0.52, depending on the caste ratio in P. guilelmimuelleri ( Wilson 1984 ). The relatively high activity level of P. pallidula majors cannot be related to a lower degree of morphological specialization as predicted by the ergonomic optimization theory ( Oster and Wilson 1978 ). Indeed, the relative head widths of the minor to the major caste are remarkably similar being 0.39 for P. guilelmimuelleri ( Patel 1990 ) and 0.42 for P. pallidula ( Detrain 1989 ).
One may question the adaptive value of maintaining a higher proportion of inactive ants among majors since, in P. pallidula , resting majors do not participate in social regulation even after large perturbations of the colony caste ratio. Part of the answer lies in the existence of replete majors with distended gaster that account for up to 32% of the total P. pallidula majors population ( Lachaud et al. 1992 ) and for nearly 50% of inner-nest majors (personal observation). This sub-caste of majors is mainly located in quiet nest areas far from the nest entrance (personal observation) or near the brood area ( Sempo et al. 2006a ), does not forage outside the nest or defend it, and is characterized by a very low activity level ( Lachaud et al. 1992 ). Replete majors were also found in other polymorphic ants ( Camponotus spp. ( Wilson 1974 ; Espadaler et al. 1990 ; Hasegawa 1993 ), Myrmecocystus mexicanus ( Conway 1990 ), P. hortensis ( Calabi et al. 1983 ), P. morrisi ( Yang 2006 ) and Solenopsis invicta ( Mirenda and Vinson 1981 )). In polymorphic ants, a distinction can thus be made between two types of inactive majors: (1) idle unspecialized majors that become active due to colony need for an additional workforce as in many Pheidole species ( Wilson 1984 ; Brown and Traniello 1998 ) and (2) replete majors that stay inactive even after large demographic perturbations (as for P. pallidula majors), acting as living reservoirs that deaden food shortages.
As shown above, P. pallidula societies do not compensate for the depletion of minor caste either by extending the behavioral repertoire of majors or by increasing their activity level. However, among the active individuals, some social regulation may also occur by switching toward activities that are essential for social cohesion (i.e. allogrooming) or for colony survival (i.e. food exchanges and brood care).
For instance, following the removal of one age-class or a traumatic change in the age structure, workers are first and foremost reallocated to social tasks ( Lenoir 1979 ; Meudec and Lenoir 1982 ; McDonald and Topoff 1985 ; Calabi and Traniello 1989 ). Similarly, in polymorphic ants, major workers can show a high sensitivity to a shift in the colony's caste-ratio (i.e. from less than 10% to 90%) majors) as they increased by 15 to 30 times their rate of social behaviors (as shown for three Pheidole species in Wilson 1984 ). Unexpectedly, in P. pallidula , the rate of social behaviors performed by majors stayed stable regardless of the colony caste ratio. However, due to their high social investment (≈ 60% of the total number of acts), majors maintain brood care and other essential tasks at a satisfactory baseline level, even in colonies almost deprived of its minor caste.
The ergonomic resiliency of an ant colony relies on its ability to cope with changes occurring over short and long time-scales ( Oster and Wilson 1978 ). In polymorphic ant species, short-term regulation can rely upon the ability of specialized majors to express new, atypical behaviors and/or to increase their performance rate of social activities ( Wilson 1984 ). This short-term behavioral flexibility of the major worker, increasing the rate of activity as well as the behavioral repertoire in case of minor depletion is well described for different species such as Pheidole guilelmimuelleri and P. pubiventris ( Wilson 1984 ), P. morrisi ( Patel 1990 ) and P. dentata ( Burkhardt 1998 ; Seid and Traniello 2006 ). However, this stand-by caste status of majors is not the common rule for all Pheidole species. Indeed, P. pallidula majors perform an almost full minors repertoire and exhibit a relatively high level of social activities under all circumstances (not only in severe crisis of minors depletion). Secondly, an adaptive demography process may occur to face long-term changes in colony needs and/or environmental constraints (season, predation, etc.). Ideally, the production of majors is expected to be finely tuned to fill-in tasks for which they are specialized, as shown by the increased number of P. pallidula majors in the presence of competitors ( Passera et al. 1996 ).
One, however, may notice that, even in similar environmental conditions, there is still a high variability in the caste ratio between colonies of the same species ( P. dentata ( Oster and Wilson 1978 ), P. morrisi (Bhatkar and Whitcomb in Patel 1990 ; Yang 2006 ), P. pallidula ( Passera 1974 )). Hence, it could be more advantageous for an ant society, as observed in P. pallidula , to dispatch each caste into almost all colony tasks except those which cannot be physiologically or morphologically achieved (for example, in P. pallidula : majors do not lay the recruitment trail ( Ali et al. 1988 ; Detrain et al. 1991 )).
Without denying the existence of behavioral flexibility among physical castes in ants, this paper has stressed alternative schemes of social regulation. Independent of any qualitative or quantitative changes in the behavioral profile of one caste, other factors, such as a spatial reorganization of ants within the nest, could participate in social regulation. In this respect, differences between castes in aggregative patterns ( Sempo et al. 2006a , b ) would deserve further investigations in order to be coupled to the efficiency and flexibility of task performance by each worker caste. | We investigated whether the physical castes of the dimorphic ant Pheidole pallidula (Nylander) (Hymenoptera: Formicidae), are involved in determining within-nest activities and how their social investment in everyday tasks is influenced by large changes in the colony's caste ratio. Although the large-headed majors are morphologically distinct from minors, they are similar in size, exhibit similar behavioral repertoires and carry out nearly the same tasks as minors. Changes, even large ones, in the colony's caste ratio have no significant effect on the repertoire size of either caste. Majors do not compensate for the depletion of minors by expanding their repertoire or increasing their activity level. Instead of being an idle stand-by caste as suggested for other Pheidole specie s, P. pallidula majors are nearly as totipotent as minors. Moreover, their performance rate of social behaviors is remarkably high and constant regardless of the colony caste ratio. Such high investment of the major caste helps the colony to keep social behaviors at a baseline even in colonies undergoing large demographic changes. Alternative schemes of social regulation in polymorphic ant species are discussed. A possible methodological bias accounting for between-species differences in the level of majors' specialization is described.
Key words | Acknowledgements
This work was supported by the Belgian Fund for Joint Basic Research (grant 2.4510.01) and funded by a F.R.I.A. PhD studentship. G. Sempo is a Postdoctoral Researcher from the FRS-FNRS. C. Detrain is a research associate from the FRS-FNRS. The experiments described in this manuscript comply with the current laws in Belgium | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Feb 18; 10:3 | oa_package/46/7f/PMC3014662.tar.gz |
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PMC3014663 | 21234097 | 1. Introduction
Guillain-Barré syndrome (GBS) is an eponym for a heterogeneous group of immune-mediated peripheral neuropathies. A feature common in all GBS variants is a rapidly evolving polyradiculoneuropathy preceded by a triggering event, most often an infection [ 1 ]. Some cases of GBS after intravenous streptokinase administration have been reported previously [ 2 – 5 ]. We describe a new case of GBS in a patient who received intravenous streptokinase therapy for acute anterior myocardial infarction. | 3. Discussion
Streptokinase is a foreign protein derived from group C streptococci which might induce an immunological reaction leading to GBS [ 6 ]. It is important to remember that outside of certain institutions with interventional capability, fibronolytic therapy is still the most common form of acute-reperfusion therapy that is used for ST-elevation myocardial infarction. Therefore, streptokinase is still being used as a fibrinolytic therapy regimen for acute myocardial infarction worldwide, especially in developing countries and in some developed countries where facilities of immediate percutaneous coronary intervention is unavailable. Clinical symptoms are thought to result from streptokinase-antibody complex mediated damage to the local blood-nerve barrier [ 7 ]. GBS after acute myocardial infarction treated with reteplase also has been reported [ 8 ]. It is proposed that high-creatine kinase from significant muscle injury might be a possible immunological precipitant.
In conclusion, GBS can be seen in the late course of acute myocardial infarction suggestive of triggered autoimmune mechanisms. After exclusion of more likely causes, diagnosis should be considered in patients who develop parasthesia and muscular weakness after 10–30 days of infarction, especially if thrombolysed with streptokinase. | Academic Editor: H. O. Ventura
Guillain-Barre syndrome (GBS) is an eponym for a heterogeneous group of immune-mediated peripheral neuropathies. We describe a case of GBS in a patient who recieved intravenous streptokinase therapy for acute anterior myocardial infarction. Clinical symptoms are thought to result from streptokinase-antibody complex mediated damage to the local blood-nerve barrier. Patient was treated with 5-days course of intravenous gammaglobulin and his outcome was good. | 2. Case Report
A man 52-year-old taxi driver has been admitted to our clinic with a 1-hour onset chest pain. On electrocardiography, signs of hyperacute anterior wall myocardial infarction were evident. Streptokinase 1500000 unit was administrated over 1 hour intravenously. Reperfusion was positive clinically. Detailed medical history of the patient revealed no exposure to any drugs or toxins. Additionally, there was no evidence of any upper respiratory or gastrointestinal infection within the last 2 months. Medical history was also negative for arterial or venous embolism, connective tissue disease, and vasculitis. He was discharged on the 7th day with oral aspirin, metoprolol, spironolactone, simvastatin, and ramipril. Eleven days after discharge, he complained of progressive weakness on his legs and distal parasthesias. He was unable to walk without aid. On neurological examination, speech was dysarthric, left peripheral type facial paralysis, glove- and stocking-type sensorial impairment and absence of deep-tendon reflexes were prominent findings. Fundoscopic examination was normal. Cranial MRI revealed nothing. Blood chemistry was normal except hyperglycemia. Cerebrospinal fluid (CSF) analysis showed elevation of protein levels (216 mg/dl). CSF cultures were negative. Electromyographic investigation revealed extensive sensorymotor demyelinized type peripheral neuropathy, supporting the diagnosis of GBS. Patient was treated with 5-days course of intravenous gammaglobulin. Patient's outcome was good and 1 month later he was able to walk without aid, and 2 months later he was able to work. | CC BY | no | 2022-01-13 01:45:13 | Cardiol Res Pract. 2010 Dec 26; 2010:315856 | oa_package/4d/59/PMC3014663.tar.gz |
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PMC3014665 | 20569126 | Introduction
Rhimphoctona ( Xylophylax ) belonging to subfamily Campopleginae of the lchneumonidae (Hymenoptera) are important parasitoids of woodborers. Based on the most current version of Taxapad ( Yu et al. 2005 ), there are 13 Palearctic, 14 Nearctic, 1 Holarctic, and 1 Oriental species of Rhimphoctona . The European species of the genus Rhimphoctona Förster were revised by K. Horstmann ( 1980 ). The Nearctic species of the subgenus Xylophylax Kriechbaumer were reported by M. Sanborne ( 1986 ). The status of the genus was elucidated by D. Wahl ( 1991 ).
The genus has not been studied thoroughly in the Oriental and Palearctic regions of China. Only one species, Rhimphoctona ( Xylophylax ) lucida ( Clément 1924 ), has been recorded ( Sheng et al. 2002 ). In the present paper, four species of subgenus Xylophylax Kriechbaumer from P. R. China are reported.
The morphological terminology follows Gauld ( 1997 ). Wing vein nomenclature follows Mason ( 1986 ).
The type specimens of R . ( Xylophylax ) rufocoxalis (Clément) were lent from Zoologische Staatssammlung München, Munich, Germany, and the specimens of R . ( Xylophylax ) lucida (Clément) for comparison were from K. Horstmann, Zoologie III, Biozentrum, Wuerzburg, Germany. | Four species of Rhimphoctona ( Xylophylax ) collected from P. R. China are reported. Two of them are new to science: Rhimphoctona ( Xylophylax ) maculifemoralis Luo and Sheng, sp.nov. reared from Tetropium castaneum (Linnaeus), and Rhimphoctona ( Xylophylax ) immaculata Luo and Sheng, sp.nov. One is a new record for China, R . ( Xylophylax ) rufocoxalis ( Clément 1924 ) reared from T. castaneum (Linnaeus). The other is R . ( Xylophylax ) lucida ( Clément 1924 ) reared from Monochamus saltuarius Gebier, Tetropium gabrieli Weise and Asemus sp. A key to species known in China is provided.
Key words | Taxonomy
Subgenus Xylophylax Kriechbaumer 1878 Xylophylax Kriechbaumer 1878:210. Type species: Pyracmon ( Parapyracmon ) rufocoxalis Clément 1924 :125. Designated by Townes (1970).
Diagnosis. Lower tooth generally much longer than upper tooth. Vein 2m-cu of the forewing meeting vein M closer to vein 3r-m than to 2r-m. Metasomal segment 1 short and stout, with large, deep glymma, its spiracle near middle. Ovipositor cylindrical, apical portion upturned. Face of male partly to entirely yellow. Clypeus of female partly to completely reddish brown.
Rhimphoctona ( Xylophylax ) maculifemoralis , sp.nov.
Diagnosis. Apical portion of antenna somewhat compressed. Hind leg black, its dorso-basal portion of femur with brownish-yellow fleck. Face of male is yellow, with a small black fleck at upper centre.
Description. Female. Body length 7.8 to 8.5 mm. Forewing length 6.0 to 6.2 mm. Ovipositor sheath length about 3 mm.
Head. Face almost flat, about 1.5 times as wide as long, with dense granulation and distinct punctures, upper margin weakly concave centrally. Clypeus ( Figure 1 ) flat, basal portion with dense punctures. Apical portion smoother, apex with a weak median projection. Mandible strong, with transverse punctures, lower tooth long and acute, 2.0 times as long as upper tooth. Malar space rough, 0.8 times as long as basal width of mandible. Gena coriaceous with very sparse punctures, hind portion weakly expanded, in lateral view 0.8 to 0.9 times as long as width of eye. Vertex evenly convex, nearly the same texture as gena. Interocellar area with median longitudinal concavity. Postero-ocellar line about as long as ocular-ocellar line. Frons concave toward centre, with fine oblique lines and a short median longitudinal carina. Antenna very thin, with 43 to 44 flagellomeres, apical portion somewhat compressed. Occipital carina complete.
Mesosoma. Pronotum slightly rough, with irregular oblique wrinkles, upper portion with fine punctures. Without epomia. Mesoscutum evenly convex, finely coriaceous and with very fine punctures. Notaulus very weak, as a vestige on front portion of mesoscutum. Scutellum smooth, with distinct, fine punctures. Mesopleuron ( Figure 2 ) and mesosternum finely coriaceous with very fine punctures and wrinkles below speculum. Speculum small. Metapleuron with indistinct punctures. Submetapleural carina complete and strong. Wing brownish hyaline, 1cu-a opposite or almost opposite 1-m. Areolet a slanting quadrangle, receiving vein 2m-cu at 0.75 to 0.8 distance from vein 2r-m to 3r-m. Vein 2-Cu as long as 2cu-a. Vein 1-cu strongly inclivous, about 3.0 times as long as cu-a. Coxae texture as mesosternum. Hind femur strongly compressed. Claw small, its base pectinate. Propodeum ( Figure 3 ) weakly rough, finely coriaceous. Costula absent. Area petiolaris with transverse wrinkles. Area superomedia separated from area basalis by strong transverse carina and combined with area petiolaris. Spiracle approximately circular (slightly elliptic).
Metasoma. Terga finely coriaceous. Hind half of metasoma compressed. First tergum 2.5 to 2.6 times as long as its apical width, slender, only postpetiole slightly wider. Median dorsal carina absent. Dorsolateral carina present, reaching to hind end of glymma. Glymma deep. Spiracle very small, slightly convex. Second tergum elongate, 1.5 times as long as its apical width. Ovipositor sheath very slim, about as long as hind tibia. Ovipositor ( Figure 4 ) without subapical dorsal notch.
Color. Black. Mandible yellow to yellowish brown. Maxillary palpus, labial palpus, tegula, front tarsus, second segment of middle trochanters, basal lower portion of middle femur, and middle tibia blackish brown. Front femur and tibia and apical portion of middle femur brown to reddish brown. Basal dorsal portion of hind femur with a yellowish brown fleck. Stigma and veins brownish black.
Male. Body length 8.5 to 10.0 mm. Forewing length 6.0 to 6.5 mm. Antenna with 43 to 45 flagellomeres. Face except median black fleck, clypeus, mandible except tooth, malar space, lower portion of gena, maxillary palpus, labial palpus, lower profile of scape, front and middle legs (except basal end of coxae black and tarsi blackish brown) yellowish brown.
Type material. Holotype ♀, CHINA: Maixiu Forestry farm, Huangnan, Qinghai Province, 2006.VI.14, L. Liu. Paratype: 31 ♂♂ 9♀♀, same data as holotype, except 2006.VII.4 to VIII.9. All types are deposited in Insect Museum, General Station of Forest Pest Management (GSFPM), State Forestry Administration, Shenyang, P. R. China.
Host. All specimens were reared from wood of Picea crassifolia Komarov from which many Tetropium castaneum (Linnaeus) (Coleoptera: Cerambycidae) emerged.
Distribution. China (Qinghai).
Etymology. The name of the new species is based on upper-basal portion of hind femur with brownish yellow fleck.
Remarks. The new species resembles R . ( Xylophylax ) lucida ( Clément 1924 ), but can be distinguished from the latter by its ovipositor approximately as long as hind tibia; hind leg, except dorso-basal portion of femur with brownish yellow fleck, black; face of male yellow, with a small black fleck at upper centre.
Rhimphoctona ( Xylophylax ) immaculata , sp.nov.
Diagnosis. Apical margin of clypeus obtuse, without median projection. First trochanter of front leg with an apical tooth on front side. Ovipositor with a small subapical dorsal notch. Clypeus and hind legs of female entirely black. Mesosternum of male entirely yellow.
Description. Female. Body length about 9.5 mm. Forewing length about 7.5 mm. Ovipositor sheath length about 3 mm.
Head. Face 2.0 times as wide as long, with fine dense granulation and punctures, slightly convex centrally, upper margin with a small median protuberance. Clypeus ( Figure 5 ) coriaceous with sparse punctures, median portion slightly concave, apical portion smoother, apical margin obtuse, without projection. Mandible strong, coriaceous with sparse fine punctures, lower tooth long and acute, 1.6 times as long as upper tooth. Malar space slightly concave and rough, 0.65 times as long as basal width of mandible. Gena coriaceous with very sparse fine punctures, in lateral view about 0.83 times as long as width of eye. Vertex evenly convex, nearly the same texture as face, but more sparse punctures. Interocellar area weakly convex, with shallow median concavity. Postero-ocellar line about 0.9 times as long as ocular-ocellar line. Frons evenly concave, the same texture as face, lower median portion with very fine transverse wrinkles. Antenna short, with 28 flagellomeres. Occipital carina complete.
Mesosoma. Front portion of pronotum with fine longitudinal wrinkles, lateral concavity with short and dense transverse wrinkles, upper portion with fine punctures. Epomia indistinct. Mesoscutum evenly convex, with even and fine punctures. Notaulus very weak, as a vestige on subanterior portion of mesoscutum. Scutellum evenly convex, smooth, with distinct fine punctures. Postscutellum slightly rough, with dense and fine punctures. Mesopleuron ( Figure 6 ) and mesosternum with even and fine punctures, the latter denser than the former, in front of speculum with fine oblique wrinkles. Speculum smooth, with unclear fine oblique lines. Metapleuron slightly rough, with dense and indistinct punctures. Submetapleural carina complete and strong. Wing brownish hyaline, 1cu-a distad of 1-m. Areolet a slanting quadrangle, receiving vein 2m-cu slightly basad of vein 3r-m. Vein 2-Cu as long as 2cu-a. Vein 1-cu slightly inclivous, about 3 times as long as cu-a. First trochanter ( Figure 8 ) of front leg with an apical tooth on front side. Claw small, distinctly pectinate. Propodeum ( Figure 7 ) weakly rough, unclearly punctured, with strong carina. Costula complete and strong. Area superomedia separated from area basalis by strong transverse carina, and combined with area petiolaris. Area superomedia slightly wider than length, connecting with costula at its middle. Apical portion of area superomedia and area petiolaris with transverse wrinkles. Spiracle approximately circular (slightly elliptical).
Metasoma. Apical portion, from third tergum to end of metasoma, strongly compressed. First tergum gradually wider behind, about 2.6 times as long as its apical width, slightly rough, apical portion with very weak longitudinal wrinkles. Median dorsal carina absent. Dorsolateral carina strong before spiracle, the rest weak. Glymma deep. Spiracle small, slightly concave. Second tergum extremely finely coriaceous, approximately as long as its apical width. Ovipositor sheath very slim, about 0.9 times as long as hind tibia. Ovipositor ( Figure 9 ) evenly upturned, with a distinct subapical dorsal notch.
Color. Black. Maxillary palpus and labial palpus unevenly blackish brown. Median portion of mandibles, front femur and outer side of middle femur reddish brown. Front and middle tibiae brown, their tarsi brownish black.
Male. Body length about 10.5 mm. Forewing length about 7.5 mm. Antenna with 41 flagellomeres. Subposterior portion of gena swollen. Face, clypeus, mandible except tooth, malar space, gena, frontal orbit, maxillary palpus, labial palpus, scape, pedicel, coxae and trochanters of front and middle legs, propleuron, lower portion of mesopleuron, mesosternum and tegula yellow. Front and middle legs and hind trochanter yellowish brown. Coxa except basal end of upper profile and femur of hind leg reddish brown. Hind tibia obscurely reddish brown. Hind tarsus blackish brown.
Type material. Holotype♀, CHINA: Baotianman Natural Preserve, Henan Province, 1280m, 2006.V.25., Xiao-Chen Shen. Paratype: 1♂, same data as holotype. All types are deposited in Insect Museum, General Station of Forest Pest Management (GSFPM), State Forestry Administration, Shenyang, P. R. China.
Distribution. China (Henan).
Etymology. This new species name is based on the color of the female body, entirely black without markings.
Remarks. This species is similar to R . ( Xylophylax ) rufocoxalis ( Clément 1924 ), but can be distinguished from the latter by the following characters: clypeus and hind legs entirely black; area superomedia of propodeum wider than long; costula complete and strong, connecting with areola at the middle of the latter. R . ( Xylophylax ) rufocoxalis (Clément): clypeus yellow; hind legs, at least coxae, trochanters and femora, reddish brown; area superomedia longer than its width; costula weak and incomplete, connecting with areola behind the middle of the latter.
Rhimphoctona ( Xylophylax ) lucida ( Clément 1924 )
Pyracmon ( Parapyracmon ) lucidus Clément 1924 . Deutsche Entomologische Zeitschrift, 1924:130.
Specimens examined: 1 ♀, CHINA: Chuo'er, Inner Mongolia, 1981.VI.18.; 9♀♀3♂♂, CHINA: Tuqian, Heilongjiang Province, 1989.VI.7., M.-L. Sheng, reared from cocoons that were collected from the galleries of Tetropium gabrieli Weise and Asemus sp. (Coleoptera: Cerambycidae); 3♀♀9♂♂, CHINA: Daxing'gou, Jilin Province, 2005. VI. 17., M.-L. Sheng, reared from cocoons that were collected from the galleries of Monochamus saltuarius Gebler (Coleoptera: Cerambycidae).
Rhimphoctona ( Xylophylax ) rufocoxalis ( Clément 1924 )
Pyracmon ( Parapyracmon ) rufocoxalis Clément 1924 . Deutsche Entomologische Zeitschrift, 1924:125.
Lectotype ( ♀ ): “Starnbg. 13.6.58 Krchb.” (München).
New record for China.
Specimens examined: 1 ♀6♂♂, Maixiu forestry farm, Huangnan, Qinghai province, June 22 to August 4, 2006, Li Liu, reared from wood of Picea crassifolia Komarov from which many Tetropium castaneum (Linnaeus) (Coleoptera: Cerambycidae) emerged (new host record).
Key to species of Rhimphoctona ( Xylophylax ) known in China
Propodeum with costula absent. Ovipositor sheath about as long as hind tibia. Hind coxa, trochanter and femur black. Basal dorsal portion of hind femur with a yellowish brown fleck R. ( Xylophylax ) maculifemoralis Luo and Sheng, sp.nov.
Propodeum with distinct costula. 2
Clypeus reddish brown. Hind coxa, trochanter and femur red or reddish brown. Costula incomplete R. ( Xylophylax ) rufocoxalis (Clément)
Clypeus black, or only apical margin slightly brownish centrally. Hind leg with at least coxa black. Costula complete. 3
Apical margin of clypeus with a weak median projection. First trochanter of front leg without an apical tooth on front side. Ovipositor sheath about 1.5 times as long as hind tibia. R. ( Xylophylax ) lucida (Clément)
Apical margin of clypeus without a weak median projection. First trochanter of front leg with an apical tooth on front side. Ovipositor sheath about 0.9 times as long as hind tibia. R. ( Xylophylax ) Immaculata Luo and Sheng, sp.nov. | Acknowledgements
The authors are deeply grateful to Dr. K. Horstmann, Zoologie III, Biozentrum, Wuerzburg, Germany, and Dr. S. Schmidt, Zoologische Staatssammlung München, Munich, Germany, for their kindness in lending specimens. Financial support from the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT0607) and the National Natural Science Foundation of China (NSFC: No. 30730075, No. 30671686) is thankfully acknowledged. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Feb 25; 10:4 | oa_package/d2/8f/PMC3014665.tar.gz |
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PMC3014666 | 20569127 | Introduction
Studies of the Turkish aphid fauna were limited until the last decade. Although preliminary studies were done at the beginning of the 1900s, most of these studies were carried out by foreign researchers and were only focused on very small parts of Turkey ( Trotter 1903 ; Fahringer 1922 ). Çanakcioğlu ( 1975 ) reviewed previous studies and listed 258 species. Tuatay ( 1988 , 1991 , 1993 ) added about 30 species as new records. Duzgunes et al. ( 1982 ) reported three additional species. Recently, many more studies have organized and added more than 40 new records ( Canakcioglu and Toper 1999 ; Kaygin and Canakcioglu 2003 ; Toros et al. 2002 ; Toros et al. 2003 ; Gorur 2002 , 2004 ; Ozdemir et al. 2005 ; Asian and Uygun 2005 ). Remaudière et al. ( 2006 ) revised studies conducted on Turkey aphid fauna and listed about 417 species, despite some controversies. Akyurek ( 2006 ) and Kaygin et al. ( 2008 ) added 11 new records. | Methods and Materials
This study was conducted between 2005 and 2006. Aphid species were collected in the field from their host plants. The study area was located in the Western Black Sea Region of Turkey, where few detailed studies have been carried out.
Collection and preparation of samples were been done according to the principles of Hille Ris Lambers ( 1950 ) and Martin ( 1983 ). Species were identified according to Bodenheimer and Swirski ( 1957 ), Canakcioglu ( 1975 ), and Blackman and Eastop ( 1994 , 2006 ). Systematic knowledge, host plants, and synonyms of determined species were taken from Blackman and Eastop ( 1994 , 2006 ) and Remaudiere and Remaudiere ( 1997 ). The taxonomic status of the species was checked according to the recent update of Fauna Europaea 1.1 ( www.faunaeur.org ). Voucher specimens were deposited at the Entomology Department of Forestry Faculty in Bartin and the Department of Biology in Niğde. | Result
Ceruraphis viburnicola (Gillette) (Hemiptera: Aphididae), Dysaphis apiifolia (Theobald) (Hemiptera: Aphididae) and Macrosiphum mordvilkoi Miyazaki (Hemiptera: Aphididae) were new records for Turkey aphid fauna. All new recorded species belonged to the Aphidinae subfamily and Macrosiphini tribe.
Ceruraphis Borner, 1926 Ceruraphis viburnicola (Gillette, 1909) NeoCeruraphis viburnicola (Gillette, 1909)
Apterous individuals were collected on Viburnum sp. at Bartin on 29 July 2005. The species was also collected on Viburnum sp. at Nigde province, which is located at the Inner Anatolia of Turkey, on 23 June 2005. They mainly feed on shoots, undersides of the leaves and flowers, such as the snowball flowers of Viburnum opulus . They heavily colonize young leaves and flowers and are attended by ants. Their primary host is V. opulus , but a secondary host is as yet unknown. Previously, it has been recorded in Canada and the USA ( Maw et al. 2000 ; www.faunaeur.org ).
Dysaphis Börner, 1931 Dysaphis apiifolia (Theobald, 1923) Aphis ferrugineastriata Essig, 1938
Apterous individuals found on Petroselinum sp. at Hendekyanı (Central Bartin), 22 May 2006. There were dense colonies at the leaf bases that were attended by a lot of ants. This aphid is yellowish-grey to greenish-grey and dusted with wax. They typically colonize heavily the leaf bases of celery and parsley, and become serious pests of these host plants ( Blackman and Eastop 2006 ). They mainly feed on Apium graveolensis , Foeniculum vulgare , Petroselinum sativum , and various Umbelliferae species.
They are essentially distributed in the Afrotropical region, Australian region, East Palaearctic, Near East, Nearctic region, Neotropical region and North Africa ( www.faunaeur.org 2007).
Macrosiphum Passerini, 1860 Macrosiphum mordvilkoi Miyazaki, 1968 Macrosiphum mulgedifolii Tashev 1967
Both apterae and alatae forms were collected on Rosa spp. at the gardens of Ulus (vicinity of Bartin) and Bartin Forest Enterprise, 31 March 2006. They were abundant in the study area and densely colonized the host plant. The species was identified from apterous individuals. This species was easily separated from other Macrosiphum species because of their black front head and antennal segment III with rhinaria extending over most of the length of segment ( Figure 1 ).
They are distributed in Japan, Korea and eastern Russia ( Blackman and Eastop 2006 ). | Discussion
Recent studies have shown that aphid species listed for Turkey's aphid fauna do not sufficiently indicate the real number, as there has been much information accumulated during the last decades. These arguments also are supported when Turkey is compared with its neighbours that are located in similar geographic areas in terms of richness of flora, geographic variability, geographic location, agricultural landscape, etc. For example, Tsitsipis et al. ( 2007 ) reviewed the known Greek aphid fauna comprising 301 species. Comparatively, the study of the fauna of some countries isn't greater, with about 340 species in Iran and only 167 species in Lebanon and Syria ( Remaudière et al. 2006 ).
The results presented have added three new species to Turkey aphid fauna, so now the Turkey aphid fauna has at least 433 species. Thus, it can be expected that with further research, in different parts of the country, the recorded Turkish aphid fauna will be considerably increased. | Editor: Wesley Eugene Hall was editor for this paper
Three aphid species were identified as new records for Turkey aphid fauna from Bartin province. These species are Ceruraphis viburnicola (Gillette) (Hemiptera: Aphididae), Dysaphis apiifolia (Theobald) (Hemiptera: Aphididae) and Macrosiphum mordvilkoi Miyazaki (Hemiptera: Aphididae). These records increase the recorded aphid-fauna of Turkey to 433 species.
Key words | Acknowledgements
Financial support from DPT Scientific Research Projects Commission Project No. 2003K121110 is gratefully acknowledged. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Feb 25; 10:5 | oa_package/e9/e9/PMC3014666.tar.gz |
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PMC3014668 | 20569137 | Introduction
In many parts of the developing world, people seek diversified sources of income, especially those that are sustainable and environmental friendly. Due to current population growth in Kenya, pressure on the Kakamega forest is growing because the forest plays an important role in satisfying the daily needs and income of local people. Wild silk farming is a unique, ecologically friendly industry with a great potential for employment generation, artisanal development and export earnings ( Kioko et al. 1999 ). African species of silkmoths provide strong silk of high commercial value ( Raina 2004 ). The Boisduval silkworm, Anaphe panda (Boisduval) (Lepidoptera: Thaumetopoeidae) shows the best potential for wild silk production since it produces a huge cocoon that is communally weaved by 20–200 silkworm larvae. Wild silk farming is among the industries that might assist resource-poor farmers of this region to escape a vicious cycle of poverty, while providing an incentive for forest conservation ( Kioko et al. 2000 ). In countries where rural communities depend on subsistence farming, wild silkmoth cultivation can be a supplementary activity for income generation while simultaneously conserving biodiversity.
Silk has been used for textiles for thousands of years ( Raina 2004 ). In Africa, most of the wild silkmoths belong to the families Saturniidae, Lasiocampidae, and Thaumetopoeidae. Anaphe species are widely distributed in the intertropical regions of continental Africa such as Nigeria, Uganda, Kenya, Cameroon, Congo and Togo. The important species used in the production of Anaphe silk are Anaphe infracta Walsingham, Anaphe venata Butler, Anaphe panda Boisduval, Anaphe reticulata Walker, Anaphe carteri Walsingham, Anaphe moloneyi Druce and Anaphe ambrizia Butler. Anaphe are polyphagous moths but Bridelia micrantha (Hochst) (Malpighiales: Euphorbiaceae) is the preferred host plant of A. panda in the Kakamega Forest. In nature, B. micrantha can be found scattered over a large area of the Kakamega forest and it flourishes in both natural tracts and those mixed with exotic species. With very little care it can be raised from seed or from cuttings that propagate rapidly and are ready for use as host plants in about a year ( Gowdey 1953 ; Jolly et al. 1979 ). In the Kakamega Forest, A. panda silkmoths lay egg clusters under the leaves of B. micrantha .
The potential of the African indigenous silkmoth species for wild silk production has been well documented in Nigeria ( Ashiru 1991 ), Uganda ( Kato 2000 ) and Kenya ( Kioko et al. 2000 ; Mbahin et al. 2008 ). However, the wild silk industry in Kenya will not be commercially viable unless technologies are developed to reduce silkworm mortality due to natural enemies and help farmers develop sustainable mass production of A. panda cocoons. Some studies have recommended the use of sleeve nets to protect young larvae ( Kioko et al. 1999 ; Raina 2000 ; 2004 ; Ngoka 2003 ), but there are no reliable data available on the benefit of using sleeve nets to improve Anaphe silkworm survival in the field. The present study was conducted in two forest habitats to test whether the survival rate of A. panda silkworms can be improved by protection with sleeve nets. | Materials and Methods
Study sites
The Kakamega Forest is a tropical rainforest that covers a total area of approximately 265 km 2 and is located between latitudes 0° 10′ and 0° 21′ North and longitudes 34° 47′ and 34° 58′ East, respectively ( Figure 1 ). It comprises several separate blocks of forest of which Isecheno (415 ha) belongs to the Lunyu sub-location whereas Ikuywa (380 ha) belongs to the Ikuywa sub-location. Two sites were used for the study: Musembe village, located in Ikuywa (a natural forest comprised exclusively of indigenous species), and Chirobani village, located in Isecheno (a modified indigenous forest comprised of a combination of native and exotic species). The exotic species are mainly pines, Acacia spp. (Fabaceae) and Eucalyptus spp. (Myrtaceae) ( Mbahin et al. 2008 ). Indigenous plants comprise about 150 species of woody trees, 90 species of dicotyledonous herbs, 80 species of monocotyledonous herbs of which about 60 are orchids, and a further 62 species of ferns, totalling to about 380 identified species of vascular plants ( KIFCON 1994 ).
Environmental data
A digital hygrothermometer (Zheda Electric Apparatus Inc., http://www.zjlab.com ) was used for recording daily temperature (maximum and minimum) and measurements of relative humidity were recorded four times daily (6 am; 12 am; 3 pm and 9 pm) at both sites throughout the period of study. A rain gauge was used for recording rainfall data.
Silkworm survival
Two naturally-laid egg clusters of A. panda were selected on each of 150 B. micrantha trees that had canopies of about 10 cubic feet ( Figure 2 ). Any additional egg clusters were removed. Each tree was divided into two experimental areas: one with a net sleeve measuring 1.5 × 1.5 × 2 m covering one cohort of larvae and a control cohort of larvae that were not covered. The net sleeves were tied closed on the branches of the host plant and a 2 m long zippered aperture \ permitted access for purpose of observation and data collection ( Figure 3 ). Branches were selected among those that bore sufficient leaves to provide adequate food. Of 300 clusters of eggs selected, eggs hatched in a total of 221 that were included in the experiment (105 protected, 116 exposed). Observations on the possible causes of mortality were made twice weekly and counts of the surviving larvae in net sleeves and on control branches from June 2005 to June 2007. Protected cohorts were moved to new branches of the same tree two or three times during the course of development as required to ensure an adequate food supply. Only cohorts that completed all seven larval instars with some survivors ( Figure 4 ) were included in the analysis. The mortality rate (by instar) was calculated as follows: With: S ini = Number of larvae alive at the beginning of the instar and S fin = Number of larvae alive at the end of the instar.
The instantaneous death rate is the boundary of the expression below when Δ t → 0. The ‘force of mortality’ (incidence rate) for each instar can be calculated as follows: Where D per = Number of dead larvae in the specific instar and S exp = Number of surviving larvae exposed to risk in that instar.
Data analysis
The data on numbers of larvae surviving to form cocoons were analyzed by factorial ANOVA ( SAS Institute, 2003 ) with ‘year’ (2005/2006/2007), ‘treatment’ (protected/unprotected), forest (natural/modified), and ‘brood’ (1 st /2 nd ) as independent variables. Analysis of survival-time data was carried out with the software Stata7 ( STATACORP, 2004 ) and a Chi-Square test was used to compare overall cohort survival between forest types. | Results
Environmental data
Data on mean monthly average temperature, relative humidity, and rainfall for the two study sites are reported in Figures 5 and 6 , respectively. Note that rainfall is bimodal in the Kakamega Forest, with a period of “long rains” from April through June, and “short rains” in August through November. During the study, the annual rainfall ranged from 180.9 to 265.9 cm in the modified forest (lsecheno), and from 188.6 to 224.7 cm in the natural forest (Ikuywa). The annual number of rainy days ranged from 196 – 219, and from 207 – 209 in the modified and natural forests, respectively. Mean monthly maximum temperature ranged from 15.5° C to 36.8° C at Isecheno, and from 16.5 – 35.6° C at Ikuywa. Mean monthly maximum humidity ranged from 45.4 – 86.2 % in the modified forest and from 35.6 – 80.9 % in the natural forest.
Larval survival
There was no effect of ‘year’ on the number of larvae pupating ( F = 0.09; df = 2,177; P = 0.910) so data were pooled across years for further analysis. In both forest types, mortality rates tended to be higher for young larvae (1 st to 4 th instar) than for older instars (5 th to 7 th ) ( Table 1 ), but the main effect of forest type was not significant ( F = 2.49; df = 1, 178; P = 0.117). However, the effect of the sleeve net protection treatment was highly significant ( F = 229.26; df = 1,178; P < 0.001) and cohorts eclosing in the wet season (2 nd brood) had significantly lower survival than those eclosing in the dry season (1 st brood) ( F = 12.49; df = 1,178; P = 0.02). The forest type*treatment interaction was not significant ( F = 0.24; df = 3,176; P = 0.624), nor was the treatment*brood interaction ( F = 2.34; df = 3,176; P = 0.128), nor the forest*brood interaction ( F = 0.10; df = 3,176; P = 0.757), nor the three-way interaction ( F = 2.34; df = 7,172; P = 0.440).
The overall survival of silkworm cohorts was significantly higher in the natural forest compared to the modified forest (c2 = 36.6, P < 0.001). In the Ischeno modified forest, 38 out of 48 cohorts (79.2%) survived to spin cocoons in the sleeve net treatment, compared to 15 out of 38 (39.5 %) for unprotected controls. In the Ikuywa natural forest 76 of 78 (97.4 %) protected cohorts survived, compared to 51 of 57 (89.5 %) control cohorts. Considering total numbers of larvae, only 2,557 of 15,198 (16.8%) unprotected silkworms survived to pupation in the modified forest, whereas 7,757 out of 12,447 (62.3%) survived when protected with sleeve nets. In the natural forest, 3,511 out of 17,213 (20.4%) unprotected silkworms survived, compared to 8,888 out of 13,124 (67.7%) in the sleeve nets. Thus, protection with sleeve nets increased survival 3.7 and 3.3 fold in the modified and natural forests, respectively. The Nelson-Aalen cumulative hazard function ( Figure 7 ) reveals that the protective environment provided by net sleeves significantly reduced ( F = 202.04; df = 1,125; P < 0.001) silkworm larval mortality across all instars, although mortality was greater in early instars than in later ones. | Discussion
The ‘force of mortality’ (incidence rate) for each instar can be calculated as follows: Where D per = Number of dead larvae in the specific instar and S exp = Number of surviving larvae exposed to risk in that instar.
Data analysis
The data on numbers of larvae surviving to form cocoons were analyzed by factorial ANOVA ( SAS Institute, 2003 ) with ‘year’ (2005/2006/2007), ‘treatment’ (protected/unprotected), forest (natural/modified), and ‘brood’ (1 st /2 nd ) as independent variables. Analysis of survival-time data was carried out with the software Stata7 ( STATACORP, 2004 ) and a Chi-Square test was used to compare overall cohort survival between forest types. | Associate Editor: J.P. Michaud was editor of this paper
Prospects for development of a wild silk industry in Africa would be improved if silkworm survival during mass production could be improved. A study on the survival of the Boisduval silkworm, Anaphe panda (Boisduval) (Lepidoptera: Thaumetopoeidae) was conducted with and without protection by net sleeves in two different forest habitats (natural and modified) in the Kakamega forest of western Kenya. Overall, cohort survival was significantly higher ( P < 0.001) in the natural than in the modified forest, but larval survival was improved over three-fold by protection with net sleeves in both habitat types. In the modified forest, only 16.8% of unprotected larvae survived to the pupal stage and formed cocoons, whereas 62.3% survived in the same environment when they were protected with net sleeves. In the natural forest, 20.4% of unprotected larvae survived, whereas 67.7% survived in net sleeves. There was also a significant effect of season; cohorts of larvae that eclosed in the wet season had significantly lower survival than those eclosing in the dry season (P = 0.02). Sources of mortality appeared to be natural enemies (parasites, predators and diseases) and climatic factors.
Key words | Results
Environmental data
Data on mean monthly average temperature, relative humidity, and rainfall for the two study sites are reported in Figures 5 and 6 , respectively. Note that rainfall is bimodal in the Kakamega Forest, with a period of “long rains” from April through June, and “short rains” in August through November. During the study, the annual rainfall ranged from 180.9 to 265.9 cm in the modified forest (Isecheno), and from 188.6 to 224.7 cm in the natural forest (Ikuywa). The annual number of rainy days ranged from 196 – 219, and from 207 – 209 in the modified and natural forests, respectively. Mean monthly maximum temperature ranged from 15.5° C to 36.8° C at Isecheno, and from 16.5 – 35.6° C at Ikuywa. Mean monthly maximum humidity ranged from 45.4 – 86.2 % in the modified forest and from 35.6 – 80.9 % in the natural forest.
Larval survival
There was no effect of ‘year’ on the number of larvae pupating ( F = 0.09; df = 2,177; P = 0.910) so data were pooled across years for further analysis. In both forest types, mortality rates tended to be higher for young larvae (1 st to 4 th instar) than for older instars (5 th to 7 th ) ( Table 1 ), but the main effect of forest type was not significant ( F = 2.49; df = 1, 178; P = 0.117). However, the effect of the sleeve net protection treatment was highly significant ( F = 229.26; df = 1,178; P < 0.001) and cohorts eclosing in the wet season (2 nd brood) had significantly lower survival than those eclosing in the dry season (1 st brood) ( F = 12.49; df = 1,178; P = 0.02). The forest type*treatment interaction was not significant ( F = 0.24; df = 3,176; P = 0.624), nor was the treatment*brood interaction ( F = 2.34; df = 3,176; P = 0.128), nor the forest*brood interaction ( F = 0.10; df = 3,176; P = 0.757), nor the three-way interaction ( F = 2.34; df = 7,172; P = 0.440).
The overall survival of silkworm cohorts was significantly higher in the natural forest compared to the modified forest (c2 = 36.6, P < 0.001). In the Ischeno modified forest, 38 out of 48 cohorts (79.2%) survived to spin cocoons in the sleeve net treatment, compared to 15 out of 38 (39.5 %) for unprotected controls. In the Ikuywa natural forest 76 of 78 (97.4 %) protected cohorts survived, compared to 51 of 57 (89.5 %) control cohorts. Considering total numbers of larvae, only 2,557 of 15,198 (16.8%) unprotected silkworms survived to pupation in the modified forest, whereas 7,757 out of 12,447 (62.3%) survived when protected with sleeve nets. In the natural forest, 3,511 out of 17,213 (20.4%) unprotected silkworms survived, compared to 8,888 out of 13,124 (67.7%) in the sleeve nets. Thus, protection with sleeve nets increased survival 3.7 and 3.3 fold in the modified and natural forests, respectively. The Nelson-Aalen cumulative hazard function ( Figure 7 ) reveals that the protective environment provided by net sleeves significantly reduced ( F = 202.04; df = 1,125; P < 0.001) silkworm larval mortality across all instars, although mortality was greater in early instars than in later ones.
Discussion
The climatic conditions observed in this study ( Figures 5 & 6 ) were consistent with reports by Muriuki and Tsingalia ( 1990 ) and Kokwaro ( 1988 ). As Poikilothermie organisms, the life cycle, activity, distribution and abundance of Lepidoptera are influenced by temperature ( Hill et al. 1999 ). Pollard and Yates ( 1985 ) found that temperature and rainfall were likely to influence the survival of butterflies directly and indirectly through the effects on plant growth, disease, predation or other factors. In light of the present study, further work is warranted to understand why a forest insect like A. panda periodically develops high populations in certain well-defined types of forest habitat, but not in all habitats where it occurs.
Predation likely contributed to mortality of young larvae, and disease to mortality of older instars. The larval parasitoids Exorista cardinalis F. (Diptera: Tachinidae) and Cryptus leucopygus Granenhorst (Hymneoptera: Ichneumonidae) were recovered in mummified larvae during the course of the study. These findings are consistent with parasitoids recovered from silkmoths by Kioko et al. ( 1999 ) and Raina ( 2004 ) and indicate that sleeves nets can be used to reduce silkworm mortality due to natural enemies, especially during the rainy season when larval mortality tends to be higher. This simple technology has the potential to improve the commercial viability and sustainability of the wild silk industry in Africa. The introduction of wild silk production to the Kakamega forest may offer important economic incentives to farmers surrounding the forest. More than 12,400 ha are suitable for silkworm food plants in the Kakamega Forest and could be utilized for the cultivation of B. micrantha ( Mbahin et al. 2008 ). Although the use of sleeve nets greatly improved the survival rate of silkworms in both forest habitats, overall cohort survival was somewhat higher in the natural forest than in the modified forest that contained introduced tree species. Thus, reforestation with indigenous species such as B. micrantha will not only favor the conservation of indigenous biodiversity, but also enhance the productivity of the wild silkmoth industry. | Acknowledgments
The authors express their sincere thanks to Deutscher Akademischer Austauschdienst (DAAD) for providing the scholarship and to International Fund for Agricultural Development (IFAD) and GEF (Global Environmental Facility) for logistical assistance. Thanks are also due to ICIPE for providing facilities. | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Feb 25; 10:6 | oa_package/a6/a0/PMC3014668.tar.gz |
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PMC3014669 | 20575745 | Introduction
Parasitoids spend part of their life as endo or ectoparasites of other invertebrates. During this process, their eggs and larvae contend with the immune defense responses of the hosts. Several mechanisms have evolved that enable parasitoids to escape or suppress host immune responses, which vary depending on the parasitoid species ( Beckage and Gelman 2004 ; Renault et al. 2005 ; Pennacchio and Strand 2006 ). These include co-injection of polydnavirus (PDVs) or maternal factors, such as venom or ovarian protein with their eggs at the time of oviposition ( Lanzrein et al. 1998 ; Beckage and Gelman 2004 ; Espagne et al. 2004 ; Moreau and Guillot 2005 ; Asgari 2006 ). These factors have often been shown to mediate suppression of the host immuneresponse. However, in certain parasitoid species, venom plays a major role in ensuring the survival and development of the parasitoid progeny in vivo ( Dani et al. 2005 ). Despite an increasing number of peptides, proteins, and enzymes reported from the venoms of ichneumonid, braconid or eucoilid wasps ( Moreau and Guillot 2005 ), understanding venom composition remains fragmental.
Parasitoid venoms seem to be rich in enzymes, and these enzymes vary among different parasitoid species that have highly specialized adaptations to their host range ( Whitfield 1998 ; Schmidt et al. 2001 ; Dani et al. 2005 ). About thirty of them have been identified by enzymatic assays, immunostaining or molecular sequence. Notably, some of them have been reported to possess interesting physiological functions involving in destroying the host's immune system. For example, a serine proteinase homolog of Cotesia rubecula venom can interfere with the prophenoloxidase activation cascade, which inhibits melanization of host hemolymph ( Asgari et al. 2003 ); phenoloxidase of Nasonia vitripennis venom is critical in the pathway leading to cell death ( Abt and Rivers 2007 ); and γ-glutamyl transpeptidase of Aphidius ervi venom can induce apoptosis in host ovarioles by generating an alteration of glutathione metabolism and consequent oxidative stress ( Falabella et al. 2007 ). The detailed exploration of diverse enzymes of parasitoid venoms is useful to contribute to the definition of the molecular bases of host-parasitoid interactions. Nevertheless, just few of them have been revealed. Moreover, among the available enzymes, there has been little information on the study of their exact biochemical and molecular natures.
Alkaline phosphatase (ALPase) (E.C. 3.1.3.1) has been characterized as an important component of venoms of some snakes and spiders ( Rodrigues et al. 2006 ). In insects, several ALPases have been characterized in the brains of Bombyx mori, Drosophila melanogaster, Ceratitis capitata, Culex tarsalis, Schistocerca americana and Bemisia tabaci , as well as the Malpighian tubules, salivary glands, saliva, etc ( Houk and Hardy 1984 ; Bourtzis et al. 1993 ; Chang et al. 1993 ; Itoh et al. 1999 ; Yang et al. 2000 ; Funk 2001 ), but there have been only few reports of ALPase from insect venoms. In these cases, ALPase was detected in ant venoms of Paraponera cribrinodis and Pogonomyrmex badius ( Schmidt et al. 1986 ) and this enzyme was also reported to be present in honeybee venom ( Hoffman 1977 ). In parasitoid wasps, ALPase has been reported in venom of the pupal endoparasitoid, Pimpla hypochondriaca , using the API ZYM, a semiquantitative colourimetric kit ( Dani et al. 2005 ).
Our aim is to describe components of parasitoid wasp venom and better understand its enzymatic diversity. In the present study, the presence of an ALPase in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae) is described. This enzyme was partially characterized at the biochemical level, its coding gene cloned and its mRNA expression levels investigated. | Methods and Materials
Insect rearing
The colony of P. puparum was maintained as described by Cai et al. ( 2004 ). Following the eclosion of wasps, female adults were collected in glass vials (50 × 230 mm) and fed with 20% (v/v) honey solution absorbed on cotton at 25 ± 1° C with a photoperiod of 10:14 (L:D).
Enzyme histochemistry
5-bromo-4-chloro-3′-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) (Promega, www.promega.com ) staining followed the method of Yang et al. ( 2000 ) with some modifications. Briefly, the venom apparatus was soaked in 4% (v/v) paraformaldehyde for 20 min. The venom apparatus was then treated with 0.1% Triton in PBS for 30 min and washed 3 times for 5 min with PBS. Finally, the venom apparatus was labeled using the chromogenic substrate of BCIP/NBT in the dark until the color developed. It was then washed 3 times for 5 min with PBS. The BCIP/NBT labeling reaction contained 0.17 mg/ml BCIP and 0.33 mg/ml NBT in reaction buffer (100 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl 2 , pH 9.5). The control venom apparatus was processed in the same way with the exception that NBT/BCIP was not added. Color development was viewed under a dissecting microscope and photographed with an Auto-Montage camera system.
For electron microscopy, the method was performed as described by Sarathchandra et al. ( 2005 ) modified from Rees and Ali ( 1988 ). The procedures for treating the samples were as follows: (1) fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4° C for 1 h; (2) washed in 0.1 M sodium cacodylate buffer for 3 times for 10 min; (3) incubated with 40 mM Tris/HC1 buffer (pH 9.0) containing 9 mM sodium β glycerophosphate, 5 mM magnesium chloride and 3.6 mM lead Nitrate for 4 h at room temperature; (4) washed briefly in 0.1 M sodium cacodylate buffer (pH 7.4); (5) post-fixed in OSO4 in 0.1 M cacodylate buffer for 2 h; and (6) dehydrated in a graded 50–100% acetone series and then embedded in Epon 812. In control samples, β-glycerophosphate was not added to the incubation medium. The ultrathin sections were observed using a JEX-1230 transmission electron microscope (JEOL, Tokyo) at an accelerating voltage of 80 kV.
Enzyme Assay
The crude venom was prepared in sterile distilled water as described by Zhang et al. ( 2005 ). The enzyme activity was determined according to the method of Dani et al. ( 2005 ) using p -nitrophenyl phosphate ( p -NPP) as the substrate at 45° C with one modification that the acid buffer was instead by 0.1 M glycine-NaOH alkaline buffer (pH 8.5). The amount of p -nitrophenol ( p -NP) released was measured using a microplate reader (Bio-Tek Instruments) at 405 nm. The thermal denaturalization of enzyme was analyzed at 40, 50 and 60° C with incubation times of 5, 10, 20 and 30 min. To study the effects of metals (Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ ), the final concentrations ranged from 0 to 5 mM. To determine the time-course of enzyme activity, samples of crude venom from the adults 0 to 7 days after eclosion were assayed, and 100 venom reservoir equivalents (VREs) (one VRE being defined as the supernatant from one torn venom reservoir in 1 μl sterile distilled water) per assay was used. The specific activities for one VRE were calculated using a nitrophenol calibration curve produced using a standard solution of p -NPP (Sigma, www.sigmaaldrich.com ). The assays not added venom samples were used as controls.
Molecular cloning of ALPase
Venom apparatus SMART cDNA library ( Zhu et al. 2008 ) was used as the template in all subsequent PCR amplifications. Degenerate primers, forward primer (5′-GTNGARGGNG GNAARATHGA-3′) and reverse primer (5′TCYTCNCCNCCRTGNGTYTC-3′) were designed based on highly conserved amino acid sequences VEGGKID and ETHGGED found in several insects. They were used to amplify a cDNA fragment in a PCR reaction with an initial denaturation step at 94° C for 3 min, 40 cycles running each with 94° C for 30 s, 55° C for 30 s, and 72° C for 1 min, and 10 min at 72° C for extension. For full length cDNA cloning, the RACE protocol was performed according to the instructions of the SMARTTM RACE cDNA amplification kit (Clontech, www.clontech.com ). Gene specific primers, (5′-CGACGAGAACAGGAAGGACCCGTTTTAT-3′) and (5′-GTTGCCTCGTTCTGGGTAGCCGTTCAT-3′) were designed to amplify the 3′- and 5′-ends of the cDNA fragment, respectively in combination with CDS III/3′ PCR primer and SMART IV oligonucleotide primer.
The PCR products were gel extracted (QIAquick-Gel Extraction Kit, www.qiagen.com ) and sequenced according to the dideoxy method with CEQ Dye Terminator using an ABI Prism 377 DNA sequencer (Applied Biosystems, www.appliedbiosystems.com ). Sequence similarity was assessed using BLASTP against the NCBI database ( Altschul et al. 1997 ). Multiple alignments were performed with CLUSTAL X, version 1.83 ( Thompson et al. 1997 ). Signal peptide predictions were made using the SignalP ( Bendtsen et al. 2004 ), and motifs were searched with Motifscan ( Falquet et al. 2002 ).
Semiquantitative RT-PCR
Venom apparatus from 0 to 7 days old P. puparum females were dissected and their RNA samples were extracted using Trizol reagentTM (Invitrogen, www.invitrogen.com ). Total RNA samples were quantified to the same content and were treated with RQI RNase-free DNase (Promega, www.promega.com ) to eliminate possible trace amounts of DNA contamination according to the manufacturer's instructions. The cDNA templates were synthesized using a RevertAid TM First Strand cDNA Synthesis Kit (MBI, Fermentas, www.fermentas.com ). To control for genomic DNA contamination, the total RNA samples that were not reverse transcribed into cDNA were used as the control templates. Two gene-specific primers were (forward, 5′-CATCACCAGAACCACGCAC-3′ and reverse, 5′- TCTTTGAAGCAAGCCGCAT-3′). PCRs were standardized using 18S rRNA specific primers (forward, 5′CGAGCGATGAACCGACAGC-3′ and reverse, 5′-ATTGGAGGGCAAGTCTGGTG-3′). PCR cycling conditions were as follows: 94° C (3 min) followed by 40 cycles (30 cycles for 18s rRNA) at 94° C (30 s), 52° C (30 s) (50° C for 18s rRNA), and 72° C (1 min), and then 1 cycle of 72° C for 10 min. PCR products were then visualized with ethidium bromide on 1% agarose gels. | Result
Histochemical detection of ALPase
Using chromogenic substrates BCIP and NBT, the localization of ALPase activity was examined in the venom apparatus of P. puparum by light microscopy ( Figure 1 ). Within the venom gland, ALPase activity was detected in whole mount of this tissue, while color development was weak ( Figure 1A ). In contrast, the venom reservoir stained strongly positive for ALPase activity, indicating the high amount of ALPase contained in this tissue ( Figure 1B ). In addition to light microscopy, the presence of ALPase in the venom apparatus was also detected as depicted in Figure 2 using the p -NPP technique for histochemical localization by electron microscopy. The black deposit of lead phosphate indicative of phosphatase activity was observed along the inner margin of the secretory vesicles and within the nuclei ( Figures 2A and B ). Controls showed no positive reaction, as no black deposit was produced ( Figures 2C and D ).
Thermal stability of ALPase
In order to determine a possible modulation of the ALPase activity by thermal denaturalization, the activity was measured by preincubation of the crude venom at 40, 50 and 60° C for 5, 10, 20 and 30 min and then p -NPP was added as substrate to start the reaction. Figure 3A shows the time course of the ALPase thermal stability. There was only a very slight decrease in p -NPP hydrolysis with time after pre-incubation at 40° C, but higher temperatures markedly inactivated the enzyme. The ALPase retained 71.88% of its initial activity after pre-incubation for 30 min at 40° C, 53.87% activity at 50° C and 26.70% at 60° C.
Effect of divalent cations on ALPase activity
The effects of different divalent cations at different concentrations of ALPase activity were studied ( Figure 3B ). Mg 2+ , Ca 2+ and Mn 2+ affected the activity of ALPase but to differing extents. At concentrations of 0 to 5 mM, ALPase activity was steadily activated by these three cations. With 5 mM Mg 2+ and Ca 2+ , the enzyme activity increased to 164.18% and 144.41% compared with the control, respectively. In contrast to Mg 2+ and Ca 2+ , the effect of Mn 2+ on the activity of ALPase was higher with an increase to 224.36%. However, the presence of Zn 2+ resulted in inactivation of the enzyme activity. The activity of ALPase markedly decreased with increasing Zn 2+ concentrations. The activity was reduced to 62.75% with 5 mM Zn 2+ .
cDNA clone of ALPase
The amplification of venom ALPase of P. puparum using two degenerate primers designed based on the conserved amino acid sequences yielded the expected PCR product of approximately 400 bp in length. The sequence of the cloned fragment showed significant similarity to reported ALPases available in the NCBI database. In order to obtain the full-length sequence, two primers were then designed as the gene-specific primers from the known fragment for 3′ and 5′ RACE. PCR of 3′ and 5′ RACE resulted in amplicons of 721 and 1787 bp, respectively. After assembling these three fragments, the full length ALPase was a total of 2645 bp long (Accession number: EU726269). The open reading frame (ORF) was 1623 bp long beginning with the methionine start codon ATG and ending with translation stop codon TAG at nucleotide positions 615 and 2,237, respectively. The cDNA contained a 5′ untranslated region (5′-UTR) of 614 bp and a 3′ untranslated region (3′-UTR) of 408 bp, including a poly (A) tail of 28 bp. The predicted molecular mass of the ALPase deduced amino acid sequence was calculated to be 59.83 kDa and the predicted calculated isoelectric point was 6.98. A BLASTP ( Altschul et al., 1997 ) search revealed that the deduced protein sequence had strong homology with ALPase of the other insect species ( N. vitripennis 91%, Apis mellifera 66%, Tribolium castaneum 48%, Anopheles gambiae 48%, D. melanogaster 46%). The alignment of the venom ALPase of P. puparum with that of these insects is reported in Figure 4 . Analysis of the deduced amino acid sequence by Signal P ( Bendtsen et al. 2004 ) showed the presence of a signal peptide of 22 predominantly hydrophobic amino acids (MKNTEALAIIGALLMCSSLCSG) at its N-terminus. Motifscan ( Falquet et al. 2002 ) was used to find profiles of the ALPase protein. There was a long, highly conserved ALPase gene family signature at amino acid positions 56–495. Moreover, there were three potential N-glycosylation sites, seven potential casein kinase II phosphorylation sites, nine potential N-myristoylation sites, four potential protein kinase C phosphorylation sites, one potential tyrosine kinase phosphorylation site and several metal ligand sites.
Time-course related expression
Semiquantitative RT-PCR analysis was employed to compare the levels of ALPase transcripts in venom apparatus of P. puparum from 0 to 7 days after adult eclosion ( Figure 5A ). A low ALPase expression was found at days 0 and 1. Thereafter expression levels increased gradually, and remained high on days 6 to 7. Subsequently, ALPase activity was measured from 0 day post female adult eclosion and continued until 7 days ( Figure 5B ). A clear time related modulation of enzymatic activity in the venom apparatus of P. puparum was evident in different days. ALPase activity was observed to be increasing at 0 to 6 days. The maximum peak was seen at day 6. Then, the level showed a slight decrease at day 7. The results revealed that the time-dependent changes in the gene expression of ALPase mRNA that corresponds to the alterations in ALPase enzymatic activity. | Discussion
In the present study, the histochemical results demonstrated that ALPase is present in the venom apparatus of P. puparum . From the P. puparum venom apparatus ultrastructural study ( Zhu et al. 2007 ), we know that the main function of the venom gland is to produce the venom that is stored in the venom reservoir. According to the BICP/NBT staining, a slight color development in the venom gland was observed, while the venom reservoir was deeply stained suggesting that ALPase was produced in the venom gland and then accumulated in the venom reservoir. ALPase activity was located in the secretory vesicles and nuclei of venom gland using p -NPP technique and electron microscopy.
ALPase has been shown to be located on the cell surface, linked to the cell membrane via a phosphatidylinositol glycan linkage ( Low and Saltiel 1988 ). This is not the case in the venom gland secretory cells of P. puparum as ALPase was located in all parts of the nuclei and in periphery of the secretory vesicles. Therefore, we suggest that P. puparum venom ALPase is an intracellular enzyme.
ALPase has multifunctional roles in cell biology. In bacterial cells, this enzyme was reported to be located in the periplasmic space as a homodimer involved in the acquisition of phosphate from esters ( Suzuki et al. 2005 ). In human cells, ALPase is important for regulating B cell differentiation and maturation ( Hossain and Jung 2008 ). In rat intestinal epithelial cells, ALPase activity is associated with morphological alterations ( Wood et al. 2003 ). In insects, acid phosphatase activity is commonly related to cellular pathways leading to cell secretion ( Eguchi 1995 ). Although the role of ALPase located in the organelles of P. puparum venom gland secretory cells remains to be established, we believe that it could be involved in the venom secretion cycle based on its presence in nuclei and secretory granules that are indicative of protein synthesis and secretion.
It is known that the response of ALPase to heat treatment can vary widely. In P. puparum , the thermal denaturalization results indicated that this venom enzyme was weakly resistant to heat treatment, with a 30 min exposure to 60°C resulting in 73.3% inhibition. This observation is consistent with the other reports of the thermal stability of a number of ALPases from mammalian intestine and insect salivary gland which were resistant to heat treatment ( Goldstein et al. 1980 ; Funk 2001 ). As ALPase is a dimeric metalloenzyme ( Eguchi 1995 ), the effect of different divalent cations on its activity was evaluated. The results showed that venom ALPase activity was reduced by Zn 2+ agreeing with the observation of Rodrigues et al. ( 2006 ) who reported that Zn 2+ is an inhibitor of ALPase from spider venom. ALPases in a variety of organisms demonstrate activity dependence on Zn 2+ ( Mazorr et al. 2002 ). A series of studies on insect ALPases showed that it was activated by Mg 2+ and Ca 2+ ( Terra et al. 1979 ; Eguchi 1995 ). Similar results were found for the ALPase from P. puparum venom. This enzyme was also highly activated by Mn 2+ . The above results suggest that P. puparum venom ALPase is temperature sensitive and sensitive to some metal ions that may play important roles in the enzyme activity. Since only tiny amount of venom is available for recovering from the parasitoid wasps, purification of venom ALPase from whole body homogenates is required. This partial biochemical information is useful for providing some basis to isolate this enzyme.
The venom ALPase gene was cloned from P. puparum using a venom apparatus cDNA library as template, providing the first evidence for a clone of this venom proteinase gene from the venom gland of parasitoids. Generally, ALPases in insects are present in different isozymes ( Eguchi 1995 ). In the silkworm, B. mori , two ALPase isozymes of mALPase (membrane bound type) and sALPase (soluble type) are known from the larval midgut epithelium ( Itoh et al. 1999 ). The possibility that there are ALPase isozymes present in the venom apparatus of P. puparum is raised. It is possible that the ALPase gene cloned here would be just one of the isozymes that are responsible for the enzyme activity detected during the biochemical characterization described above. To solve this problem, efforts to isolate other clones having different nucleotide sequences and southern blot analyses to identify the gene copy number of ALPase in the venom apparatus of P. puparum are needed. With regard to the clone isolated in this study, its calculated molecular weight of 59.83 kDa and pi of 6.98 is similar to values previously reported for other insect ALPases ( Itoh et al. 1991 , 1999 ). In all reported parasitoid venom proteins ( Moreau and Guillot 2005 ), signal peptides are located in their sequences. P. puparum venom ALPase also has this structure.
It is known that signal peptides are functional for directing polypeptides into the endoplasmic reticulum to initiate secretion ( Leader 1979 ). This suggests that venom ALPase of P. puparum is derived from the venom apparatus secretory system. This strengthens the above conclusion obtained from histochemical observations that this enzyme is as an intracellular enzyme produced in the venom gland secretory cells and finally secreted into the venom reservoir for storage.
ALPases from insects are post-translationally modified by glycosylation ( Itoh et al. 1999 ; Asgeirsson et al. 2003 ). In addition to N-glycosylation sites, venom ALPase from P. puparum had different numbers of potential phosphorylation and N-myristoylation sites. Concerning the effect of divalent cations on ALPase, residues for metal ligand sites were present in its sequence. Furthermore, sequence alignment analysis demonstrated that the venom protein exhibited features typical of ALPase from other species including the long conserved ALPase gene family signature sequence, suggesting conservation of function.
In living organisms ALPase activity serves a variety of essential functions that include nutrition, phosphate metabolism, intracellular signaling as well as modification and transport of metabolites across biological membranes ( Cho-Ngwa et al. 2007 ). For example, in crabs, ALPase is important in the absorption of phosphate and calcium from seawater and for the shell formation and as potential biochemical indicator of stress ( Pinoni et al. 2005 ). In mammals, there is some experimental evidence that ALPase is involved in tissue development and cellular differentiation, although its precise role in these cellular events is not known ( Ali et al. 2005 ). In bacteria, ALPase plays a key role providing inorganic phosphate from the growth medium ( Torriani 1990 ). It has been suggested that ALPases are involved with nutrient absorption involved in the processes of digestion in insect midgut ( Eguchi 1995 ; Yi and Adams 2001 ). In keeping with this hypothesis, it is worth suggesting that ALPase in parasitoid venom could assist parasitoid absorption of nutrients from host insects. Moreover, parasitoid venom ALPase could be potentially involved in many other functions as occurs in other organisms. New experiments with these speculations are needed to understand the function of ALPase in parasitoid venom.
In parasitoid wasps, two venom genes, phenoloxidase and acid phosphatase, had specific expression patterns that responded during development ( Parkinson et al. 2001 ; Zhu et al. 2008 ). It is important to investigate the changes of venom ALPase gene expression after female adult wasp eclosion. During days 0 to 7 after eclosion, RT-PCR analysis showed that gene transcription of venom ALPase increased. By comparison with the mRNA level, the change profiles of enzymatic activities found in the venom apparatus of P. puparum were in accord with gene transcription during the time interval observed. Like venom phenoloxidase and acid phosphatase ( Parkinson et al. 2001 ; Zhu et al. 2008 ), we suggest that transcriptional activity of venom ALPase would be also correlated with the oviposition process. | Using chromogenic substrates 5-bromo-4-chloro-3′-indolyl phosphate and nitro blue tetrazolium, alkaline phosphatase (ALPase) was histochemically detected in the venom apparatus of an endoparasitoid wasp, Pteromalus puparum L. (Hymenoptera: Pteromalidae). Ultrastructural observations demonstrated its presence in the secretory vesicles and nuclei of the venom gland secretory cells. Using p-nitrophenyl phosphate as substrate to measure enzyme activity, the venom ALPase was found to be temperature dependent with bivalent cation effects. The full-length cDNA sequence of ALPase was amplified from the cDNA library of the venom apparatus of P. puparum , providing the first molecular characterization of ALPase in the venom of a parasitoid wasp. The cDNA consisted of 2645 bp with a 1623 bp open reading frame coding for 541 deduced amino acids with a predicted molecular mass of 59.83 kDa and pI of 6.98. Using multiple sequence alignment, the deduced amino acid sequence shared high identity to its counterparts from other insects. A signal peptide and a long conserved ALPase gene family signature sequence were observed. The amino acid sequence of this venom protein was characterized with different potential glycosylation, myristoylation, phosphorylation sites and metal ligand sites. The transcript of the ALPase gene was detected by RT-PCR in the venom apparatus with development related expression after adult wasp emergence, suggesting a possible correlation with the oviposition process.
Key words | Acknowledgements
We thank Dr. Peng Xu in Institute of Insect Sciences, Zhejiang University, Hangzhou, China, for how to use the Auto-Montage camera system and Mrs. Ying Xu in the Facility Center of Zhejiang University, Hangzhou, China, for technical assistance with transmission electron microscopy. This work was supported by grants from the National Basic Research and Development Program of China (Grant No. 2006CB102005), the National Nature Science Foundation of China (Grant Nos. 30571251 and 30671825), the program for New Century Excellent Talents in University of the Ministry of Education of China (Grant No. NCET-05-0513), and the Innovation Research Team Program of the Ministry of Education of China (Grant No. IRT0535). We are grateful to the two anonymous referees for their very constructive remarks. | CC BY | no | 2022-01-12 15:40:23 | J Insect Sci. 2010 Mar 4; 10:14 | oa_package/00/9a/PMC3014669.tar.gz |
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PMC3014670 | 20578880 | Introduction
South Bohemian peatbogs are isolated, paleorefugial habitats with unique flora and fauna characteristic of oligotrophic mires. These habitats have developed under specific conditions, predominantly within a “forest tundra climate” ( Spitzer 1994 ). Parasitic braconid wasps (Hymenoptera: Braconidae) are among the most often encountered components of these ecosystems, and their diversity in peatlands implies the presence of complex interactions among plants, hosts, and parasitoids. Despite previous work (e.g. Enderlein 1908 ; Krogerus 1960 ; Finnamore 1994 ; Papp 1982 ; Tereshkin 1996 ; Lozan and Tobias 2002 ), faunistic and taxonomic analyses of peatbog braconids remain limited. Little is known about host-parasitoid linkages in peatbogs ( Krogerus 1960 ; Chalmers-Hunt 1969 ; Havel 1970 ; Bourn and Warren 1997 ), despite their potential importance to bog ecosystems and implications for bogland conservation.
Typically, insects inhabiting peatbogs are classed ecologically as tyrphobionts, tyrphophiles, or tyrphoneutrals (e.g. Spitzer 1994 ; Spitzer et al. 1999 ; Spitzer and Danks 2006 ), the former being of conservation interest because of their strict dependence on the bog environment. To date, there is no existing ecological classification of Braconidae inhabiting peatbogs. A critical problem is that taxonomic descriptions and records from Central Europe (and elsewhere) do not always include information about habitat, and the records may be false as misidentifications and misinterpretations are rather common in the many groups that are not properly revised. Additionally, limited, and sometimes controversial, data on species ranges and habitat preferences often hinder our understanding of species' bog affinity (for members of the genus Microchelonus as an example, see Telenga 1941 ; Shenefelt 1973 ).
Here we sampled the braconid parasitoid fauna from 7 sites in South Bohemia and used curated specimens from 7 museums in order to compile a list of tyrphobiontic species (full list of species provided in the Appendix ) based on all available geographical information, field data, museum material (Palaearctic only), and direct correspondence with specialists, collection curators and collectors. This is the first large-scale survey of braconid parasitoids in Central Europe, with particular emphasis on the bogs of South Bohemia in the Czech Republic. | Materials and Methods
Site descriptions
The 7 examined bogs span an altitudinal gradient from lowland (470 m) to mountain raised peatbogs (1120 m) and are dominated by Sphagnum mosses, ericaceous Vaccinium shrubs, and, to some degree, forest trees, particularly mountain and bog pine (complex of Pinus mugo s.l.) ( Spitzer 1994 ; Spitzer and Jaros 1993 , 1998 ; Spitzer et al. 2003 ; Bezděk et al. 2006 ; Kučera 1995 ). Some of the investigated bogs of South Bohemia, particularly in the Treboň Basin, were partially eroded by former human activities that left a succession of vegetation in clearings and hollows after peat exploitation. Following is a brief description of the bogs used in the study (see Figures 1 – 2 ): (1) Červené Blato (472 m, 331 ha), in the Třeboň Basin near Š almanovice, is a transient peatbog forested by bog pine ( Pinus rotundata ) and shrubs of Vaccinium spp. and Ledum palustre; (2) Mrtvý Luh (740 m, 310 ha) near Volary is a core zone of Š umava National Park. It is a valley peatbog, surrounded by forest and relatively isolated by mountains. Sphagnum spp., Vaccinium uliginosum, and Eriophorum vaginatum comprise the unforested parts of the bog, with some areas of dwarf forest of P. mugo s. lat; (3) Velká Niva bog (750 m, 120 ha) is near Lenora in the Sumava Mountains. In outer areas, it is a waterlogged spruce forest, and the central area is open forest of P. mugo s. lat. with patches of Betula pubescens; (4) Chalupská Slat' bog (900 m, 116 ha) near Borová Lada is also a core zone of Šumava National Park. It is an intermediate between valley bog and mountain raised bog, with a central lake, with Betula spp. and Carex spp. in the bog margins, and with an outer ring of mountain pine forest; (5) Jezerní Slat' is a montane upland peatbog (1050 – 1075m, 190 ha), a core zone in the Š umava National Park, covered with large islands of Pinus mugo s. lat. and treeless areas of shrubs of Betula nana and Vaccinium spp. in cotton-grass layers; (6) Rokytská Slat' (1073 – 1119 m, over 250 ha), a core zone in the Sumava Mts., is a typical mountain-type of raised peatbog, where small, central bog-lakes are surrounded by mountain pines ( P. mugo s. lat.), with spruce trees and dwarf birches of B. nana and Vaccinum spp. in opened areas; (7) Luzenská (= Hranični) Slat' (1130 – 1120m), a core zone in the Sumava National Park, is a complex of several small, raised peatbogs, with several small bog-lakes and shrubs of Vaccinium spp. and P. mugo s. lat., surrounded by norway spruce forest ( Picea excelsa ). All but Luzenská (= Hranični) Slat' are National Nature Reserves.
Data are included for three additional peatbog areas in Central Europe using the following museum collections: the National Nature Reserves of Velke Dařko in the Czech-Moravian highlands (material of MMB); the Skřítek peatbog ( ca. 166 ha) in the Jeseníky Mountains of Silesia (material of MMB); the Pürgschachenmoor bog in Styria, Austria, which is an Naturschutzgebiet and the World Wildlife Fund Nature Reserve and one of the southernmost outposts of boreal peatland ecosystems in Central Europe (see Spitzer et al. 1996 ) (material of MNHV). | Results and Discussion
Diversity and association to peatbogs
A total of 350 species from 19 subfamilies and 76 genera were recorded in the samples from peatbogs of South Bohemia during 2001–2007 (see the Appendix for a complete list). Most of the Braconidae, 222 species (64%), are new records in the Czech Republic, although almost all species are known from neighbouring countries ( Čapek and Lukáš 1989 ; Čapek and Hofmann 1997; Van Achterberg 2002 ; Belokobylskij et al. 2003 ; Belokobylskij 2004 ; Papp 2005 ). The most numerous taxa belong to the subfamilies Alysiinae (94 new country records) and Microgastrinae (44 new country records), comprising approximately 63% of all new faunistic records for the country.
The vast majority of species were also found in adjacent or “non-bog” areas and as a result, only 5 species (1.4%) are proposed here as tyrphobiont taxa ( Table 1 ): Microchelonus basalis (Curtis), M. koponeni Tobias, Coloneura ate (Nixon), C. danica Griffiths and Myiocephalus niger Fischer. These species were never present outside of bog habitat in Central Europe and thus appear to be obligatory components of peatlands. M. niger occurs in northern Europe and Asia but the remaining four species are recorded only from Europe. They are typically boreal and arctic/cold-adapted species, and here display a clear, narrow association with bogs in Central Europe, as well as a probable dependence on edaphic conditions of the bog habitat.
Four additional species (1.1% of the total), Microchelonus pedator (Dahlbom), M. subpedator Tobias, M. karadagi Tobias and M. gravenhorstii (Nees) are here classified as tyrphophiles, being frequently encountered within peatbogs and only rarely in wet meadows or forests nearby. The remaining 341 species (97.5%) are considered tyrphoneutrals, being more or less widely distributed and not only found in peatlands. Most of them are eurytopic or opportunistic species, often abundant and locally dominant.
Taxonomic composition of samples
In peatbogs, the cyclostome complex of subfamilies is represented by the Braconinae with three genera and 23 species, the Rogadinae by 15 species of only one genus ( Aleiodes Wesmael), the Exothecinae s. 1. by five genera and ten species, the Doryctinae by three species, and the Rhyssalinae by two species. Many of these are associated with forest areas in boreal or temperate zones, being widely distributed in Palaearctic and/or Holarctic realms. The polymorphic Euphorinae, present in all studied bogs with 16 genera and 40 species, were locally abundant and also found in other wetlands throughout the country. The carapace-bearing Cheloninae wasps were present in peatbogs with four genera and 20 species ( Lozan 2006 ; Lozan et al. unpublished data). Seemingly, most members of the genus Microchelonus Szépligeti are obviously confined to bog habitat ( Lozan and Tobias 2002 , 2006 ), however, their real diversity and ecological affinity to peatbogs are still poorly understood.
The Microgastrinae is the second speciose subfamily, with 8 genera, 63 species and containing koinobiont endoparasitoids of Lepidoptera. Several of the species were the most abundant braconids among all samples. Remarkably, the species Cotesia tibialis (with a trans-Palaearctic distribution) presented almost everywhere and was extremely abundant in Mrtvý Luh and Chalupská Slat'.
There were 142 species (40% of all collected braconids) within the clade of subfamilies Opiinae + Alysiinae, koinobiont endoparasites of cyclorrhaphous Diptera. Opiinae were represented by 6 genera and 26 species, while 24 genera and 124 species of Alysiinae were found in investigated peatbogs. Most of these are newly recorded here for the fauna of Czech Republic. Such genera as Alysia Latreille, Idiasta Förster, Anisocyrta Förster, and Phaenocarpa Förster are known to occur in the northern part of the northern hemisphere (most taxa are boreal with Holarctic range) ( Wharton 1986 ) and were present in almost all of the investigated bogs.
Nocturnality
Crepuscular and/or nocturnal flight activity is well-known in several subfamilies of braconids ( Gauld and Huddleston 1976 ; Jakimavičius 1979 ; Čapek and Lukaš 1981 ; Huddleston and Gauld 1988 ; Quicke 1992 ; Lozan 2002 , 2004 ). From 26 species in our light trap samples, only one tyrphobiont ( M. niger ) was present, while the rest seem to be tyrphoneutrals, being widely distributed and not restricted to the bog habitat ( Lozan 2002 ). Only two species are egg-larval idiobionts ( Diospilus Haliday) of coleopteran larvae ( Belokobylskij 1996 ), and the rest of the species are endoparasitoid koinobionts of either Lepidoptera or Coleoptera, or, rarely, Diptera.
Tyrpho-classification of Braconidae
There is a general lack of ecological data, particularly habitat affinity, for most Hymenoptera Parasitica; therefore, the tyrpho-classification is an attempt to distinguish taxa with different degrees of association to peatbogs. In the samples there were many species, somewhat locally abundant, overwhelmingly dominant in peatbogs and rarely (if at all) present in other habitats within the country; however, recorded habitat data from elsewhere range from forests to meadows and various wetlands (including marshes and peatbogs).
Quantitative indexes in bog samples have not always been reflecting the true connection to peatbogs, as many eurytopic species were numerous within rather than outside and as other species have only been encountered in investigated sites. Several Holarctic species ( Ontsira imperator Haliday, Ichneutes reunitor Nees, Anisocyrta perdita Haliday), ranging around the circumboreal forests in the northern hemisphere have also been recorded from peatbogs and, likewise, the other forest type species in the Palaearctic region ( Macrocentrus resinellae (L.), Bracon hylobii Ratzeburg, Coeloides abdominalis (Zetterstedt) etc.), which are widely distributed and not connected with peatbogs, could easily be mistreated as highly associated to bogs. Usually, these species are cold-adapted and rather abundant in northern areas ( Van Achterberg 1986 ; Koponen 2000 ; Hance et al. 2007 ), so their habitat pictures include not only forests, but also wetlands (swamps, marches, peatbogs), boggy forested areas and upland (alpine) meadows southward, being ecologically confined to their potential forest hosts. Such boreo-montane elements as Alysia fuscipennis Haliday, which is a European species occurring in uplands and montane meadows (sometimes abundant), has also been found in peatbogs; however, most specimens were collected in the wet meadows nearby. The Holarctic euphorine species Myiocephalus boops Wesmael is recorded from the Pinetum-sphagnosum community (collected by Malaise trap, abundant) in peatlands of Belarus, swept also from B. nana in boggy areas of Finland ( Koponen and Vikberg 1984 ) and in taiga-forest of Russia (Buryatia, Yakutia, Kamchatka) ( Belokobylskij and Tobias 2000 ). Nevertheless, all these species are not typical for peatlands, so they are incuded in the tyrphoneutral category.
Tyrphobiontic Braconidae: Species breakdown and habitat affinity
This is a very specific and characteristic ecological group restricted to bogs, including five braconid species in our samples, with no evidence of occurrence in other habitats in Central Europe ( M. niger is recorded also from high mountain, however, boggy areas). All these species seem to require microclimatic conditions typical for peatbogs. There is no obvious evidence these species are associated closely with bogs through their hosts (and it should not be obligatory), but edaphic conditions are probably among basic ecological determinants in their successful development. All these species are cold adapted with affinities to boreal and subarctic areas in Europe and Asia ( Spitzer and Danks 2006 ). When matching data of localities and/or regions where species were collected, it was discovered that all those areas were either boggy forests (partly open lands with lakes and peatbogs, in Sealand and Jutland [Denmark], for instance, peatbogs within sandy dunes) or areas with severe climate (mountains, boreal forest, tundra). Despite some taxonomic, geographical and ecological uncertainties while working out various series elsewhere, their tyrphobiontic connection is obvious. Many biotopic data used in this classification are from either field observation or from information provided by other collectors. Thus, the species' actual distributional and habitat characteristics within their virtual ranges, including Central Europe, are as follows (also see Table 1 ):
1) Microchelonus basalis. European species (north Europe according to Fahringer 1930 ): England (southwest, hilly open moorland with blanket bogs of the Exmoor National Park), Scotland (Moulin Moor peatbogs), Finland (northern Lapland, highlands: bogs, forests and lakes), Sweden (bogs of Tyresta National Park, plains of southermost Skĺne province, Degaberga and Hoor [with no details]), Denmark (East Jutland: in dry sandy areas with peatbogs), Germany (in Kettner 1965 , but no ecological data; however, the geographical locality corresponds with peatbog areas in northwest Germany, somewhat similar to habitat data from Denmark), Poland (peatbogs with sandy dunes in northeastern parts of the country, somewhat similar habitats to Denmark and Germany; see Enderlein 1908 ), Austria (a boreal valley peatbog, 632 m, closed by Alps in Styria; see Spitzer et al. 1996 ), Czech Republic (only from three peatbogs). Host unknown.
Shenefelt ( 1973 ), based on Telenga's data ( 1941 ), indicates Palaearctic distribution for M. basalis: England, Sweden, Russia, Kazakhstan, Iran, Hungary, Finland and Germany. The material is missing and we consider it just a misidentification. Analysis of long series of M. basalis in the collection of BMNH (collected in England and Sweden) showed there were several species in fact ( M. basalis, M. pusillus (Szépligeti) and M. atripes (Thomson). We are not sure about the geographical data of labels of some material, so these specimens are excluded (collected in 1931–1935, Marshal coll.' specimen dates 1904). There are two additional specimens from Germany in the collection of BMNH with only label numbers and a locality of a rather common name without any details. Another old specimen in the collection of ZIN is labeled only as “Germany”. The species is included in the checklist of the Braconidae of Germany ( Belokobylskij et al. 2003 ) with reference to Shenefelt ( 1973 ), which reffers to Telenga ( 1941 ), but the material is missing. No specimens from Russia and Asia were found at all, but it does not exclude the species might occur in boggy areas there.
2) Microchelonus koponeni. European species. Described from southeast Sweden in forest and bog areas ( Tobias 1995 ), it has recently been discovered in several peatbogs of Czech Republic ( Lozan and Tobias 2002 ). Long series of this species also have been found in MMB collection from other peatbogs throughout the country in the Czech-Moravian highlands and east-northern Moravia (Silesia, Jeseniky Mts.) (old collection [Cheloninae], 1944–1947, leg. Hoffer, unpublished data). Newer data also come from Sweden (Tyresta National Park, Ungfars mosse bog - materials of the Swedish Malaise Trap Project). Host unknown.
3) Myiocephalus niger. Trans-Palaearctic distribution: Austria (boggy areas in mountains, 1000–1150m in Alps), Netherlands (sandy area with lake [“fen”] surrounded by forest), Byelorussia (boggy forests), Russia (northwestern Murmansk region; and Siberia - northern taiga, Kamchatka - boggy forests with lakes). In Czech Republic, only from an upland peatbog (1075 m). Host: presumably ants; however, no direct evidence ( Belokobylskij 2000 ).
4) Coloneura ate. European species. It was described from Sweden, the southernmost Skåne province ( Nixon 1943 ) with a maritime climate where the habitat picture relates to large forests, mountains and bogs/peatbogs within. In eastern Denmark (Sealand) its habitat covers boggy areas, similar to that of south Sweden (in Griffiths 1968 ). In Czech Republic only from peatbogs. It is also mentioned in the checklist of Braconidae of Hungary ( Papp 2005 ), but the species occurrence has not been confirmed. Known host: puparia of Liriomyza Mik or Metopomyza Enderlein species (Diptera: Agromyzidae) ( Griffiths 1968 ).
5) Coloneura danica. European distribution (similar habitat requirements as previous species): Denmark (Sealand, in boggy areas), Czech Republic (only in peatbogs). Known host: Metopomyza nigrohumeralis Hendel (Diptera: Agromyzidae), miners on Carex ( Griffiths 1968 ).
These five species are highly stenotopic taxa: typically boreal-alpine species, well confined to peatbogs in southern temperate zones and covering various areas northward, and for M. niger somewhat extended southward in Siberian or Far Eastern north-south mountain chains or forests and peatbogs.
Tyrphophilous behaviour
While tyrphobiont braconids are ecologically well characterized by the conditions of the bog habitat, the tyrphophiles are not typical for peatbogs and, therefore, not an easily distinguishable category. Members of the subgenus Stylochelonus Hellén species group of the genus Microchelonus were quite abundant in peatlands, but found were also in wet meadows nearby. Comparatively widely distributed in central-northern Europe, Microchelonus ( S. ) pedator, that is considered a rare species, has been collected in abundance in several peat bogs and surrounding meadows (collection of RMNH, Leiden, Netherlands). Hellén ( 1958 ) reported Aphelia paleana (Hübner) (Lepidoptera: Tortricidae) as host for this species; however, there is no reliable evidence of parasitism ( Papp 1997 ). Taxonomically close to previous species, but lesser known in Europe, is M. ( S. ) subpedator, which is defined ecologically by the same habitat requirements. M. ( S. ) karadagi, described from mountain forest-steppe (wood, ‘grass + Stipa’, in Tobias 1995 ) area of Crimea peninsula (Kara-Dag) in Ukraine, was recorded from several peatbogs of Czech Republic (but interestingly never outside the bog habitat at all, see Lozan and Tobias 2002 ) and from bogs of Tyresta National Park in Sweden (materials of Swedish Malaise Trap Project). Another chelonine species, Microchelonus ( Parachelonus ) gravenhorstii, is likely to be tyrphophilous, occurring in boggy forests and other peatlands. Long series of this species have been in the collections of RMNH (collected in Netherlands and Spain [peatlands of Galicia]) and MIZW (from peatbogs and boggy forests).
Of course, the list can be extended, and further field data are needed to properly evaluate species belonging to certain categories (see Spitzer and Danks 2006 ). This is another study case, as tyrphophilous categorization would require long-term and detailed statistical analysis. Not excluded, some mentioned tyrphophiles are in fact tyrphobionts in Central Europe and further complex investigations may change their status.
Tyrphoneutrals
The rest of the studied Braconidae are considered tyrphoneutrals, most of them having a more general distribution (e.g. generalists, see Lozan 2002 ; Spitzer and Danks 2006 ) and/or eurytopic components of various habitats. In peatlands they could be either abundant (especially some Cotesia Cameron, Apanteles Förster, Microgaster Latreille, Dacnusa Haliday etc.) or rare species, but never characteristic to bogs. For many of them the bog habitat may be a true refuge, where they can survive and/or find an alternative habitat as a result of changes in environment. However, a certain degree of tyrphobiontic and/or tyrphophile tendency can be found in various groups among Braconidae. As there exists lots of taxonomic issues over species validity and many parasitoid species awaiting discovery, understanding the “shifts” in ecological preference of parasitoids and the mechanisms driving it, e.g. generalist/specialist versus habitat/host affinity, are sometimes very problematic.
Parasitoids and Bog Landscape
It is already beyond question that the bog environment is rather heterogeneous and provides important habitat conditions for insects ( Spitzer and Danks 2006 ). Presence of patches of trees/shrubs in an opened, treeless area is just emphasizing the role of bog habitat in enhancing parasitoid diversity and raising affinity to the habitat (see Roland 2000 ). Being mostly niche specific, parasitoids are searching for particular microhabitat or host food niche (see Hawkins 1994 ), which can be found in a host-rich environment such as peatbogs. Hawkins ( 1994 ) also pointed out that hymenopterous generalist (idiobiont) and specialist (koinobiont) parasitoids respond differentially to plant architecture, and plant effects are strongest in natural habitats. Despite the fact that parasitoids may actually respond differentially to vegetation assemblages, the results show that the bog habitat adequately supports this high clade of species richness, where koinobiont braconids are predominant. | Results and Discussion
Diversity and association to peatbogs
A total of 350 species from 19 subfamilies and 76 genera were recorded in the samples from peatbogs of South Bohemia during 2001–2007 (see the Appendix for a complete list). Most of the Braconidae, 222 species (64%), are new records in the Czech Republic, although almost all species are known from neighbouring countries ( Čapek and Lukáš 1989 ; Čapek and Hofmann 1997; Van Achterberg 2002 ; Belokobylskij et al. 2003 ; Belokobylskij 2004 ; Papp 2005 ). The most numerous taxa belong to the subfamilies Alysiinae (94 new country records) and Microgastrinae (44 new country records), comprising approximately 63% of all new faunistic records for the country.
The vast majority of species were also found in adjacent or “non-bog” areas and as a result, only 5 species (1.4%) are proposed here as tyrphobiont taxa ( Table 1 ): Microchelonus basalis (Curtis), M. koponeni Tobias, Coloneura ate (Nixon), C. danica Griffiths and Myiocephalus niger Fischer. These species were never present outside of bog habitat in Central Europe and thus appear to be obligatory components of peatlands. M. niger occurs in northern Europe and Asia but the remaining four species are recorded only from Europe. They are typically boreal and arctic/cold-adapted species, and here display a clear, narrow association with bogs in Central Europe, as well as a probable dependence on edaphic conditions of the bog habitat.
Four additional species (1.1% of the total), Microchelonus pedator (Dahlbom), M. subpedator Tobias, M. karadagi Tobias and M. gravenhorstii (Nees) are here classified as tyrphophiles, being frequently encountered within peatbogs and only rarely in wet meadows or forests nearby. The remaining 341 species (97.5%) are considered tyrphoneutrals, being more or less widely distributed and not only found in peatlands. Most of them are eurytopic or opportunistic species, often abundant and locally dominant.
Taxonomic composition of samples
In peatbogs, the cyclostome complex of subfamilies is represented by the Braconinae with three genera and 23 species, the Rogadinae by 15 species of only one genus ( Aleiodes Wesmael), the Exothecinae s. 1. by five genera and ten species, the Doryctinae by three species, and the Rhyssalinae by two species. Many of these are associated with forest areas in boreal or temperate zones, being widely distributed in Palaearctic and/or Holarctic realms. The polymorphic Euphorinae, present in all studied bogs with 16 genera and 40 species, were locally abundant and also found in other wetlands throughout the country. The carapace-bearing Cheloninae wasps were present in peatbogs with four genera and 20 species ( Lozan 2006 ; Lozan et al. unpublished data). Seemingly, most members of the genus Microchelonus Szépligeti are obviously confined to bog habitat ( Lozan and Tobias 2002 , 2006 ), however, their real diversity and ecological affinity to peatbogs are still poorly understood.
The Microgastrinae is the second speciose subfamily, with 8 genera, 63 species and containing koinobiont endoparasitoids of Lepidoptera. Several of the species were the most abundant braconids among all samples. Remarkably, the species Cotesia tibialis (with a trans-Palaearctic distribution) presented almost everywhere and was extremely abundant in Mrtvý Luh and Chalupská Slat'.
There were 142 species (40% of all collected braconids) within the clade of subfamilies Opiinae + Alysiinae, koinobiont endoparasites of cyclorrhaphous Diptera. Opiinae were represented by 6 genera and 26 species, while 24 genera and 124 species of Alysiinae were found in investigated peatbogs. Most of these are newly recorded here for the fauna of Czech Republic. Such genera as Alysia Latreille, Idiasta Förster, Anisocyrta Förster, and Phaenocarpa Förster are known to occur in the northern part of the northern hemisphere (most taxa are boreal with Holarctic range) ( Wharton 1986 ) and were present in almost all of the investigated bogs.
Nocturnality
Crepuscular and/or nocturnal flight activity is well-known in several subfamilies of braconids ( Gauld and Huddleston 1976 ; Jakimavičius 1979 ; Čapek and Lukaš 1981 ; Huddleston and Gauld 1988 ; Quicke 1992 ; Lozan 2002 , 2004 ). From 26 species in our light trap samples, only one tyrphobiont ( M. niger ) was present, while the rest seem to be tyrphoneutrals, being widely distributed and not restricted to the bog habitat ( Lozan 2002 ). Only two species are egg-larval idiobionts ( Diospilus Haliday) of coleopteran larvae ( Belokobylskij 1996 ), and the rest of the species are endoparasitoid koinobionts of either Lepidoptera or Coleoptera, or, rarely, Diptera.
Tyrpho-classification of Braconidae
There is a general lack of ecological data, particularly habitat affinity, for most Hymenoptera Parasitica; therefore, the tyrpho-classification is an attempt to distinguish taxa with different degrees of association to peatbogs. In the samples there were many species, somewhat locally abundant, overwhelmingly dominant in peatbogs and rarely (if at all) present in other habitats within the country; however, recorded habitat data from elsewhere range from forests to meadows and various wetlands (including marshes and peatbogs).
Quantitative indexes in bog samples have not always been reflecting the true connection to peatbogs, as many eurytopic species were numerous within rather than outside and as other species have only been encountered in investigated sites. Several Holarctic species ( Ontsira imperator Haliday, Ichneutes reunitor Nees, Anisocyrta perdita Haliday), ranging around the circumboreal forests in the northern hemisphere have also been recorded from peatbogs and, likewise, the other forest type species in the Palaearctic region ( Macrocentrus resinellae (L.), Bracon hylobii Ratzeburg, Coeloides abdominalis (Zetterstedt) etc.), which are widely distributed and not connected with peatbogs, could easily be mistreated as highly associated to bogs. Usually, these species are cold-adapted and rather abundant in northern areas ( Van Achterberg 1986 ; Koponen 2000 ; Hance et al. 2007 ), so their habitat pictures include not only forests, but also wetlands (swamps, marches, peatbogs), boggy forested areas and upland (alpine) meadows southward, being ecologically confined to their potential forest hosts. Such boreo-montane elements as Alysia fuscipennis Haliday, which is a European species occurring in uplands and montane meadows (sometimes abundant), has also been found in peatbogs; however, most specimens were collected in the wet meadows nearby. The Holarctic euphorine species Myiocephalus boops Wesmael is recorded from the Pinetum-sphagnosum community (collected by Malaise trap, abundant) in peatlands of Belarus, swept also from B. nana in boggy areas of Finland ( Koponen and Vikberg 1984 ) and in taiga-forest of Russia (Buryatia, Yakutia, Kamchatka) ( Belokobylskij and Tobias 2000 ). Nevertheless, all these species are not typical for peatlands, so they are incuded in the tyrphoneutral category.
Tyrphobiontic Braconidae: Species breakdown and habitat affinity
This is a very specific and characteristic ecological group restricted to bogs, including five braconid species in our samples, with no evidence of occurrence in other habitats in Central Europe ( M. niger is recorded also from high mountain, however, boggy areas). All these species seem to require microclimatic conditions typical for peatbogs. There is no obvious evidence these species are associated closely with bogs through their hosts (and it should not be obligatory), but edaphic conditions are probably among basic ecological determinants in their successful development. All these species are cold adapted with affinities to boreal and subarctic areas in Europe and Asia ( Spitzer and Danks 2006 ). When matching data of localities and/or regions where species were collected, it was discovered that all those areas were either boggy forests (partly open lands with lakes and peatbogs, in Sealand and Jutland [Denmark], for instance, peatbogs within sandy dunes) or areas with severe climate (mountains, boreal forest, tundra). Despite some taxonomic, geographical and ecological uncertainties while working out various series elsewhere, their tyrphobiontic connection is obvious. Many biotopic data used in this classification are from either field observation or from information provided by other collectors. Thus, the species' actual distributional and habitat characteristics within their virtual ranges, including Central Europe, are as follows (also see Table 1 ):
1) Microchelonus basalis. European species (north Europe according to Fahringer 1930 ): England (southwest, hilly open moorland with blanket bogs of the Exmoor National Park), Scotland (Moulin Moor peatbogs), Finland (northern Lapland, highlands: bogs, forests and lakes), Sweden (bogs of Tyresta National Park, plains of southermost Skĺne province, Degaberga and Hoor [with no details]), Denmark (East Jutland: in dry sandy areas with peatbogs), Germany (in Kettner 1965 , but no ecological data; however, the geographical locality corresponds with peatbog areas in northwest Germany, somewhat similar to habitat data from Denmark), Poland (peatbogs with sandy dunes in northeastern parts of the country, somewhat similar habitats to Denmark and Germany; see Enderlein 1908 ), Austria (a boreal valley peatbog, 632 m, closed by Alps in Styria; see Spitzer et al. 1996 ), Czech Republic (only from three peatbogs). Host unknown.
Shenefelt ( 1973 ), based on Telenga's data ( 1941 ), indicates Palaearctic distribution for M. basalis: England, Sweden, Russia, Kazakhstan, Iran, Hungary, Finland and Germany. The material is missing and we consider it just a misidentification. Analysis of long series of M. basalis in the collection of BMNH (collected in England and Sweden) showed there were several species in fact ( M. basalis, M. pusillus (Szépligeti) and M. atripes (Thomson). We are not sure about the geographical data of labels of some material, so these specimens are excluded (collected in 1931–1935, Marshal coll.' specimen dates 1904). There are two additional specimens from Germany in the collection of BMNH with only label numbers and a locality of a rather common name without any details. Another old specimen in the collection of ZIN is labeled only as “Germany”. The species is included in the checklist of the Braconidae of Germany ( Belokobylskij et al. 2003 ) with reference to Shenefelt ( 1973 ), which reffers to Telenga ( 1941 ), but the material is missing. No specimens from Russia and Asia were found at all, but it does not exclude the species might occur in boggy areas there.
2) Microchelonus koponeni. European species. Described from southeast Sweden in forest and bog areas ( Tobias 1995 ), it has recently been discovered in several peatbogs of Czech Republic ( Lozan and Tobias 2002 ). Long series of this species also have been found in MMB collection from other peatbogs throughout the country in the Czech-Moravian highlands and east-northern Moravia (Silesia, Jeseniky Mts.) (old collection [Cheloninae], 1944–1947, leg. Hoffer, unpublished data). Newer data also come from Sweden (Tyresta National Park, Ungfars mosse bog - materials of the Swedish Malaise Trap Project). Host unknown.
3) Myiocephalus niger. Trans-Palaearctic distribution: Austria (boggy areas in mountains, 1000–1150m in Alps), Netherlands (sandy area with lake [“fen”] surrounded by forest), Byelorussia (boggy forests), Russia (northwestern Murmansk region; and Siberia - northern taiga, Kamchatka - boggy forests with lakes). In Czech Republic, only from an upland peatbog (1075 m). Host: presumably ants; however, no direct evidence ( Belokobylskij 2000 ).
4) Coloneura ate. European species. It was described from Sweden, the southernmost Skåne province ( Nixon 1943 ) with a maritime climate where the habitat picture relates to large forests, mountains and bogs/peatbogs within. In eastern Denmark (Sealand) its habitat covers boggy areas, similar to that of south Sweden (in Griffiths 1968 ). In Czech Republic only from peatbogs. It is also mentioned in the checklist of Braconidae of Hungary ( Papp 2005 ), but the species occurrence has not been confirmed. Known host: puparia of Liriomyza Mik or Metopomyza Enderlein species (Diptera: Agromyzidae) ( Griffiths 1968 ).
5) Coloneura danica. European distribution (similar habitat requirements as previous species): Denmark (Sealand, in boggy areas), Czech Republic (only in peatbogs). Known host: Metopomyza nigrohumeralis Hendel (Diptera: Agromyzidae), miners on Carex ( Griffiths 1968 ).
These five species are highly stenotopic taxa: typically boreal-alpine species, well confined to peatbogs in southern temperate zones and covering various areas northward, and for M. niger somewhat extended southward in Siberian or Far Eastern north-south mountain chains or forests and peatbogs.
Tyrphophilous behaviour
While tyrphobiont braconids are ecologically well characterized by the conditions of the bog habitat, the tyrphophiles are not typical for peatbogs and, therefore, not an easily distinguishable category. Members of the subgenus Stylochelonus Hellén species group of the genus Microchelonus were quite abundant in peatlands, but found were also in wet meadows nearby. Comparatively widely distributed in central-northern Europe, Microchelonus ( S. ) pedator, that is considered a rare species, has been collected in abundance in several peat bogs and surrounding meadows (collection of RMNH, Leiden, Netherlands). Hellén ( 1958 ) reported Aphelia paleana (Hübner) (Lepidoptera: Tortricidae) as host for this species; however, there is no reliable evidence of parasitism ( Papp 1997 ). Taxonomically close to previous species, but lesser known in Europe, is M. ( S. ) subpedator, which is defined ecologically by the same habitat requirements. M. ( S. ) karadagi, described from mountain forest-steppe (wood, ‘grass + Stipa’, in Tobias 1995 ) area of Crimea peninsula (Kara-Dag) in Ukraine, was recorded from several peatbogs of Czech Republic (but interestingly never outside the bog habitat at all, see Lozan and Tobias 2002 ) and from bogs of Tyresta National Park in Sweden (materials of Swedish Malaise Trap Project). Another chelonine species, Microchelonus ( Parachelonus ) gravenhorstii, is likely to be tyrphophilous, occurring in boggy forests and other peatlands. Long series of this species have been in the collections of RMNH (collected in Netherlands and Spain [peatlands of Galicia]) and MIZW (from peatbogs and boggy forests).
Of course, the list can be extended, and further field data are needed to properly evaluate species belonging to certain categories (see Spitzer and Danks 2006 ). This is another study case, as tyrphophilous categorization would require long-term and detailed statistical analysis. Not excluded, some mentioned tyrphophiles are in fact tyrphobionts in Central Europe and further complex investigations may change their status.
Tyrphoneutrals
The rest of the studied Braconidae are considered tyrphoneutrals, most of them having a more general distribution (e.g. generalists, see Lozan 2002 ; Spitzer and Danks 2006 ) and/or eurytopic components of various habitats. In peatlands they could be either abundant (especially some Cotesia Cameron, Apanteles Förster, Microgaster Latreille, Dacnusa Haliday etc.) or rare species, but never characteristic to bogs. For many of them the bog habitat may be a true refuge, where they can survive and/or find an alternative habitat as a result of changes in environment. However, a certain degree of tyrphobiontic and/or tyrphophile tendency can be found in various groups among Braconidae. As there exists lots of taxonomic issues over species validity and many parasitoid species awaiting discovery, understanding the “shifts” in ecological preference of parasitoids and the mechanisms driving it, e.g. generalist/specialist versus habitat/host affinity, are sometimes very problematic.
Parasitoids and Bog Landscape
It is already beyond question that the bog environment is rather heterogeneous and provides important habitat conditions for insects ( Spitzer and Danks 2006 ). Presence of patches of trees/shrubs in an opened, treeless area is just emphasizing the role of bog habitat in enhancing parasitoid diversity and raising affinity to the habitat (see Roland 2000 ). Being mostly niche specific, parasitoids are searching for particular microhabitat or host food niche (see Hawkins 1994 ), which can be found in a host-rich environment such as peatbogs. Hawkins ( 1994 ) also pointed out that hymenopterous generalist (idiobiont) and specialist (koinobiont) parasitoids respond differentially to plant architecture, and plant effects are strongest in natural habitats. Despite the fact that parasitoids may actually respond differentially to vegetation assemblages, the results show that the bog habitat adequately supports this high clade of species richness, where koinobiont braconids are predominant. | Conclusions
A total of 350 species of Braconidae that were properly identified is only a part of the entomofauna of Hymenoptera Parasitica of Central European peatbogs. While most of the collected braconid parasitoids are tyrphoneutral taxa (341 species, 97.5%), with many of them giving obvious preference to peatbogs, a fewer number of them are associated to bogs, e.g. five tryphobiontic (1.4%) and at least four tyrphophilous (1.1%) species, indicating intrinsic processes characteristic to this habitat only.
As the environment changes, by human intervention or by natural influences, the issues of biodiversity and habitat conservation are extremely important. The bog fauna of braconid parasitoids turned out to be very rich, emphasizing the fact that these habitats provide shelter to numerous guilds and groupings, some of which are unique and many of which remain unknown. No doubt some communities contain many hidden cryptic species, and their real diversity should be much higher, especially among traditionally difficult taxonomic groups such as the parasitic Hymenoptera and their hosts ( Quicke 2002 ).
These fragmented ancient patches of peatbogs harbour highly stenotopic taxa (including “geographical races” among some lepidopterans, see Šula and Spitzer 2000 ), which together with their parasitoid complexes are of great scientific and conservational values. They represent a patrimonial uniqueness and a clade of tritrophic interrelationships, being true refugiai islands for biodiversity. | An ecological overview of seven years investigation of Braconidae, a family of parasitoid wasps (Hymenoptera: Ichneumonoidea) and a tyrpho-classification of parasitoids in peatbog areas of South Bohemia, Czech Republic are given. A total of 350 species were recorded in investigated sites, but only five tyrphobionts (1.4%) are proposed: Microchelonus basalis, Microchelonus koponeni, Coloneura ate, Coloneura danica and Myiocephalus niger. All of these species have a boreal-alpine distribution that, in Central Europe, is associated only with peatbogs. Tyrphophilous behaviour is seen in at least four (1.1%) species: Microchelonus pedator, Microchelonus subpedator, Microchelonus karadagi and Microchelonus gravenhorstii; however, a number of other braconids prefer peatbogs because they were more frequently encountered within, rather than outside, the bog habitat. The rest of the braconids (342 species, 97.5%) are tyrphoneutrals, many of them being eurytopic components of various habitats throughout their current ranges. Lists of tyrphobiontic braconids and a brief commentary on species composition, distributional picture of actual ranges, and parasitoid association to bog landscape are provided. Being true refugial habitats for populations in an ever-changing world, peatbogs play a significant role in harboring insect communities.
Keywords | Parasitoid material
All newly collected specimens were obtained as part of a project studying the diversity and ecology of insects in Central European bogs from 2001–2007. In the field, braconids were collected with a sweep net (d = 40 cm), using 50 net strokes each separated by 1 m along a transect, or occasionally with haphazard sweeps. Samples were taken within bogs and in nearby meadows and forests. Light trapping was conducted in three of the peatbogs using a BL-Pennsylvania black light (8 W), serviced once a week over the growing season (March to November). At Mrtvý Luh, two light traps were used during 2000–2002, and one light trap ran at Velká Niva and Jezerní Slat' from 2005–2007.
Some braconids were obtained from leafspinning Lepidoptera on V. uliginosum, although taxonomy of the lepidopteran hosts was undetermined and remained as Tortricidae + Gelechiidae. There were hundreds of cocoon groups (from 15 to 50 white cocoons in a group, usually on Carex or Vaccinium ), mainly of the microgastrine endoparasitoid Cotesia tibialis (Curtis), distributed throughout treeless areas of the Mrtvý Luh and Chalupská Slat' bogs. They most likely originated from a rather common lepidopteran host or several hosts, but the precise relationships with the potential host(s) remain unclear. The cocoons of another microgastrine species, Cotesia gastropachae (Bouché), were found alongside the remnants of larvae of the lasiocampid moth, Macrothylacia rubi (L.), in Mrtvý Luh bog. The parasitoid guilds of bog hosts (Lepidoptera) have been investigated and results will be published elsewhere.
Approximately 7,000 specimens of Ichneumonoidea (Braconidae + Ichneumonidae) were mounted, and most of the material is in the collection of the Biology Centre, Č eské Budějovice, Czech Republic. Certain groups and species were deposited to various museums institutions (ZIM, BMNH, RMNH, MIZW, MMB).
Habitat comparative study
The degree of association to the bog habitat (i.e. status of tyrphobiont, tyrphophilous or tyrphoneutral) was evaluated by combining distributional records, available habitat data, and newly collected data from peatbogs of South Bohemia. Diagrams of soils derived from former or existing peatbogs in Europe, particulary Great Britain and Ireland ( Taylor 1983 ), were compared in order to understand historical patterns of distribution and follow presumed changes in species ranges. Old series of Braconidae from museum collections (1904–1938, BMNH; 1938–1954, MMB) were analyzed, and checklists ( Čapek and Lukáš 1981 , 1989 ; Koponen and Vikberg 1984 ; Koponen 1984 , 1989 , 1992 ; Čapek 1995 ; O'Connor et al. 1999 ; Belokobylskij et al. 2003 ; Belokobylskij 2004 ; Papp 2005 ), catalogues ( Telenga 1941 ; Thomson 1895 ; Fahringer 1930 ; Shenefelt 1973 ; Čapek and Hofmann 1996 ) and keys to species ( Fischer 1972 , 1976 , 1993 ; Papp 1977 , 1995 , 2002 , 2004 ; Tobias 1976 ; Tobias et al. 1986 ; Van Achterberg 1988 ; Tobias and Lozan 1995 ; Belokobylskij and Tobias 1998 , 2000 ) were all used in order to incorporate all available information on species range and habitat affiliation. Other works related to Braconidae of montane moors and/or highlands in Europe were considered as these areas may also harbor endemics or species with certain microclimatic preferences ( Hackman and Meinander 1974 ; Papp 1982 ; Papp and Rezbanyai-Reser 1999 ; Zeman and Vaněk 1999 ; Van Achterberg and Rezbanyai-Reser 2001 ; Tomanović et al. 2007 ).
Many species from the samples were compared with the available material (including type material and series from elsewhere) from other collections and museums to exclude taxonomical uncertainties, particularly in difficult taxa. Taxonomically unclear/difficult specimens (some Aspilota Förster and Dinotrema Förster species) or morphospecies were not included in our analysis. Series of both males and females of some species have been analysed separately to avoid the problem of linking sexes in sexually dimorphic species. | Acknowledgements
We thank K. Spitzer, J. Jaroš, A. Bezděk and H. Zikmundová (Biology Centre, České Budějovice) for their continued help and support. We are also grateful to V. I. Tobias (ZIN), D. L. J. Quicke and G. Broad (BMNH), A. Taeger (Deutsches Entomologisches Insitut im ZALF, Eberswalde), E. Haeselbarth and S. Schmidt (Bavarian State Collection of Zoology, München), J. Papp (Hungarian Natural Museum, Budapest), J. Lukaš (Comenius University in Bratislava), Tomasz Huflejt (MIZW), R. Danielsson (Entomological Museum of Lund University), the Swedish Malaise Trap Project team (Swedish Museum of Natural History, Department of Entomology) for various kinds of help: providing data, access to the Braconidae collection, sending material and samples, label data analysis. This study was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (Grants IAA600070501 and IQS500070505), European Science Foundation (BEPAR Grant Nr. 1667) and by the grant of the Russian Foundation for Basic Research (No 07-04-00454). Access to collections of BMNH and MIZW has been awarded by two EU SYNTHESYS grants (UK GB-TAF-2063 and Poland PL-TAF-3266).
Abbreviations
Natural History Museum, London;
National Museum of Natural History (Naturalis), Leiden, Netherlands;
Zoological Institute, Saint Petersburg, Russia;
Museum of Natural History (Naturhistorisches Museum), Vienna, Austria;
Moravian Museum in Brno, Czech Republic;
Museum and Institute of Zoology, Polish Academy of Sciences, Warsaw, Poland
Appendix 1
Complete list of Braconidae collected in studied peatbogs during 2001–2007.
Rhyssalinae:
Dolopsidea indagator (Haliday), Oncophanes minutus (Wesmael).
Doryctinae:
Dendrosoter middendorffi (Ratzeburg), Ecphylus silesiacus (Ratzeburg), Spathius rubidus (Rossi).
Exothecinae:
Clinocentrus exsertor (Nees), Colastes aciculatus Tobias*, Colastes braconius Haliday, Colastes flavitarsis (Thomson)*, Colastes laevis (Thomson)*, Colastes pilosus Belokobylskij*, Hormius moniliatus (Nees), Hormius similis Szépligeti, Phaenodus pallipes (Foerster), Rhysipolis meditator (Haliday).
Braconinae:
Bracon abbreviator Nees, Bracon atrator Nees*, Bracon cingulator Szépligeti*, Bracon epitriptus Marshall*, Bracon erraticus Wesmael*, Bracon exhilarator Nees*, Bracon hebetor Say, Bracon hylobii Ratzeburg, Bracon intercessor Nees*, Bracon larvicida Wesmael*, Bracon leptus Marshall*, Bracon longicollis Wesmael*, Bracon minutator (Fabricius), Bracon osculator Nees, Bracon picticornis Wesmael*, Bracon praetermissus Marshall*, Bracon stabilis Wesmael*, Bracon terebella Wesmael, Bracon trucidator Marshall*, Bracon variator Nees*, Bracon variegator Spinola*, Coeloides abdominalis (Zetterstedt), Coeloides bostrichorum Giraud.
Rogadinae:
Aleiodes bicolor (Spinola), Aleiodes circumscriptus (Nees), Aleiodes coxalis (Spinola), Aleiodes ductor (Thunberg), Aleiodes esenbeckii (Hartig), Aleiodes fortipes (Reinhard)*, Aleiodes gasterator (Jurine), Aleiodes gastritor (Thunberg)*, Aleiodes nigricornis Wesmael, Aleiodes signatus (Nees), Aleiodes similis (Curtis)*, Aleiodes unipunctator (Thunberg), Aleiodes ungularis (Thomson)*, Aleiodes abraxanae sp.n. van Achterberg*** (paratypes), Aleiodes juniperi sp.n. van Achterberg*** (paratypes).
Gnamptodontinae:
Gnamptodon decoris (Foerster)*, Gnamptodon pumilio (Nees).
Opiinae:
Apodesmia circulator (Nees)*, Apodesmia similis (Szépligeti)*, Biosteres brevisulcus (Thomson)*, Demiostoma parvulum (Wesmael)*, Opius altimontanus Fischer*, Opius ambiguus Wesmael*, Opius attributus Fischer*, Opius caricivorae Fischer*, Opius crassipes Wesmael*, Opius levis Wesmael*, Opius lugens Haliday*, Opius pallipes Wesmael, Opius piceus Thomson*, Opius pygmaeator (Nees)*, Opius singularis Wesmael, Opius staryi Fischer, Phaedrotoma aethiops (Haliday), Phaedrotoma crassicrus (Thomson), Phaedrotoma curvata (Fischer)*, Phaedrotoma depeculator Foerster, Phaedrotoma exigua (Wesmael)*, Phaedrotoma rex (Fischer), Utetes caudatus (Wesmael)*, Utetes zelotes (Marshall)*, Xynobius comatus (Wesmael)*.
Alysiinae, Alysiini:
Alloea lonchopterae Fischer*, Alysia fuscipennis Haliday*, Alysia lucicola Haliday, Alysia luciella Stelfox*, Alysia similis (Nees)*, Alysia tipulae (Scopoli)*, Alysia truncator (Nees)*, Alysia umbrata Stelfox, Anisocyrta perdita (Haliday)*, Aphaereta elegans Tobias, Aphaereta falcigera Graham*, Aphaereta major (Thomson)*, Aphaereta scaptomyzae Fischer, Asobara tabida (Nees)*, Aspilota eurugosa Fischer*, Aspilota extremicornis Fischer*, Aspilota procreata Fischer*, Aspilota tetragona Fischer*, Aspilota stenogaster Stelfox & Graham*, Aspilota vernalis Stelfox & Graham*, Cratospila circe (Haliday)*, Dinotrema betae (Bengtsson)*, Dinotrema brevicauda (Tobias)*, Dinotrema carinatum (Tobias)*, Dinotrema contracticorne (Fischer)*, Dinotrema dimidiatum (Thomson)*, Dinotrema discoideum (Fischer)*, Dinotrema lineolum (Thomson)*, Dinotrema mesocaudatum van Achterberg*, Dinotrema oleraceum (Tobias)*, Dinotrema propodeale (Tobias)*, Dinotrema speculum (Haliday)*, Dinotrema spitzzickense (Fischer)*, Dinotrema tuberculatum van Achterberg*, Dinotrema varipes (Tobias)*, Dinotrema venustum (Tobias)*, Idiasta dichrocera Königsmann*, Idiasta subannellata (Thomson)*, Orthostigma longicorne Königsmann, Orthostigma longicubitale Königsmann, Orthostigma maculipes (Haliday)*, Orthostigma pumila (Nees), Orthostigma sculpturatum Tobias*, Orthostigma sordipes Thomson, Pentapleura angustula (Haliday)*, Pentapleura fuliginosa (Haliday)*, Pentapleura pumilio (Nees)*, Phaenocarpa angustiptera Papp*, Phaenocarpa conspurcator (Haliday)*, Phaenocarpa flavipes (Haliday)*, Phaenocarpa impugnata Papp*, Phaenocarpa livida (Haliday)*, Phaenocarpa ruflceps (Nees)*, Phaenocarpa styriaca Fischer*, Synaldis concolor (Nees)*, Synaldis distracta (Nees)*, Synaldis lacessiva Fischer*, Synaldis mandibulata Fischer*, Synaldis ultima Fischer*, Tanycarpa bicolor (Nees), Tanycarpa mitis (Stelfox)*.
Alysiinae, Dacnusini:
Agonia adducta (Haliday)*, Antrusa flavicoxa (Thomson)*, Antrusa melanocera (Thomson), Chorebus affinis (Nees), Chorebus anasella (Stelfox)*, Chorebus ares (Nixon)*, Chorebus asperrimus Griffiths*, Chorebus avesta (Nixon)*, Chorebus bathyzonus (Marshall)*, Chorebus brevicornis (Thomson)*, Chorebus daimenes (Nixon)*, Chorebus dirona (Nixon)*, Chorebus fordi (Nixon)*, Chorebus freya (Nixon)*, Chorebus ganesa (Nixon)*, Chorebus gnaphalii Griffiths*, Chorebus lateralis (Haliday), Chorebus misellus (Marshall)*, Chorebus nobilis Griffiths*, Chorebus nydia (Nixon)*, Chorebus ovalis (Marshall)*, Chorebus polygoni Griffiths*, Chorebus posticus (Haliday)*, Chorebus pseudomisellus Griffiths*, Chorebus senilis (Nees)*, Chorebus siniffa (Nixon)*, Chorebus subfuscus Griffiths*, Chorebus tanis (Nixon)*, Chorebus tenellae Griffiths*, Chorebus thusa (Nixon)*, Chorebus xanthospidae Griffiths*, Coelinidea elegans (Curtis)*, Coelinidea nigra (Nees)*, Coelinius parvulus (Nees)*, Coloneura arestor (Nixon)*, Coloneura ate (Nixon)**, Coloneura danica Griffiths**, Dacnusa areolaris (Nees)*, Dacnusa austriaca (Fischer)*, Dacnusa confinis Ruthe*, Dacnusa faeroeensis (Roman), Dacnusa laevipectus Thomson, Dacnusa liopleuris Thomson*, Dacnusa longithorax (Tobias)*, Dacnusa maculipes Thomson*, Dacnusa marica (Nixon)*, Dacnusa pubescens (Curtis), Dacnusa tarsalis Thomson*, Exotela cyclogaster Foerster, Exotela hera (Nixon), Exotela sonchina Griffiths, Exotela umbellina (Nixon), Saropspopovi Tobias*, Protodacnusa tristis (Nees)*.
Helconinae:
Diospilus dilatatus Thomson, Diospilus oleraceus Haliday, Helconidea dentator (Fabricius), Lestricus secalis (Linnaeus).
Brachistinae:
Eubazus longicaudis (Ratzeburg), Eubazus pallipes Nees*, Eubazus parvulus (Ruthe)*, Eubazus semirugosus (Nees)*, Triaspis floricola (Wesmael)*, Triaspis lugubris Šnoflák, Triaspis obscurella (Nees)*, Triaspis pallipes (Nees)*.
Euphorinae:
Blacus exilis (Nees), Blacus humilis (Nees), Blacus instabilis Ruthe, Blacus longipennis (Gravenhorst)*, Blacus leptostigma Ruthe*, Blacus maculipes Wesmael, Blacus nigricornis Haeselbarth, Blacus radialis Haeselbarth, Blacus ruficornis (Nees), Centistes collaris (Thomson)*, Centistes edentatus (Haliday), Dinocampus coccinellae (Schrank), Euphorus pallidistigma Curtis*, Leiophron clypealis Tobias*, Meteorus cinctellus (Spinola)*, Meteorus consimilis (Nees)*, Meteorus heliophilus Fischer*, Meteorus ictericus (Nees), Meteorus micropterus (Haliday)*, Meteorus obsoletus (Wesmael), Meteorus pulchricornis (Wesmael), Meteorus rubens (Nees), Meteorus versicolor (Wesmael), Meteorus unicolor (Wesmael), Myiocephalus boops (Wesmael)*, Myiocephalus niger Fischer*, Perilitus brevicollis Haliday*, Perilitus rutilus (Nees), Peristenus nitidus (Curtis)*, Peristenus pallipes (Curtis)*, Peristenus picipes (Curtis)*, Pygostolus falcatus (Nees)*, Spathicopis flavocephala van Achterberg*, Streblocera flaviceps (Marshall)*, Syntretus conterminus (Nees)*, Townesilitus bicolor (Wesmael), Townesilitus deceptor (Wesmael)*, Zele albiditarsus Curtis, Zele deceptor (Wesmael).
Agathidinae:
Agathis breviseta Nees*, Agathis tibialis Nees*, Agathis montana Shestakov*, Bassus cingulipes (Nees)*, Bassus conspicuus (Wesmael)*, Bassus pumilus (Ratzeburg)*, Earinus gloriatorius (Panzer)*.
Orgilinae:
Orgilus laevigatus (Nees)*, Orgilus obscurator (Nees), Orgilus pimpinellae Niezabitowski.
Macrocentrinae:
Macrocentrus bicolor Curtis, Macrocentrus collaris (Spinola), Macrocentrus flavus Snellen van Vollenhoven*, Macrocentrus pallipes (Nees), Macrocentrus resinellae (Linnaeus).
Homolobinae:
Homolobus discolor (Wesmael), Homolobus infumator (Lyle).
Charmontinae:
Charmon extensor (Linnaeus).
Adeliinae:
Adelius subfasciatus Haliday.
Miracinae:
Mirax rufilabris Haliday.
Cheloninae:
Ascogaster abdominator (Dahlbom), Ascogaster bidentula Wesmael, Ascogaster klugii (Nees) Ascogaster rufipes (Latreille), Ascogaster similis (Nees)*, Chelonus asiaticus Telenga*, Chelonus scabrator (Fabricius), Microchelonus basalis (Curtis), Microchelonus contractus (Nees), Microchelonus elachistae Tobias, Microchelonus gravenhorstii (Nees), Microchelonus karadagi Tobias, Microchelonus koponeni Tobias, Microchelonus microphthalmus (Wesmael)*, Microchelonus pedator (Dahlbom), Microchelonus pusillus Szépligeti, Microchelonus subpedator Tobias, Microchelonus temporalis Tobias, Microchelonus vickae Lozan et Tobias, Phanerotoma bilinea Lyle.
Microgastrinae:
Apanteles arsiba Nixon*, Apanteles ater (Ratzeburg)*, Apanteles atreus Nixon*, Apanteles brunnistigma Abdinbekova*, Apanteles corvinus Reinhard*, Apanteles decorus (Haliday), Apanteles imperator Wilkinson, Apanteles infimus (Haliday), Apanteles laevigatoides Nixon, Apanteles lenea Nixon*, Apanteles longicalcar Thomson*, Apanteles mycale Nixon*, Apanteles punctiger (Wesmael)*, Apanteles sicarius Marshall*, Apanteles tedellae Nixon, Apanteles viminetorum (Wesmael), Apanteles xanthostigma (Haliday), Cotesia acuminata (Reinhard)*, Cotesia analis (Nees)*, Cotesia euryale (Nixon)*, Cotesia gastropachae (Bouché), Cotesia hyphantriae (Riley)*, Cotesia lineola (Curtis)*, Cotesia melanoscela (Ratzeburg), Cotesia ordinaria (Ratzeburg)*, Cotesia praepotens (Haliday)*, Cotesia tetrica (Reinhard), Cotesia tibialis (Curtis), Cotesia vanessae (Reinhard)*, Cotesia vestalis (Haliday)*, Cotesia zygaenarum (Marshall), Deuterixys carbonaria (Wesmael)*, Diolcogaster hinzi (Nixon)*, Diolcogaster minuta (Reinhard), Hygroplitis rugulosus (Nees)*, Microgaster acilia Nixon*, Microgaster alebion Nixon*, Microgaster meridiana Haliday*, Microgaster globata (Linnaeus)*, Microgaster hospes Marshall, Microgaster postica Nees*, Microgaster stictica Ruthe*, Microgaster subcompleta Nees, Microgaster messoria Haliday*, Microplitis deprimator (Fabricius), Microplitis fordi Nixon*, Microplitis fulvicornis (Wesmael)*, Microplitis mediator (Haliday), Microplitis spinolae (Nees)*, Microplitis stenuus Reinhard*, Microplitis tuberculifer (Wesmael), Microplitis viduus (Ruthe)*, Microplitis xanthopus (Ruthe)*, Protapanteles anchistades (Nixon)*, Protapanteles callidus (Haliday)*, Protapanteles compressiventris (Muesebeck)*, Protapanteles falcatus (Nees)*, Protapanteles fraternus (Reinhard)*, Protapanteles fulvipes (Haliday), Protapanteles liparidis (Bouché), Protapanteles pallipes (Reinhard)*, Protapanteles trinagulator (Wesmael)*, Protapanteles vitripennis (Curtis)*.
* new faunistic record for Czech Republic
** new faunistic record for Central Europe
*** two new species of the genus Aleiodes will be described by the third author (C. van Achterberg) separately. Part of paratypes are from peatbogs. | CC BY | no | 2022-01-12 16:13:44 | J Insect Sci. 2010 Mar 13; 10:16 | oa_package/c2/82/PMC3014670.tar.gz |
PMC3014672 | 21234099 | 1. Introduction
Because the demand for transplant services has always exceeded the supply of donor organs, the transplant community as well as policymakers have long recognized the need to ensure that the organ allocation system is efficient and equitable [ 1 – 6 ]. The United Network for Organ Sharing (UNOS), which oversees waitlisting and allocation guidelines in the United States, indicates that access to organs will not be based on “political influence, race, gender, religion, or financial or social status” [ 7 ]. Yet, as noted by the Institute of Medicine, the transplant process involves numerous steps and inequities can take place anywhere along the way [ 8 ].
Historically, in cases where empirical data have shown systematic differences in waiting times or in the chances of receiving a transplant, UNOS has changed its policies to improve organ allocation procedures. Examples of changes include the institution of less stringent HLA-matching requirements for renal transplantation [ 9 ], the adoption of the final rule [ 6 ], and the use of the model of end-stage liver disease (MELD) scoring system for liver transplantation [ 10 , 11 ]. For renal transplantation, researchers have access to population-based data about the early steps of the transplant process from the US Renal Data System [ 12 – 14 ]. But for other types of solid organ transplantation, including liver transplantation, information about the early steps is generally unavailable, so oversight is restricted to steps after listing.
We linked several secondary data sources to identify a statewide, population-based cohort of patients with liver-related conditions and followed the cohort through the following stages of the transplant process: disease occurrence (incidence), disease progression (natural history), disease diagnosis, referral, and evaluation by a transplant center, placement on the transplant waiting list (listing), and receipt of an organ (transplantation). We previously reported that demographics were important in determining the likelihood that patients with liver disease would be able to access the transplantation process for evaluation and listing, but not in affecting the likelihood that they would undergo transplantation once they were listed [ 15 ]. This initial analysis evaluated only whether or not patients progressed to specific stages of the transplantation process; because of missing data, it did not address matters related to timing and timeliness.
In the current paper, we estimated the relationship between sociodemographics and the time required for patients to reach specific stages of the process. Specifically, we examined waiting times experienced by subsets of patients during 2 time periods. The subsets were based on gender, race/ethnicity, and insurance status. One period was the time between a patient's diagnosis of liver disease and his or her placement on the UNOS waiting list (an interval in which there is no formal oversight or centralized data collection effort), and the other period was the time between a patient's placement on the UNOS waiting list and his or her receipt of a transplant (an interval in which there is oversight). Our main hypothesis was that gender, race/ethnicity, and insurance status would be associated with variation in waiting times before, but not after, placement on the transplant waiting list. | 2. Methods
2.1. Data Sources and Data Management
Our conceptual framework, data sources, and patient cohort have been described previously [ 15 ]. Briefly, we considered the stages in which a patient developed liver disease, was diagnosed, was referred to a transplant center and evaluated, was listed, and received a transplant. We assumed that most individuals who become sufficiently ill to be considered for a transplant were hospitalized at some point in their illness and, therefore, used hospital discharge data from the Pennsylvania Health Care Cost Containment Council (PHC4) to identify patients who had “liver-transplant potential.” Every nongovernment hospital in Pennsylvania is required by state law to submit clinically abstracted data to the PHC4 for all hospital discharges, and the accuracy of the data has been validated against chart reviews [ 16 ].
The PHC4 listed 310 participating hospitals statewide during the study period. It provided us with data for all patients with liver-related stays between 1994 and 2001. These data included the patients' age, gender, race/ethnicity, and county and ZIP code of residence, type of admission, admission and discharge diagnoses, procedures, and diagnosis-related group (DRG) codes, discharge destination, and Medical Illness Severity Grouping System (MEDISGRPS) disease category (mortality risk) and severity score. To help identify and classify patients with liver disease, we developed a detailed list of diagnostic and procedural codes related to liver problems. Based on our previous work [ 17 ], we classified patients in terms of 9 major categories of disease: viral hepatitis, alcoholic liver disease, autoimmune disorder, metabolic disease, primary sclerosing cholangitis, cancer, primary biliary cirrhosis, other chronic liver disease, and acute liver failure.
We excluded patients who were discharged in 1994 as well as those who had previously received a liver-transplant or been listed for one. This left us with a cohort of 144,507 patients with liver disease from 272 hospitals that had been newly diagnosed between 1995 and 2001. We linked the index hospitalization records of these patients to the following: the liver referral and evaluation data from the 5 predominant liver-transplant centers in Pennsylvania (Albert Einstein Medical Center, Hospital University of Pennsylvania, Thomas Jefferson University Hospital, University of Pittsburgh Medical Center, and the VA Pittsburgh Healthcare System), the listing, allocation, and transplant data from UNOS, and the mortality data from the Bureau of Health Statistics and Research of the Pennsylvania Department of Health.
For many patients in these data sets, the specific time at which some stages were reached was missing. However, the data were complete for the time of disease diagnosis, waitlisting, and transplantation. This allowed us to determine whether patients progressed to a subsequent stage and to measure the time intervals, in days, from diagnosis to listing (early waiting time) and from listing to receipt of a transplant (later waiting time). In each case, there were 3 possible outcomes: proceed to the subsequent stage of the process, remain at the current stage, or die.
The unit of analysis was the patient (not the registration). Although the data were deidentified by the honest broker, multiple listings could be identified and reconciled using a pseudoidentifier for patients. We combined the dates from all of the data sources to “timestamp” the patient's progression through the transplantation process, starting with index hospitalization (i.e., diagnosis) until either the earliest definitive outcome (i.e., transplant, death) or the end of the study period (i.e., still waiting).
In pooling the data sources, we applied 2 strategies for creating the longitudinal records for our patient cohort. First, we did not adjust the waiting times to account for periods when the patient was inactive (a special status category for patients on the waiting list), which increases the estimates of later waiting times from listing to transplantation. Second, for patients with multiple listings, we took the earliest listing date available. Both of these conventions minimize early waiting time from diagnosis until listing and maximize later waiting times from listing to transplant, serving to bias against our hypothesis that early waiting times vary with socioeconomic variables (but later waiting times do not).
Our study was funded by the National Institute of Diabetes and Digestive and Kidney Diseases and approved by the institutional review boards at the University of Pittsburgh and other participating transplant centers. We protected patient confidentiality by having the PHC4 serve as an honest broker to link records across data sources and provide our team with deidentified versions of the files.
2.2. Statistical Analyses
To characterize patients in terms of sociodemographic and clinical data, stage of the allocation process (diagnosis, listing, and receipt of a transplant), waiting times, and outcomes, we used descriptive statistics.
To compare the characteristics of subsets of patients at each stage of the process, we used univariable and multivariable survival models that included the following covariates: age, gender, race/ethnicity (white, black, Hispanic/Asian/other, and unknown), insurance status (commercial only, Medicaid only, Medicare only, combined Medicare plus commercial, combined Medicare plus Medicaid, uninsured, and unknown) based on the index hospitalization, type of liver disease using the diagnostic categories listed above, and severity of illness at the time of diagnosis, based on the MediQual severity scale [ 18 ] and ranging from 0 to 4 (representing none, minimal, moderate, severe, or maximal) or coded as unknown. Given our focus on state-level data, there was no variation in terms of geographic region, but we did include location of transplant center (Pennsylvania versus non-Pennsylvania) as a covariate in the models to account for Pennsylvania residents who were listed and/or transplanted at other centers. We included year of index hospitalization and also tested for interaction variables (e.g., interaction between diagnosis and gender).
In the unadjusted case, we looked at differences in early waiting times based on the proportion of diagnosed patients who were placed on the transplantation waiting list within a specified period (i.e., 1, 3, 5, and 8 years) using Kalbfleisch and Prentice's cumulative incidence technique [ 19 ]. Similarly, we looked at differences in later waiting times by examining the proportion of listed patients who received transplants at these same intervals. In both cases, we controlled for the competing risk of death.
In the adjusted models, we tested for differences in early waiting times by estimating the time to listing for the entire cohort, and we tested for differences in later waiting times by estimating time to transplant for patients on the waiting list. We used Fine and Gray's survival model that takes death and other competing risks into account [ 20 ]. We compared differences in the magnitude and significance of our primary covariates (gender, race/ethnicity, and insurance status) while adjusting for the other covariates.
We retained all covariates with P < .05, and we used SAS, version 9.2 (SAS Institute Inc., Cary, NC) and STATA, version 8 (Stata Corp., College Station, TX) for all analyses. | 3. Results
Table 1 shows data for 3 groups—the full cohort, the subset of patients listed for transplantation, and the final subset of patients who received transplants—stratified by sociodemographic and clinical characteristics. In all 3 groups, the largest proportions of patients were male, were 40–64 years of age, had commercial health insurance only, had a diagnosis of viral hepatitis, alcoholic liver disease, or autoimmune disorder, and had a moderate or severe level of illness. Because of the large sample size, between-group differences were statistically significant for all covariates. In general, however, differences between the first and second groups (full cohort and listed patients) were larger than differences between the second and third groups (listed patients and transplant recipients).
Of the 144,507 adults in our final cohort of patients with liver-transplant potential, 3,071 (2.1%) were placed on the transplant waiting list. Of these 3,071 patients, 1,537 (50.0%) went on to receive a liver transplant by December 2003. Based on raw waiting times, 61% ( n = 1,879) of all transplant candidates were placed on the waiting list within 1 year of diagnosis, and 85.5% ( n = 2,626) were listed within 3 years of diagnosis. Among transplant recipients, 26.7% ( n = 1,130) received transplants within 1 year of listing and 95% ( n = 1,468) received transplants within 3 years of listing. A total of 57,020 patients (39.5%) died during the study period.
3.1. Univariable Analyses
Because waiting times are censored for patients who do not progress to the next stage of the process (e.g., diagnosed patients who are not listed, and listed patients who do not receive transplants), mean waiting times cannot be computed. Instead, Table 2 reports the cumulative percentage of patients who progressed to the next stage of the transplantation process within specific time intervals, first among diagnosed patients and then among listed patients, as well as the cumulative percentage of patients who died (the competing risk) during those same intervals.
Among the 144,507 diagnosed patients in the cohort, 1.3% were listed within 1 year of the index hospitalization and 2.1% were listed within the 8-year study period ( Table 2 , top panel). During the same period, 21.9% of the patients died within 1 year and 38.6% died within 8 years. In the stratified results, women diagnosed with liver disease were less likely than their male counterparts to be listed but were also less likely to die. At every interval, black patients had lower probabilities of being listed, and patients in the Hispanic/Asian/other category had lower probabilities of death than the overall cohort did. In terms of insurance status, patients with commercial insurance were more likely to be listed and less likely to die whereas patients with any form of Medicare insurance coverage exhibited the opposite pattern and were less likely to be listed and more likely to die than the overall cohort.
For later waiting times after listing (until transplantation) ( Table 2 , bottom panel), women continued to receive transplants at lower rates than men and had greater risks of dying on the waiting list. Black patients showed the same pattern, with fewer transplants and more deaths than white patients. Both the Medicare patients and the uninsured patients (including self-pay patients) were listed at higher-than-average rates, but the Medicare patients had higher-than-average risks of death while the uninsured patients had lower-than-average risks of death.
3.2. Multivariable Analyses
The adjusted competing risks models included the primary covariates (gender, race/ethnicity, and insurance status), all other covariates mentioned above, interaction terms for diagnosis and gender, and interaction terms for diagnosis and race/ethnicity for both the early waiting period and the later waiting period models. The main effect of insurance status was nonsignificant in the later period (after listing), so interaction terms between diagnosis and insurance were only included in the model to estimate early waiting times. Full regression results for the models are provided in Table 3 .
Graphs of the findings for gender, race/ethnicity, and insurance status are shown in Figures 1 , 2 , and 3 , respectively. The information is analogous to that presented in Table 2 for the univariable analyses. Of note, in competing risk models, if the longest noncensored followup time in the observed data set coincides with a patient death, then the estimated cumulative incidence function for death (i.e., the competing risk) converges to 1.0 in the graph. This is the case for all of the competing risk graphs (right-hand side) in Figure 1 through 3; this artifact is similar to the way in which Kaplan-Meier graphs show no survivors at the end of the followup period. Diagnosis-specific interaction results for gender are provided in Figure 4 .
3.2.1. Gender
In terms of the main effect of gender, early waiting times (from diagnosis to listing) were similar for men and women (beta = 0.052, P = .31; Table 3 ), and men were more likely to die without ever being listed (beta = −0.2175, P < .0001).
However, the overall effect of gender on early waiting time was slightly different when the interactions between gender and diagnosis and the distribution of men and women in the diagnosis categories were taken into account ( Figure 4 ). Three diagnosis categories—hepatitis, cancer, and metabolic diseases—affected 65% of the total cohort, and larger percentages of women than men were in these categories. Women in these categories were less likely to be listed for transplants than were men in the total cohort. As a result, women experienced slightly longer waiting times in the early period than men overall, as shown in Figure 1(a) .
Later waiting times (from listing to transplant) were longer for women, and women were less likely to ever receive a transplant (beta = −0.2165, P = .0021; Table 3 ). Among patients listed for transplantation, the likelihood of death was similar for men and women. The disease-specific interactions did not significantly change the overall effect of gender in the later period.
3.2.2. Race/ethnicity
In the early period (Figures 2(a) and 2(b) ), race/ethnicity continued to be important even after adjustment for covariates in the multivariable models. Compared with white patients (the referent group), black patients waited longer and were less likely to be listed for transplantation (beta = −0.7324, P < .0001; Table 3 ), and patients in the Hispanic/Asian/other group had substantially better survival times without listing (beta = –0.29, P < .0001). The interaction terms indicate that only black patients with metabolic disorders showed a different pattern from the overall trend in that they tended to be listed sooner and to die sooner ( Table 3 ).
In the later period, the time from listing to transplantation was similar among the racial/ethnic groups and unaffected by the disease-specific interactions ( Figure 2(c) ). In terms of the competing risk ( Figure 2(d) ), black patients were more likely than other patients to die on the waiting list without receiving a transplant. The risks of death were affected by disease-specific interactions in that they were lower for Hispanic/Asian/other patients with cancer (beta = −8.938, P < .0001; Table 3 ), higher for Hispanic/Asian/other patients with metabolic disease (beta = 1.197, P = .028), lower for patients of unknown race with cancer (beta = −8.964, P < .0001), and higher for patients of unknown race with acute liver failure (beta = 1.048, P = .01).
3.2.3. Insurance Status
Time to listing was similar for most insurance status groups (Figures 3(a) and 3(b) ). The exceptions were patients with combined Medicare/Medicaid coverage, who had longer waiting times (beta = −0.411, P = .0043; Table 3 ), and patients with commercial insurance, who had shorter waiting times (beta = 0.6716, P < .0001). Among individuals who were not listed for transplants, the highest risks of dying were in those with combined Medicare/Medicaid (beta = 0.1222, P = .0011) and those with Medicaid alone (beta = 0.0809, P = .023), whereas the lowest risks of dying were in commercially insured patients (beta = −0.267, P < .0001) and uninsured patients (beta = −0.1487, P = .018). These trends were also apparent in the disease-specific interactions, where the lowest risks of dying were again in commercially insured patients and uninsured patients ( Table 3 ).
After listing, there was no variation related to insurance status: both time to transplant and time to death without transplant were similar for all payer groups (Figures 3(a) and 3(b) ). | 4. Discussion
Our analyses of a statewide population-based data set for adults who had liver-related hospitalizations showed that sociodemographics were associated with variation in early waiting times (before being listed for transplant) as well as risk of death. Although the overall experiences were similar for men and women before listing, there was substantial variation related to both race and insurance status. Black patients were less likely to be listed for transplant upon diagnosis. Insurance status also mattered in the early period, in terms of both the likelihood of being listed for transplant and the likelihood of death without ever being listed. Whereas commercially insured patients tended to do better, those covered by Medicare/Medicaid combined were disadvantaged. These patterns may be indicative of disease progression when patients present with symptoms (in this case, when patients are hospitalized), but our analyses did adjust for disease severity at the time of diagnosis.
Once patients are placed on the transplant waiting list, gender appeared more significant as women waited longer to receive a transplant; black patients were more likely to die on the waiting list without a transplant, but insurance status played no role in later waiting time differences. All in all, the timing differences were most pronounced before listing, but were not completely eliminated after listing.
Our study had several limitations that deserve mentioning. First, the study depended on hospitalization data from only one state (Pennsylvania) to identify patients with transplant potential. Second, although the study linked information from 5 participating transplant centers, this information varied across centers in terms of format, detail, and completeness. As a result, we could only explore disparities for 2 periods (diagnosis to listing and then listing to transplant). A more comprehensive analysis of early disparities requires standardizing the data that are collected at these earlier transitions in the transplantation process prior to listing (diagnosis to referral, referral to evaluation, and evaluation to listing). Third, insurance status was based on the index hospitalization only; any potential changes in payer information were not observed. Fourth, the study period predates the MELD scoring system, though it is worth noting that our main finding (i.e., that race/ethnicity and insurance status are associated with variability in early waiting times) refers to stages of the organ allocation process that are unaffected by MELD. Fifth, given the study's retrospective nature and the lack of information about patient preferences for transplantation, we cannot infer causality.
To our knowledge, our study is the first population-based study of the timing of being listed for transplant services. Previously, we reported differences in the overall likelihood of moving through the allocation and transplant process [ 15 ]. The results of the study reported here confirm those earlier findings and provide strong evidence that socioeconomic factors play a role in access to the stages of transplant services in which there is no formal oversight.
With the persistent gap between demand for transplant services and supply of available donor organs, much effort by policymakers and the transplant community is devoted to ensuring the fairness of the transplant system. Where this system is visible and the process is accountable—namely, after individuals are listed by a transplant center—researchers have demonstrated marked improvements in recent years, attributed in part to UNOS oversight and reforms such as the MELD scoring system. Still lacking, however, are centralized data sources to accurately measure the denominator population—that is, the population of all individuals who have end-stage liver disease and are potentially eligible for a transplant. Only with these data can researchers and policymakers measure the true demand for liver-transplant services, assess the fairness of the process, and optimize the allocation of available donor organs. | Academic Editor: Paul C. Kuo
Fair allocation of organs to candidates listed for transplantation is fundamental to organ-donation policies. Processes leading to listing decisions are neither regulated nor understood. We explored whether patient characteristics affected timeliness of listing using population-based data on 144,507 adults hospitalized with liver-related disease in Pennsylvania. We linked hospitalizations to other secondary data and found 3,071 listed for transplants, 1,537 received transplants, and 57,020 died. Among candidates, 61% ( n = 1,879) and 85.5% ( n = 2,626) were listed within 1 and 3 years of diagnosis; 26.7% ( n = 1,130) and 95% ( n = 1,468) of recipients were transplanted within 1 and 3 years of listing. Using competing-risks models, we found few overall differences by sex, but both black patients and those insured by Medicare and Medicaid (combined) waited longer before being listed. Patients with combined Medicare and Medicaid insurance, as well as those with Medicaid alone, were also more likely to die without ever being listed. Once listed, the time to transplant was slightly longer for women, but it did not differ by race/ethnicity or insurance. The early time period from diagnosis to listing for liver transplantation reveals unwanted variation related to demographics that jeopardizes overall fairness of organ allocation and needs to be further explored. | Support and Disclaimers
This work was supported in part by Grant No. K25 DK002903 from the National Institute for Diabetes and Digestive and Kidney Disorders (NIDDK), which supported C. L. Bryce's Career Development Award during the study period, and Grant No. UL1 RR024153 from the National Center for Research Resources (NCRR) and the National Institutes of Health (NIH) Roadmap for Medical Research. The contents of the work are solely the responsibility of the authors and do not necessarily represent the official view of the NIDDK, NCRR, or NIH. Data used in this paper were supplied by the Pennsylvania Health Care Cost Containment Council (PHC4) and the United Network for Organ Sharing (UNOS). The PHC4 is an independent state agency that has provided data in an effort to further its missions of educating the public and containing health care costs in Pennsylvania. The PHC4, its agents, and staff have made no representation, guarantee, or warranty (express or implied) that the data provided are error-free or that the use of the data will avoid difference of opinion or interpretation, and they bear no responsibility or liability for the results of the analysis, which are solely the opinion of the authors. UNOS is the contractor for the Organ Procurement and Transplantation Network (OPTN). The interpretation and reporting of UNOS data are the responsibility of the authors and in no way should be seen as an official policy of or interpretation by the OPTN or the US government. The authors of the paper have no conflict of interests, including financial interests and relationships and affiliations relevant to the subject of the paper. | Acknowledgments
The authors acknowledge assistance from the staff at Pennsylvania Health Care Cost Containment Council, Maureen McBride and Sarah Taranto (the United Network for Organ Sharing), Craig Edelman (the Pennsylvania Department of Health), Howard Nathan (Gift of Life Donor Program), Mical S. Campbell, MD (Coastal Digestive Diseases, New London, CT), Elliot Kozin (University of Pennsylvania), Ignazio R. Marino (Jefferson Medical College, Thomas Jefferson University), Cosme Manzarbeitia (Thomas Jefferson University), Joe Donaldson (Starzl Transplant Institute), and D. Renae Geraci (University of Pittsburgh).
Abbreviations
Diagnosis-related group
Medical illness severity grouping system
Model for end-stage liver disease
Pennsylvania Health Care Cost Containment Council
United Network for Organ Sharing. | CC BY | no | 2022-01-13 02:56:45 | J Transplant. 2010 Dec 23; 2010:467976 | oa_package/90/c8/PMC3014672.tar.gz |
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PMC3014674 | 21234101 | 2. Discussion
Right-sided infective endocarditis (RSIE) is a rare condition that constitutes only 10% of cases of IE [ 1 ]. It is usually associated with intravenous illicit drug use (IVDU) or central catheter use, with Staphylococcus aureus being the most common infectious agent in all cases [ 2 ].
The incidence of RSIE is on the rise due to the increased number of patients using central venous lines, as well as pacing and other intravascular devices [ 3 ]. This incidence is also high among renal patients receiving haemodialysis throughout a long-standing central catheter. The ever increasing number of patients on dialysis requiring central catheters makes RSIE a frequent complication with greater morbidity and mortality than the general population [ 4 ].
Most cases of RSIE involve the tricuspid valve. By contrast, pulmonary valve endocarditis is extremely rare, especially in structurally normal hearts. Structural cardiac abnormalities such as PVS and ASD were present in our patient and serve as additional risk factors. PVS appears as a rare congenital heart disease in adults, and it is even rarer in association with ASD [ 5 ].
Another remarkable aspect of this case is the presence of a significant left-to-right shunt through the ASD in a patient with a longstanding severe PVS, due to anticipated changes in right ventricular compliance a smaller shunt would be expected. However, we face the case of an elderly, hypertensive patient with chronic renal failure and associated left ventricular hypertrophy. All of these factors could increase the afterload and lead to a reduced left ventricular compliance. This impaired left compliance could explain the left-to-right shunt severity.
The case of pulmonary valve IE reported here, presents a combination of predisposing factors for this entity, such as severe congenital PVS, the presence of a central venous catheter, and haemodialysis. The fact that it was an older patient with severe congenital PVS and associated with a previously undiagnosed ASD, is also an unusual feature of this case, making it even more interesting. Interatrial communication has been reported in some cases of RSIE; however, these involved normal native pulmonary valves [ 6 ]. Anatomically normal, bicuspid, or even unicuspid pulmonary valves have been found in another series of nine autopsies of patients with isolated pulmonary valves IE, but they were not associated with neither PVS nor ASD [ 7 ]. To our knowledge, the association of both structural heart diseases leading to pulmonary valve IE had not been previously reported.
RSIE usually presents without the classic signs and symptoms of IE, and therefore, the diagnosis is often more challenging [ 8 ]. Other possible septic sources as well as other septic clinical complications such as clinical or radiological septic pulmonary embolism, which may be associated with RSIE, were reasonably ruled out in this case. The diagnosis of pulmonary valve IE was made based on the presence of two major and two minor modified Duke criteria [ 3 ]. The challenge in diagnosing pulmonary valve IE, as compared to other valvular locations, which may lead to a low clinical suspicion and cause it to be overlooked. The initial TTE was performed in another institution, so we cannot determine whether the valvular vegetation was simply overlooked or was not present. Importantly, in this particular case our initial clinical suspicion and a detailed echocardiography imaging study allowed us to establish the definitive diagnosis and led us to initiate a successful treatment of IE and obtain a favourable outcome.
In conclusion, the pulmonary valve should be properly assessed in TTE and TEE examinations of all patients with clinical suspicion of IE, especially in the presence of prolonged fever associated with pulmonary infectious processes, IVDU, central catheters, or haemodialysis, and particularly if they also have predisposing heart disease. The diagnosis of pulmonary valve IE should no longer be missed in cases similar to this one. Failure of this diagnosis could be the main reason for such low frequency of RSIE. | Academic Editor: J. Brugada
A hypertensive 76-year-old man with severe pulmonary valve stenosis (PVS) and recent initiation of haemodialysis was referred with fever, chills, and asthenia. One month prior, he had been admitted with similar symptoms. Transthoracic echocardiography (TTE) had shown a PVS and no valve vegetations were observed. Following discharge, he was readmitted with fever and blood cultures positive for Staphylococcus haemolyticus . A new TTE revealed two pulmonary valve vegetations and a previously undetected ostium secundum-type atrial septal defect (ASD), confirmed by transesophageal echocardiography. The clinical course was uneventful with intravenous antibiotic treatment and the patient was safely discharged. This is a case of pulmonary valve infective endocarditis (IE). The incidence of right-sided IE is on the rise due to the increased number of patients using central venous lines, pacing, haemodialysis and other intravascular devices. Pulmonary valve IE is extremely rare, especially in structurally normal hearts. The case reported here, presents a combination of predisposing factors, such as severe congenital PVS, the presence of a central venous catheter, and haemodialysis. The fact that it was an older patient with severe congenital PVS and associated with a previously undiagnosed ASD, is also an unusual feature of this case, making it even more interesting. | 1. Case Report
A hypertensive 76-year-old man with severe pulmonary valve stenosis (PVS) and recent initiation of haemodialysis for chronic kidney disease was referred to our hospital with fever, chills, and asthenia.
One month prior, he had been admitted to another hospital with similar symptoms. He was found to have bacteraemia with serial blood cultures positive for Staphylococcus haemolyticus , appropriate antibiotic therapy was initiated, and a previously placed right internal yugular hemodialysis catheter was removed. Although transesophageal echocardiography (TEE) was not available in that facility, transthoracic echocardiography (TTE) examination had shown a thickened and calcified pulmonary valve with a reduced opening. Continuous wave Doppler measured a peak systolic gradient of 75 mmHg and mean systolic gradient of 42 mmHg across the pulmonary valve. No valve vegetations were observed. The patient was discharged in good condition 15 days later. Two weeks following discharge, he was readmitted with fever, blood cultures were positive for Staphylococcus haemolyticus, and the patient was subsequently referred to our institution. Physical examination on admission was unremarkable, however ultrasonographic examination by TTE revealed two pulmonary valve vegetations ( Figure 1 ). Also noted, were dilated right heart cavities with abnormal interventricular septal motion and normal right ventricular systolic function. A significant left-to-right flow across a previously undetected ostium secundum-type atrial septal defect (ASD) was shown by colour flow Doppler imaging. This findings were confirmed by TEE ( Figure 2 ).
Blood cultures drawn on admission to our hospital remained positive for Staphylococcus epidermidis . He received appropriate intravenous antibiotic treatment for six weeks, and haemodialysis continued throughout an arteriovenous fistula. The clinical course was uneventful and the patient was safely discharged after this time. At present, the patient has refused any intervention on his heart disease, but has remained asymptomatic and without further recurrences. | CC BY | no | 2022-01-13 01:45:13 | Cardiol Res Pract. 2010 Dec 26; 2010:798956 | oa_package/4d/01/PMC3014674.tar.gz |
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PMC3014676 | 21234103 | 1. Introduction
Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity reaction to Aspergillus species. In children, it is mostly encountered in the context of cystic fibrosis (CF).
The reported prevalence of ABPA complicating CF lung disease varies from 6 to 25% depending on the age and the diagnostic criteria used [ 1 – 3 ]. The clinical diagnosis is difficult because symptoms are nonspecific and resemble bacterial CF airway infection. Different diagnostic criteria have been established based on a combination of clinical and radiological signs together with the following biochemical parameters, again none of them being very specific: elevated IgE (>500–1000 IU/l or a 4-fold rise), eosinophilia, specific IgE for A. fumigatus or recombinant antigens (or positive skin prick test), and precipitins to A. fumigatus . [ 3 ].
Systemic corticosteroids are still the cornerstone of therapy starting at a dose of 1-2 mg/kg/day for 2-3 weeks [ 4 ], but the pace of tapering is highly variable and individualized. Adjunctive antifungal agents like itraconazole (200–400 mg/day for several months) may reduce corticosteroid need. However, few studies involving only a small number of CF patients have been published [ 3 , 5 ].
In our CF center, ABPA is usually treated with a combination of systemic steroids and itraconazole. In patients with frequent ABPA flares or failure of steroid tapering, inhaled amphotericin B (iAMB) is considered as alternative antifungal therapy. Because only scarce data exist [ 6 , 7 ], we report our experience with this treatment in 7 CF children with a difficult-to-treat ABPA course. | 2. Methods
Inhaled AMB is considered in case of one or more of the following: (1) insufficient response to initial therapy with corticosteroids and itraconazole (2) frequent ABPA relapse during/after steroid withdrawal despite itraconazole treatment (3) failure to taper systemic steroids (4) intolerance for itraconazole. Data were retrospectively collected on 7 CF patients treated for ABPA with inhaled AMB.
In a first step AMB deoxycholate (AMB-d) (Fungizone Bristol-Meyers Squibb Belgium) was administered as a test dose (20 mg in a concentration of 1 mg/ml; nebulization for 10–15 minutes with a PARI turboboy nebulizer or Aeroneb Go) [ 8 ]. If tolerated, therapy was given 3 times a week. In case of intolerance for AMB-d (cough, wheeze, and shortness of breath), AMB-lipid complex (ABLC) was used (Abelcet Wyeth 50 mg in a concentration of 5 mg/ml, 2 ×/week; nebulization time 10–15 minutes). Duration of therapy was individualized (see cases and figures). Choice of the dosage scheme was mainly based on doses used in other indications [ 9 ] and the reported long half-life of iAMB in the lung in animal models [ 10 ], especially when using a lipid formulation. Because ABLC has a 1/1 molar ratio for the active product versus the phospholipids (see Table 2 ), the weekly dose was about the double compared to AMB-d.
Treatment was considered a success if systemic steroids could be stopped without ABPA relapse for at least 12 months after steroid withdrawal. Prednisolone was started at a dose of 1-2 mg/kg/day. After 3 weeks, the patient was seen in clinic, and tapering was started in case of clinical improvement and fall in IgE. If clinically indicated steroids were tapered with 5 mg every 2 weeks. However, final decision for speed of tapering was made by the treating CF physician. Improvement of lung function is not used as an absolute criterion for treatment success in this small patient series because important comorbidities influence lung function evolutions.
Specific IgE for different recombinant Aspergillus antigens were measured (f1, f2, f3, and f4) (ImmunoCAP 1000, Phadia; cut-off >0.7 Ua/ml).
Patient 1 (WS °21-12-2001) This CF girl had a difficult respiratory course from young age with frequent infectious exacerbations treated with oral and IV antibiotics. ABPA was first diagnosed at age 5 based on a respiratory exacerbation not improving with IV antibiotic treatment combined with a rise in IgE to 1089 kU/l and positive RAST to the recombinant Aspergillus antigen f6 (4–6.5 Ua/ml). Aspergillus was growing from her sputum. Treatment with oral steroids and itraconazole (Sporanox Janssen-Cilag 1 × 100 mg) was started ( Figure 1 ). Because of persisting atelectasis of the left upper lobe after 2 weeks of oral steroids and itraconazole, inhaled AMB-d was added after which the atelectasis cleared. Sputum remained negative for Aspergillus . One year after steroid stop and 8 months after inhaled AMB-d was stopped, ABPA recurred and was successfully treated with the same combination of prednisolone and antifungal therapy (itraconazole and AMB-d). She is now 9 month off steroids, and inhaled AMB-d was changed to ABLC and will be continued until at least 1 year free of ABPA. IgE is however rising again, and a new flare is suspected.
Patient 2 (BL °13-4-1998) From diagnosis, the respiratory course of this girl was unstable with frequent infections and lung infiltrates treated with IV antibiotics. Because of severe gastro-esophageal reflux, a Nissen fundoplication was performed at the age of 5 years. She developed asthmatic symptoms (in the context of familial atopy and asthma) for which she has been treated with inhaled steroids. She was diagnosed with a first episode of ABPA based on a respiratory exacerbation not improving with IV antibiotics, IgE rising from 230 kU/l to 500 kU/l, eosinophilia (1200/ μ l), and a positive RAST for recombinant Aspergillus antigen f4 (between 9 and 43 Ua/ml). Aspergillus was cultured from sputum. She was treated with oral steroids and itraconazole (Sporanox Janssen-Cilag 1 × 100 mg, later 2 × 100 mg) ( Figure 2 ). Two years later there was a relapse of ABPA. Only slow clinical improvement was seen under oral steroids, and itraconazole was replaced by inhaled ABLC. Because of decreasing lung function and repeated Scedosporium prolificans in sputum cultures, voriconazole (Vfend Pfizer 2 × 125 mg increased to 2 × 200 mg/d based on subtherapeutic blood levels) was added. Sputum was initially not clear; however, she finally became free of Aspergillus (from march 2009). Only under combined antifungal therapy, steroids could be stopped and lung function improved. She is now free of oral steroids since 1 year.
Patient 3 (BW °30-10-1995) This boy developed chronic lung infection with B. cepacia ( multivorans ) from the age of 8. He was diagnosed with ABPA in the same year after a prolonged respiratory exacerbation being not resolved despite repeated antibiotic administration. IgE titer was 1635 kU/l; specific IgE for Aspergillus recombinant antigens were positive only for f3 (1 Ua/ml), not for f4 nor f6. He was treated with oral steroids and itraconazole (Sporanox Janssen-Cilag 2 × 100 mg). One year later, he developed an ABPA relapse with prolonged course and difficult steroid tapering ( Figure 3 ). After start of inhaled AMB-d (later switched to ABCL), steroids could be tapered and finally stopped. Inhaled ABCL was stopped after he was free of steroids and ABPA relapse for 1 year. Sputum cultures became negative for Aspergillus since start of iAMB. Despite the resolution of the ABPA, the overall evolution in this CF patient was unfavorable. He developed a chronic lung abscess in the right lung for which a right lower lobe resection was performed at the age of 12. However, chronic B. cepacia suppurative infection of the remaining right lung persisted with development of collapse and a functional right lung. He received a lung transplant at the age of 13.5.
Patient 4 (DK °13-1-1993) This girl developed severe obstructive lung disease and bronchiectasis despite intensive treatment and frequent courses of IV antibiotics. The first episode of ABPA at the age of 10 ( Figure 4 ) was treated with oral steroids and itraconazole (Sporanox Janssen-Cilag 2 × 100 mg). Several attempts to taper the steroids failed. From the age of 11 (2004), Mycobacterium avium-intracellulare (MAC) infection has been documented. Several combination treatments have been given with limited if any success (ethambutol, rifampicin, clarithromycin or azithromycin, IV amikacin, ciprofloxacin or levofloxacin, interferon gamma 1-b). Lung function continued to decline. Sputum cultures mainly grew Candida and Aspergillus species despite longstanding treatment with itraconazole. Hair loss was reported. Therefore, treatment with voriconazole (Vfend Pfizer 2 × 120 mg) was started. After 1 month of therapy, slight improvement of lung function and decreased cough were seen. Voriconazole had to be stopped because of severe diarrhoea. Posaconazole (Noxafil Schering-Plough 2 × 400 mg) was given for a certain period but stopped because of reimbursement issue (see Figure 3 ). Inhaled AMB-d was initiated without clear response and therefore stopped again. ABLC was only given for short periods of time but was disliked by the patient. Sputum initially cleared; however, Aspergillus was isolated again after stop of iAMB. Voriconazole was successfully reintroduced at a higher dose (2 × 240 mg daily) based on subtherapeutic blood levels, but IgE remains high ( Figure 4 ) and lung function fails to improve.
Patient 5 (BP °25-7-90) ABPA was diagnosed based on bilateral lung infiltrates not improving under adequate antibiotic therapy combined with raised IgE (3295 kU/l) together with positive RAST and precipitins for A. fumigates . She was treated with oral steroids and itraconazole (Sporanox Janssen-Cilag 2 × 200 mg) but had frequent relapses over the years ( Figure 5 ). Only after starting inhaled ABLC, steroids could be tapered as she has been free of ABPA relapse for almost 2 years although her IgE levels remained high. No more Aspergillus was cultured from her sputum.
Patient 6 (VM °24-7-1989) Childhood years were characterized by rather stable respiratory disease, but since adolescence, respiratory exacerbations became frequent. A Nissen fundoplication was performed at the age of 13 because of severe gastro-oesophageal reflux. ABPA was diagnosed at the age of 11 based on a respiratory exacerbation not improving with IV antibiotics and a high IgE of 3890 kU/l. She was treated successfully with oral steroids and itraconazole (Sporanox Janssen-Cilag 2 × 100 mg later 2 × 200 mg) ( Figure 6 ). After a relapse 3 years later, tapering of steroids resulted in frequent ABPA exacerbations. Sputum cultures were intermittently positive for A. fumigatus , C. albicans and S. prolificans . After initial start of inhaled ABLC, compliance was reported as low. Insisting on the importance of the antifungal therapy finally resulted in better adherence resulting not only in successful weaning (more than 2 years off steroids) of therapy but also improved lung function. She remained free of Aspergillus in her sputum under treatment with iAMB.
Patient 7 (ZB °13-10-89) Diagnosis of ABPA was first made at the age of 9 based on a respiratory exacerbation with a new lung infiltrate not clearing with antibiotics. IgE was 881 kU/l, with positive specific RAST for A. fumigatus as well as positive skin prick test. Aspergillus was never grown from her sputa. She was first treated with oral steroids only. At the time of the 3rd ABPA episode, itraconazole (Sporanox Janssen-Cilag 2 × 200 mg) was started, but steroid tapering was difficult. At the age of 17, inhaled ABLC was started because of failure to stop the systemic steroids. Steroids were slowly tapered and successfully stopped after 8 months. She has been free of ABPA since 2.5 years with more stable lung function evolution ( Figure 7 ). | 3. Discussion
We report the use of inhaled AMB in the treatment of 7 CF children with difficult-to-treat ABPA. For some patients AMB was used in combination with itraconazole or voriconazole. For 5 of the 7 patients treated with inhaled AMB, treatment was considered a success: patients were weaned from systemic steroids without ABPA relapse for at least 12 months. For 2 of these 5 patients, clinical evolution may well be influenced by cotreatment with voriconazole although plasma levels were subtherapeutic. The patient that remained steroid dependent (patient 4) had complex lung disease with MAC infection. In 4 out of 5 successes, lung function improved as well. The patient that had progressive lung function decline, despite successful ABPA treatment, had B. cepacia lung infection.
A consensus guideline on management of ABPA in CF has been published [ 3 ]. To reduce the burden of A. fumigatus in the respiratory tract it may be worthwhile to treat with antifungal medication [ 4 , 11 , 12 ] . Itraconazole is however lipophilic and may not be well absorbed in CF patients. Use of proton pump inhibitors may further interfere with itraconazole absorption. Other possible drug interactions include CYP3A4 inhibition leading to QT prolongation and decreased methylprednisolone metabolization. Voriconazole, approved for the treatment of invasive aspergillosis, has a better oral bioavailability. It is however expensive, has a high potential for drug interactions, and has been associated with a number of adverse effects [ 13 , 14 ], and no formal data exist on its absorption in CF.
In the light of these concerns, use of inhaled medication like AMB is considered. Data on clinical use mainly concern prevention and treatment of invasive Aspergillus infections in neutropenic or lung transplant patients [ 9 ]. AMB has been the treatment of choice for most invasive fungal infections since its introduction in the 1950s. It has a broad spectrum of activity including Aspergillus fumigatus , Candida albicans, and Cryptococcus neoformans . Lipid formulations have been developed to reduce nephrotoxicity after parental administration while retaining the drug's activity. Main differences between these formulations are listed in Table 2 . Clinically they differ mainly in risk for toxicity, dosing, and tissue concentration after parental administration [ 15 ].
The nebulization of AMB was first studied in a rat model of pulmonary Aspergillus infection. Prophylactic treatment before fungal inoculation with either formulation resulted in a significantly prolonged survival for all. Mean concentrations of AMB in the lungs were significantly higher and had a longer half-life with ABLC compared to a similar dose of AMB-d [ 16 ]. The liposomal formulation had the longest half-life [ 10 ].
In lung transplant patients, ABLC and L-AMB are better tolerated than conventional AMB-d. The deoxycholate salt used in conventional AMB-d acts as a detergent, impairing the surfactant function which is a potential toxic effect [ 17 , 18 ]. In contrast, the liposomal carrier in L-AMB exhibits a pulmonary surfactant-like function.
The clinical use of inhaled AMB has been recently reviewed [ 9 ]. Several data indicate that nebulizing AMB is possible and lung deposition can be obtained. Lipid formulations nebulize better than AMB-d. Few reports are found on the use of inhaled AMB in the treatment of ABPA. The use of nebulized AMB-d in the treatment of acute ABPA in a non-CF boy was reported [ 19 ]. A dose of 2.5 mg was given 3 times daily, but duration of therapy was not mentioned. Data on 5 CF patients treated weekly with L-AMB was published in abstract form [ 20 ]. A dose of 50 mg was given diluted in 8 mL aqua. Duration of nebulization was reported as 150 minutes on average which makes this treatment burdensome. Systemic steroids could be stopped in 4 of the 5 patients. In one patient, ABPA relapsed 7 months after stop of L-AMB. Recently, a short report describes the successful use of nebulized AMB (5 mg two times daily) in 3 CF children with ABPA in combination with inhaled budesonide [ 7 ].
Based on pharmacokinetic data, we used a 2- or 3-time per week administration of inhaled AMB. AMB-d was used when tolerated. Based on possible effects on pulmonary surfactant, we try however to avoid longstanding use of AMB-d and switch to ABLC.
With AMB-d, main adverse event was cough and wheeze induced by the nebulization. These side effects were far less prominent with ABLC. None of the patients had to stop the treatment with ABLC because of side effects. Patients however dislike the treatment because of the bad taste.
We acknowledge that this paper is only a retrospective case series. Prospective interventional trails will be needed in order to evaluate the place of inhaled antifungal medication in the treatment of ABPA.
We are aware that recently several case reports have been published on the use of omalizumab in ABPA treatment in CF [ 21 – 24 ]. A prospective randomized trial is currently conducted in Europe. Even if this therapy proves to be efficient for this indication, omalizumab will not be suitable for all CF patients with ABPA, merely because of side effects but also because of high cost and the necessary 2 weekly in-hospital administration. | Academic Editor: Ian T. Adatia
Background . Systemic steroids and adjunctive antifungal therapy are the cornerstone in treating allergic bronchopulmonary aspergillosis (ABPA) in the context of CF. Aim . Evaluate the use of inhaled amphotericin B (iAMB) as antifungal agent in this context. Methods . Report of 7 CF patients with recurrent or difficult to treat ABPA and failure to taper systemic corticosteroids treated with AMB deoxycholate (AMB-d) (Fungizone 25 mg 3× a week) or AMB lipid complex (ABLC) (Abelcet 50 mg twice weekly). Successful therapy was defined as steroid withdrawal without ABPA relapse within 12 months. Results . Therapy was successful in 6 of 7 patients treated with iAMB. In 5/6, lung function improved. The patient with treatment failure has concomitant MAC lung infection. Conclusion . Inhaled AMB may be an alternative to commonly used adjunctive antifungal therapy in the treatment of ABPA. More data are needed on safety and efficacy. | 4. Summary
We report on the use of inhaled AMB in the treatment of 7 CF patients with difficult-to-treat ABPA. In 6 of the 7 patients treated, steroids could be stopped and patients remained free of relapse after several years of recurrent ABPA episodes.
Based on this limited experience, inhaled AMB may be considered as antifungal therapy in the context of ABPA treatment in CF. More data are needed for efficacy and long-term safety.
Conflict of Interests
All authors state that there is no conflict of interests. | Abbreviations
(inhaled) Amphotericin B
Amphotericin B deoxycholate
Amphotericin B lipid complex
Liposomal amphotericin B
Amphotericin B colloidal dispersion
Cystic fibrosis
Allergic bronchopulmonary aspergillosis. | CC BY | no | 2022-01-13 02:39:08 | Int J Pediatr. 2010 Dec 23; 2010:376287 | oa_package/87/a1/PMC3014676.tar.gz |
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PMC3014678 | 21234105 | 7. Conclusion
PPARs are lipid-activated transcription factors that have emerged as key regulators of both lipid metabolism and inflammation. They exert positive and negative controls over the expression of a range of inflammatory genes. The anti-inflammatory properties of PPARs make them attractive targets for intervention in human autoimmune diseases, including MS. A growing body of literature suggested that PPAR agonists could be used therapeutically in autoimmune diseases such as MS as a preliminary clinical study has suggested. Further studies will be required to fully understand the complicated mechanisms of PPAR regulation of immune responses and autoimmune encephalomyelitis. | Academic Editor: Bèatrice Desvergne
PPARs are members of the steroid hormone nuclear receptor superfamily and play an important role in regulating inflammation as well as lipid metabolism. The PPAR subfamily has been defined as PPAR α , PPAR β / δ , and PPAR γ , each with different ligands, target genes, and biological roles. PPARs regulate the expression of target inflammatory genes through mechanisms involving both transactivation and transrepression. The anti-inflammatory properties of PPAR agonists have led to the investigation of PPAR functions in regulating autoimmune encephalomyelitis. This paper will summarize some of the general mechanisms by which PPARs regulate inflammatory gene expression and focus on the recent advances of PPAR regulation of autoimmune encephalomyelitis. | 1. Peroxisome Proliferator-Activated Receptors (PPARs)
The nuclear receptor superfamily integrates both inflammatory and metabolic signals to maintain homeostasis in immune cells such as macrophages, dendritic cells, and lymphocytes [ 1 , 2 ]. PPARs are nuclear receptors activated by fatty acids and control the expression of genes involved in both lipid metabolism and inflammation. So far, there are three isoforms that have been identified and cloned, including PPAR- α , PPAR- β / δ , and PPAR- γ , and they exhibit different tissue distribution as well as different ligand specificities. PPAR α was the founding member of the PPAR subfamily and was first cloned in rodents. It was shown to be activated by a diverse class of rodent hepatocarcinogens that causes proliferation of peroxisomes [ 3 ]. Subsequently, two other family members were discovered, PPAR β / δ and PPAR γ [ 4 , 5 ]. Natural ligands for the PPARs include native and modified polyunsaturated fatty acids and eicosanoids [ 6 – 8 ]. Additionally, the PPARs have a large ligand-binding pocket that can accommodate a diverse range of synthetic ligands [ 9 – 11 ].
All PPARs have four main domains named A/B, C, D, and E/F. The N-terminal A/B domain has a transcriptional activating function (AF-1). The C domain, or DNA binding domain (DBD), is formed by two zinc finger-like motifs that can recognize a peroxisome proliferator response element (PPRE) on target genes. PPREs are specific DNA sequences of the repetition of a consensus hexanucleotide sequence (AGGTCA), separated by one or two nucleotides. The D domain is a hinge region that can modulate the DNA binding ability and is involved in cofactor interaction. The E/F domain is the ligand-binding domain (LBD), which is responsible for the ligand binding and is involved in the dimerization with the 9-cis retinoic acid receptor (RXR) [ 12 ].
PPARs are expressed by several different immune cells, including macrophages [ 13 – 15 ], T cells [ 16 – 19 ], B cells [ 20 ], and dendritic cells [ 21 – 23 ]. Other than regulation of lipid metabolism, PPARs have also been shown to play an important role in regulating immune responses and inflammation, by programming inflammatory gene expression in immune cells, including macrophages, dendritic cells, and lymphocytes [ 8 , 24 , 25 ]. All three members of the PPAR family have been shown to exert anti-inflammatory effects in vitro and in vivo. The anti-inflammatory effects of PPAR agonists have been observed in autoimmune diseases, including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Although the detailed mechanisms by which PPARs regulate inflammatory responses and autoimmune encephalomyelitis are still not well established, recent studies have broadened our understanding on the transcriptional regulation of inflammatory target genes by PPARs and shed light on the mechanism of PPAR regulation of autoimmune encephalomyelitis. The positive regulation of target gene transcription by PPARs was through direct binding to the PPRE on the promoter of target genes, whereas negative regulation of target gene expression was mostly indirect, through a mechanism termed transrepression [ 6 , 26 ]. This paper will summarize some general mechanisms by which PPARs regulate inflammatory gene expression and focus on the recent advances of PPAR regulation of autoimmune encephalomyelitis.
2. General Mechanisms of PPAR Action
2.1. Positive Regulation of Target Gene Expression
PPARs can both positively and negatively regulate their target gene expression. One of the mechanisms by which PPARs exert their function is through binding to a PPRE as a heterodimer with RXR in a ligand-dependent manner. Ligand-dependent activation is linked to the recruitment of coactivator complexes that modify chromatin structure and facilitate assembly of the general transcriptional machinery at the promoter [ 27 , 28 ].
In the unliganded state, PPARs are associated with a nuclear receptor corepressor. NCoR (nuclear receptor corepressor) is among the most studied corepressors. In addition, heat shock protein-90 and the hepatitis virus B X-associated protein 2 have been shown to be associated with PPAR- α , which negatively regulates subsequent gene activation [ 29 , 30 ]. Upon ligand activation, the PPARs undergo a conformational change that results in the dissociation from the corepressor, enabling the PPARs to bind nuclear receptor coactivators. These coactivators then act to reorganize the chromatin templates allowing the basal transcription machinery to gain access to the promoter regions and drive the transcription of target genes [ 24 ]. One example of positive regulation of inflammatory gene expression in autoimmune encephalomyelitis is the regulation of IL-4 gene by the PPAR α agonist gemfibrozil. Gemfibrozil induced immune deviation and protected mice from EAE. PPAR- α was shown to regulate the IL-4 and IL-5 genes and bind to the IL-4 promoter in the presence of steroid receptor coactivator-1 (SRC-1), suggesting transactivation of the IL-4 gene [ 31 ].
2.2. Negative Regulation of Target Gene Expression by Transrepression
The ligand-dependent gene repression by PPARs is mediated through an indirect regulatory mechanism, termed ligand-dependent transrepression, which is distinguished from active repression of target genes in that the repression does not depend on the binding of PPARs to PPREs in target gene promoters. The general mechanism of transrepression involves protein-protein interactions between PPARs and their target transcription factors or coregulatory complexes. Transrepression is widely used to negatively regulate gene expression but the detailed mechanism by which different nuclear receptors exert their specific transrepression varies from receptor to receptor. It is difficult to identify a unified mechanism of repression by all PPARs, since signal, cell, and/or promoter-specific mechanisms exist for all three PPAR family members.
2.2.1. Tether Transcription Factors Away from Their Binding Sites
One of the mechanisms by which PPARs transrepress their target inflammatory gene expression is to tether transcription factors away from their DNA binding sites in the target gene promoter region, which in turn inhibits gene transcription of the target inflammatory genes. This process involves physical interaction between PPARs and their target transcription factors. The inhibition of phorbol ester-induced IL-2 expression by PPAR γ was mediated by this mechanism. PPAR γ ligands, troglitazone and 15 deoxy Delta (12, 14) prostaglandin J (2) (15d-PGJ2), inhibited IL-2 production and phytohemagglutinin-inducible proliferation in human peripheral blood T cells in a dose-dependent manner. The activated PPAR γ physically associates with the transcription factor, nuclear factor of activated T cells (NFAT), regulating the IL-2 promoter by blocking NFAT DNA binding and transcriptional activity. The ligand-dependent binding of PPAR γ to NFAT correlates with the dissociation of NFAT from the IL-2 promoter [ 32 ].
2.2.2. SUMOylation-Dependent Pathway
Another important transrepression pathway is the SUMOylation-dependent pathway. SUMOylation-dependent targeting of a nuclear receptor to corepressor complexes to prevent their signal-dependent clearance is likely to represent a general molecular strategy for transrepression of proinflammatory target genes [ 33 , 34 ]. The SUMOylation-dependent pathway mediated transrepression of inflammatory response genes was first identified for PPAR γ and then extended to PPAR α and two additional members of the nuclear receptor family, LXR α and LXR β . In macrophages, the SUMOylation-dependent pathway was initiated by ligand-induced SUMOylation of the PPAR γ ligand-binding domain. This modified PPAR γ in turn bound to NCoR complexes associated with the promoters of target inflammatory genes, which were marked by the presence of NCoR-HDAC3-TBL corepressor complexes. These complexes prevented Ubc5 recruitment in response to lipopolysaccharide (LPS) signals, which supposedly led to the clearance of NCoR and HDAC3, and the switch from repression to transcriptional activation. As a result, NCoR complexes were not cleared from the promoter, and target genes were maintained in a repressed state [ 33 ].
Collectively, recent studies have defined some molecular mechanisms by which PPARs positively or negatively regulate their target inflammatory gene expression. However, the molecular mechanisms by which PPARs regulate autoimmune encephalomyelitis have not been well defined. So far, no study has been published demonstrating the detailed molecular mechanism of negative regulation of PPAR in the EAE model although Gocke et al. showed the molecular pathway of positive regulation of IL4 and IL5 gene by PPAR- α in EAE . More studies are needed in the future to elucidate the detailed mechanisms by which PPARs regulate autoimmune encephalomyelitis.
3. MS and EAE
MS is the leading cause of neurologic disability in the United States in young adults after trauma; thus, most patients suffer from the effects of MS for most of their adult life. MS is thought to be a T cell-mediated autoimmune disease of the central nervous system (CNS) with a complex genetic background. Although the precise etiology of MS is still unknown, it is generally accepted that MS begins with the formation of acute inflammatory lesions which are mediated by autoreactive T cells and B cells. The demyelinating plaques are dominated by activated T cells and macrophages associated with oligodendrocyte and myelin destruction ( Figure 1 ).
CD4 T cells are at the center of MS pathogenesis and are the focus of MS research, given their important role in mediating disease. CD4 T cells differentiate into different types of T effector cells in the periphery in response to different pathogenic microorganisms as a result of recognition of these organisms by the innate immune system. It has been suggested for more than two decades that there are two different types of CD4 T helper cells, Th1 and Th2 cells. The Th1 cell subset mainly produces IFN- γ , IL-2 and GM-CSF, while the Th2 cell subset produces IL-4, IL-5 and IL-13 [ 35 – 38 ]. Th1 cells have been implicated in a variety of autoimmune diseases, including MS [ 39 – 41 ]. Conversely, Th2 cells control infections by extracellular microbes, and cytokines produced by Th2 cells mediate helper T cell functions for antibody production and mediate the immunopathology of allergic responses. Early studies suggested that the IFN- γ producing Th1 CD4 T cells, which were driven by IL-12, played an essential role in mediating disease, while Th2 cytokines such as IL-4 were associated with amelioration of EAE and remission in MS. More recently, Th17 cells have been identified as a new CD4 T cell lineage. In vivo, Th17 cells were driven by IL-23 although, in vitro, they were induced by TGF- β and IL-6. Th17 cells also have been shown to be critical in the development of autoimmune diseases. Studies have been done to define the roles of different T cell subpopulations in MS pathogenesis and focus on how to manipulate pathogenic Th1 and Th17 cells and related cytokines to suppress disease.
EAE is an inflammatory demyelinating disease mediated by myelin-specific Th1 and Th17 CD4 lymphocytes. EAE is characterized by relapsing paralysis, CNS inflammation, and demyelination. It has been used as a model for MS for several decades, since it shares clinical and immunopathological similarities to MS. EAE can be induced in mice by immunization with various myelin proteins or peptides emulsified in CFA or by the transfer of activated myelin-specific CD4 Th1 lymphocytes into naive recipients.
The anti-inflammatory properties of PPAR agonists have led to the investigation of PPAR functions in regulating autoimmune encephalomyelitis, hoping to develop new therapeutic strategies for MS. Many studies have been performed to test the effects of different PPAR agonists in regulating EAE and have shown very promising results. Some of the targets of PPARs in MS pathogenesis are shown in Figure 1 . Based on these promising animal data, a PPAR agonist has been tested in a clinical trial as an adjunctive treatment for MS patients. In the next three sections, we are going to discuss the regulation of autoimmune encephalomyelitis by PPAR γ , PPAR α , and PPAR β / δ , respectively.
4. Regulation of Autoimmune Encephalomyelitis by PPAR γ
4.1. PPAR γ Agonists Suppress EAE
Several PPAR- γ agonists have been shown to ameliorate EAE. Troglitazone was shown to ameliorate MOG 35-55-induced EAE in C57BL/6 mice, and troglitazone treatment during the effector phase is more effective than when it is given during the induction phase [ 42 ]. Administration of another PPAR γ agonist, 15d-PGJ2, before and at the onset of clinical signs of EAE significantly reduced the severity of disease in B10 PL mice. More importantly, culture of encephalitogenic T cells with 15d-PGJ2 reduced their ability to adoptively transfer EAE, suggesting that PPAR- γ ligands may regulate T cell encephalitogenicity in vitro [ 43 ]. Furthermore, the combination of 15d-PGJ2 and 9-cis-retinoic acid (RA), the ligand for RXR, resulted in enhanced amelioration of disease, suggesting that combination of RXR-specific ligands and PPAR γ ligands may be highly effective in the treatment of autoimmune demyelinating diseases such as MS [ 44 ]. Similarly, in SJL/J mice, 15d-PGJ2 or Ciglitazone decreased the duration and clinical severity of active immunization and adoptive transfer models of EAE [ 45 ]. Orally administered pioglitazone was also shown to reduce the incidence and severity of monophasic, chronic disease in C57BL/6 mice and of relapsing disease in B10.PL mice. Pioglitazone also reduced clinical signs when it was provided after disease onset. The suppression of clinical signs was paralleled by decreased lymphocyte infiltration, lessened demyelination, reduced chemokine and cytokine expression, and increased inhibitor of κ B (I κ B) expression in the brain [ 46 ]. Another PPAR γ aonist, rosiglitazone, when used to treat DCs, was able to prevent EAE development in mice [ 47 ].
On the other hand, PPAR γ antagonists exacerbated EAE. Treatment with PPAR γ antagonists, Bisphenol A diglycidyl ether (BADGE), or 2-Chloro-5-nitro-N-(4-pyridyl)benzamide (T0070907) increased the severity and duration of EAE in C57BL/6 wild-type and PPAR γ -deficient mice. The exacerbation of EAE was associated with an augmented neural antigen-induced T cell proliferation, IFN γ production, and Th1 differentiation [ 48 ]. Furthermore, BADGE and benzamide (T0070907) reversed the inhibition of EAE by the PPAR γ agonists, Ciglitazone and 15d-PGJ2, in C57BL/6 wild-type and PPAR γ +/− mice. The reversal of EAE was associated with restoration of neural antigen-induced T cell proliferation, IFN γ production, and Th1 differentiation inhibited by Ciglitazone and 15d-PGJ2 [ 49 ].
Together, these data demonstrated that PPAR γ played an important role in regulating autoimmune encephalomyelitis in vivo and suggested that PPAR γ agonists might be a new therapeutic treatment for autoimmune demyelinating diseases such as MS.
4.2. EAE in PPAR γ -Deficient Mice
It is controversial whether PPAR γ agonists such as 15d-PGJ2 require PPAR γ for their anti-inflammatory function, because there are studies showing PPAR γ -independent mechanisms in the induction of anti-inflammatory effects by 15d-PGJ2 [ 50 ]. PPAR γ -deficient heterozygous mice were used to demonstrate the function of endogenous PPAR γ in EAE. In the endogenous state, the PPARs are likely occupied by their fatty acid ligands, which may be produced at sites of inflammation. PPAR γ -deficient heterozygous mice developed an exacerbated course of EAE with prolonged clinical signs compared to wild-type littermates. The exacerbation was associated with an increased expansion of CD4 and CD8 T cells and expression of CD40 and MHC class II molecules in response to antigen, confirming PPAR γ as a critical regulator of EAE and perhaps MS [ 51 ].
4.3. Role of PPAR γ in Regulating Immune Cells in EAE
Since studies in vivo showed PPAR γ agonists inhibited CNS inflammation and demyelination in EAE, studies were done to elucidate the potential therapeutic mechanisms. Troglitazone has been shown to attenuate the inflammation and decreased the clinical signs through the attenuation of proinflammatory cytokine gene expression in the spinal cord [ 42 ]. In addition to this, additional studies have been published to show the different effects of PPAR γ agonists on immune cells and CNS resident cells in EAE as described below.
4.3.1. Antigen-Presenting Cells
PPAR γ agonists were shown to regulate the function of antigen-presenting cells, including monocyte/macrophages and dendritic cells. 15d-PGJ2 was shown to inhibit phorbol ester-induced nitric oxide (NO), TNF- α , IL-1, and IL-6 production by cells of the monocyte/macrophage lineage, in part by antagonizing the activities of transcription factors such as AP-1 and NF- κ B [ 14 , 15 ]. Another PPAR γ agonist, rosiglitazone, was shown to interfere with NF- κ B activation in murine DCs. As a result, treated DCs showed impaired maturation and a reduced capacity to activate antigen-specific T cells and were able to prevent EAE development in mice [ 47 ].
4.3.2. T Cells
Since MS and EAE are suspected T cell-mediated autoimmune diseases, studies have been conducted to determine how PPARs regulate T cell function. 15d-PGJ2 was shown to inhibit the proliferation of Ag-specific T cells from myelin basic protein Ac1-11 TCR-transgenic mice and suppress IFN- γ , IL-10, and IL-4 production by lymphocytes. Similarly, the disease inhibition with 15d-PGJ2 or Ciglitazone in SJL/J mice was associated with a decrease in IL-12 production and differentiation of neural antigen-specific Th1 cells. Treatment of activated T cells with PPAR γ agonists in vitro inhibited IL-12-induced activation of the JAK-STAT signaling pathway and Th1 differentiation [ 45 ]. Orally administered pioglitazone was also shown to reduce the antigen-dependent IFN- γ production from EAE-derived T cells [ 46 ].
In addition to pathogenic Th1 cells, Th17 cells have also been shown to be pathogenic in MS and EAE. One recent study identified PPAR γ as a key negative regulator of human and mouse Th17 differentiation. PPAR γ activation in CD4 T cells selectively suppressed Th17 differentiation through inhibition of TGF- β /IL-6 induced ROR γ t expression, but not differentiation into Th1, Th2, or regulatory T cells. More importantly, human CD4 T cells from healthy controls and MS patients were strongly susceptible to PPAR γ -mediated suppression of Th17 differentiation, suggesting that PPAR γ is a promising molecular target for specific immunointervention in Th17-mediated autoimmune diseases such as MS [ 52 ].
4.4. Role of PPAR γ in CNS-Resident Cells in EAE
Other than regulating immune responses, PPAR γ agonists were also shown to regulate the functions of CNS-resident cells, including microglia and astrocytes. It has been shown that PPAR γ agonists modulate EAE, at least in part, by inhibiting the activation and cytokine production of microglia and astrocytes. 15d-PGJ2 together with 9-cis retinoic acid potently inhibited microglial cell activation and inhibit EAE development in mice [ 53 ]. Three TZDs, rosiglitazone, pioglitazone, ciglitazone, and 15d-PGJ2, were all effective in inhibiting production of NO, the proinflammatory cytokines TNF- α , IL-1 β , and IL-6, and the chemokine MCP-1 from microglia and astrocytes [ 54 , 55 ]. 15d-PGJ2 and rosiglitazone inhibited the induction of IL-12p40, IL-12p70 (p35/p40), IL-23 (p19/p40), and IL-27p28 proteins by LPS-stimulated primary microglia. 15d-PGJ2 potently suppressed IL-12p40, IL-23, and IL-27p28 production by primary astrocytes, whereas rosiglitazone suppressed IL-23 and IL-27p28, but not IL-12p40 in these cells [ 56 ]. These effects on CNS-resident cells might contribute to the suppression of EAE by PPAR γ agonists.
4.5. PPAR γ Agonists and MS
PPAR γ agonist pioglitazone was tested as an add-on therapy with interferon- β in a small cohort of relapsing remitting MS (RRMS) patients. RRMS patients taking IFN β -1 α were randomized to treatment with pioglitazone (30 mg daily, p.o.) or placebo and monitored clinically by EDSS and by MRI for 1 year. After 1 year, there were no significant differences in clinical signs as assessed by EDSS; however, MRI showed a significant reduction in gray matter atrophy and a trend for reduced lesion burden in the treatment group. These data suggested some beneficial effects for RRMS patients, and further trials need to be performed to establish clinical efficacy [ 57 ].
PPAR γ involvement in autoimmune encephalomyelitis was also implicated by a population-based study in MS patients. The Ala allele of the PPAR γ Pro12Ala polymorphism was strongly associated with delayed disease onset (44.1 ± 5.3 years versus 34.5 ± 4.2 years; P = .006). This study demonstrated that the Pro12Ala polymorphism resulting in an amino acid exchange in the N-terminal sequence of PPAR γ may influence the onset of MS [ 58 ].
In summary, data from EAE studies showed that PPAR γ agonists were able to suppress disease severity by regulating the functions of both immune cells and CNS-resident cells ( Table 1 ), supporting PPAR γ agonists as an effective treatment of autoimmune demyelinating diseases such as MS. Small-scale clinical data further confirmed that the PPAR γ agonist pioglitazone maybe beneficial for RRMS patients. More clinical studies are needed to further establish clinical efficacy.
5. Regulation of Autoimmune Encephalomyelitis by PPAR α
PPAR α is expressed in different immune cells, including monocytes/macrophages, T cells, and B cells and plays an important role in regulating inflammation and cytokine production. PPAR α agonists have also been tested in the treatment of EAE and shown to be protective [ 24 ]. However, recent studies showed, other than sharing the common anti-inflammatory effects of all three PPAR subtypes, PPAR α had specific effects in inducing immune deviation in EAE. This specific regulation of immune deviation made these PPAR α agonists very attractive candidates to be used therapeutically in treating Th1-mediated autoimmune diseases, including MS, in addition to their excellent track history as oral agents used to treat hypertriglyceridemia. Here we are going to focus on several recent studies demonstrating PPAR α regulation of immune deviation, gender differences, and its role in CNS-resident cells ( Table 2 ).
5.1. Immune Deviation Induced by PPAR α Agonists
5.1.1. Immune Deviation
Th1 and Th2 cells are two distinct CD4 T cell lineages, and they play different roles in autoimmune diseases, including MS and EAE. Autoimmune diseases can be divided into those mediated by Thl cells with primarily inflammatory manifestations and those mediated by Th2 cells whose manifestations are secondary to autoantibody containing immune complexes [ 66 ]. Immune deviation was a term used to characterize an immune response where Th2 cells predominate, and one approach to the immunotherapy of inflammatory autoimmune disease, including MS, was the antigen-specific deviation of an immune response dominated by a Th1 response to a Th2 response.
5.1.2. PPAR α Agonists Ameliorate EAE by Inducing Immune Deviation
The PPAR α agonist gemfibrozil was shown to regulate immune responses by promoting the deviation of immune responses dominated by a pathogenic Th1 response to a nonpathogenic Th2 response [ 31 , 60 , 67 ]. Lovett-Racke et al. demonstrated that PPAR α agonists induced a shift in cytokine production from Th1 cytokines to Th2 cytokines in both mouse and human T cells and protected mice from EAE. PPAR α agonists increased the production of the Th2 cytokine, IL-4, and suppressed proliferation by TCR transgenic T cells specific for the myelin basic protein Ac1-11 peptide. Oral administration of PPAR α agonists gemfibrozil and fenofibrate inhibited the clinical signs of EAE. More importantly, the PPAR α agonist gemfibrozil shifted the cytokine secretion of human T cell lines from IFN γ secretion to IL-4 secretion [ 59 ]. Gocke et al. studied the molecular mechanisms by which PPAR α agonists induce immune deviation and protect mice from EAE. They demonstrated that PPAR α agonists directly activated Th2 cytokine IL-4 gene expression by directly binding to the IL-4 promoter region. Gemfibrozil treatment increased Th2 transcription factor GATA-3 expression and decreased Th1 transcription factor T-bet expression in vitro and directly ex vivo. For the first time, they showed that PPAR α regulated the IL-4 and IL-5 genes and bound the IL-4 promoter in the presence of the steroid receptor coactivator-1 [ 31 ].
5.1.3. Is the Effect Receptor Dependent or Not?
Gocke et al. showed that the protective effects of PPAR α agonists in EAE occurred in a receptor-dependent manner [ 31 ]. Dasgupta et al. observed similar effects with the PPAR α agonist Gemfibrozil in mice, including switching of a Th1 profile to a Th2 profile, inhibiting T-bet expression, stimulating GATA3 expression, and inhibiting the encephalitogenicity of antigen-primed T cells. However, they suggested the switch of immune response from a Th1 to a Th2 profile by PPAR α agonists was receptor independent, as the drug was equally effective in treating EAE in PPAR α -deficient and well as wild-type mice [ 60 ]. Similarly, Cunard et al. showed that treatment with WY14,643 and other fibrates led to marked increases in supernatant concentrations of IL-4. They also showed that this effect on IL4 production was largely through a PPAR α -independent mechanism, since WY14,643 induced IL-4 expression in splenocytes from PPAR α -deficient mice [ 19 ]. Further studies are needed to elucidate the receptor dependency of PPAR α agonists.
5.2. PPAR α and Gender Differences
PPAR α could be one of the genes mediating gender differences in EAE. PPAR α expression in T cells is higher in male mice compared to female mice, and this expression is reduced by castration and increased by α -DHT treatment. The deficiency of PPAR α gene expression resulted in higher IFN- γ and TNF α production by T cells in male mice. Male but not female PPAR α -deficient mice developed more severe EAE that was restricted to the acute phase of disease. These findings provide a molecular basis for why males may be less prone to developing Th1-mediated autoimmunity [ 67 ].
Women are more susceptible than men to develop autoimmune diseases, including MS. In MS, twice as many women as men develop the disease. This may be related to the fact that women have more robust immune responses than men although the exact mechanism is not understood [ 68 ]. A study showed women to be more prone than men to develop Th1-polarized responses directed against myelin antigens during MS [ 69 ]. Whether PPAR α is responsible for the gender differences in MS susceptibility remains to be determined.
5.3. PPAR α Regulation of CNS-Resident Cells in EAE
Moreover, PPAR α agonists were also shown to regulate CNS-resident cells and the protective effects of PPAR α agonists in EAE were in part through effects on CNS cells. Microglia cells are resident CNS cells that may serve as antigen-presenting cells. Activated microglia exhibit increased pathogenic cytokine production and increased synthesis of NO, which may contribute to axonal degradation in MS. Xu et al. showed that PPAR α agonists inhibited microglia production of NO, IL-1 β , and TNF- α , which were potentially toxic to cells including myelin-producing oligodendrocytes. In addition, these agonists inhibit microglial production of Th1- and Th17-promoting cytokines, IL-12 and IL-23 [ 61 ]. PPAR α agonists also suppressed microglia production of MCP-1, a chemokine that plays an important role in modulating monocyte infiltration into the CNS in MS [ 70 ]. Similar effects were observed in LPS-stimulated astrocytes. A combination of 9-cis RA and the PPAR α agonists fenofibrate or gemfibrozil cooperatively inhibited NO, TNF- α , IL-1 β , IL-6, and MCP-1 production by these cells [ 62 ]. Thus, PPAR α agonists could also modify cytokine expression in the CNS during inflammation such as that observed in EAE or MS.
6. Regulation of Autoimmune Encephalomyelitis by PPAR β / δ
PPAR β / δ is the predominant PPAR isotype in brain. However, the exact functions of PPAR β / δ are not yet well understood, but it is likely to play a role in cell proliferation [ 71 ], differentiation, survival, lipid metabolism, and development [ 72 , 73 ].
6.1. PPAR- β / δ -Specific Agonists and EAE
A protective effect in EAE was reported for a PPAR- β / δ -specific agonist, and this protection was suggested to be due to a reduction in glial inflammation. Polak et al. showed that oral administration of the selective PPAR δ agonist GW0742 reduced clinical signs in actively immunized C57BL/6 mice, especially when it was administered during disease progression [ 63 ]. The protective effect of GW0742 was receptor dependent, since no amelioration of EAE clinical scores was observed in PPAR δ -deficient mice [ 64 ]. RT-PCR analysis showed that GW0742 increased expression of some myelin genes. GW0742 reduced astroglial and microglial inflammatory activation and IL-1 β levels in EAE brain.
Other than GW0742, two other PPAR β / δ agonists, GW501516 and L165041 were shown to ameliorate MOGp35-55-induced EAE in C57BL/6 mice by blocking IFN- γ and IL-17 production by Th1 and Th17 cells [ 65 ]. GW 501516 was also tested for its capacity to protect from antibody-mediated demyelination. However, GW 501516 did not protect against antibody-mediated demyelination although it showed some anti-inflammatory activity [ 74 ].
The regulation of EAE by PPAR β was further confirmed by a study in Steroid receptor coactivator-3-(SRC-3) deficient mice. SRC-3 is a member of the p160 family of coactivators that interact with nuclear receptors to enhance their transactivation in a ligand-dependent manner. SRC-3 deficiency significantly inhibited the disease severity of EAE. However, these effects are not caused by inhibition of peripheral T cell response, but by upregulation of PPAR β in the CNS, which induced an alternative activation state of microglia in SRC-3 deficient mice. These alternatively activated microglia inhibited CNS inflammation through inhibition of proinflammatory cytokines and chemokines, such as TNF- α , IFN- γ , CCL2, CCL3, CCL5, and CXCL10 as well as upregulation of the anti-inflammatory cytokine IL-10 and opsonins [ 75 ].
6.2. PPAR β / δ Regulation of Immune Responses
Treatment of T-cells with GW0742 either in vivo or in vitro did not reduce Th1 cytokine IFN γ production [ 63 ]. However, a study showed that the PPAR δ agonists, GW501516 and L165041, ameliorated EAE by blocking IFN γ and IL-17 production by Th1 cells and Th17 cells and was associated with a decrease in IL-12 and IL-23 and an increase in IL-4 and IL-10 expression in the CNS and lymphoid organs [ 65 ].
The PPAR β / δ regulation of immune cells was further confirmed by one recent study in PPAR β / δ -deficient mice. PPAR- β / δ -deficient mice developed a severe inflammatory response during EAE characterized by a striking accumulation of IFN- γ + IL17A- and IFN- γ + IL-17A+ CD4+ cells in the spinal cord, which resulted from immune system aberrations including enhanced Th cell expansion, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. This data strongly suggests that PPAR- δ serves as an important molecular brake for the control of autoimmune inflammation [ 76 ] ( Table 3 ).
6.3. PPAR β / δ and Oligodendrocyte (OL) Maturation
The special feature of PPAR δ function is that PPAR δ agonists are more effective when administered during later stages of disease and they increase myelin gene expression [ 63 ], which suggested that they might affect OL maturation. Vittoria Simonini et al. demonstrated that PPAR δ played a role in OPC maturation. GW0742 was shown to increase the number of myelin-producing OLs in OPCs, and this was receptor dependent, since OLs were reduced in PPAR-deficient OPCs [ 64 ]. | CC BY | no | 2022-01-13 03:14:01 | PPAR Res. 2010 Dec 22; 2010:104705 | oa_package/04/a5/PMC3014678.tar.gz |
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PMC3014679 | 21234106 | 1. Introduction
The present study is concerned with the design and experimental testing of intelligent control systems for temperature control in the precipitation plant of bromelain enzyme recovery. This biotechnological process may be considered the first step in the downstream processing of the protein. It is motivated by the high commercial value of this enzyme, the increasing demand for bromelain in pharmaceutical and industrial applications [ 1 , 2 ], and the fact that bromelain can be recovered from kitchen waste (pineapple stem and rind).
The aim of the precipitation process is to achieve separation of solutes by conversion to solids. Precipitants can be chosen which do not denature the biological product, and the precipitate is often more stable than the dissolved form. Although precipitation is a simple operation, in the recovery of bromelain from pineapple, temperature control is crucial to avoid irreversible protein denaturation and hence improve the precipitation yield and the enzyme activity of the product [ 3 ].
Despite that automation and process control can significantly influence the yield and final quality of bioproducts, there are few experimental studies on the application of automatic controllers in the bioprocesses. Most works focus on results obtained from computational simulations, which indeed do not represent these processes in all their complexity. The transient behavior and nonlinearities of these processes make the design of classical control dependent on trial-and-error methodology, showing limited performance. On the other hand, intelligent controllers based on fuzzy logic and neural networks can be applied successfully to both linear and nonlinear systems. The main advantage of intelligent controllers is that modeling, based on first principles, is not required. Implementation of these controllers in small-scale pilot plants is essential to evaluate their potential value.
The wide range of existing fuzzy control applications indicates that this technique is gaining considerable importance in the control of complex processes and represents a promising approach to solving industrial control problems [ 4 – 9 ]. Some successful fuzzy logic experimental applications in biotechnological processes are reported in the literature by Steyer et al. [ 10 ], Martínez et al. [ 11 ], Sousa and Almeida [ 12 ], Babuska et al. [ 13 ], Horiuchi and Kishimoto [ 14 ], Traoré et al. [ 15 ], and Fileti et al. [ 16 ]. The computational simulation of a semibatch reactor stationed at a pharmaceutical company was used by Dovžan and Škrjanc [ 17 ] for testing a predictive functional control based on an adaptive fuzzy model. The goal was to control the temperature of the ingredients stirred in the reactor's core, so that they synthesize optimally into the final product. In order to achieve this, the temperature had to follow, as accurately as possible, the prescribed reference trajectory, with as low overshoot as possible.
Castañeda-Miranda et al. [ 18 ] developed a greenhouse intelligent climate control system that uses a fuzzy controller, based on a field programmable with a great potential for use in agricultural technology development due to its characteristics to produce fast prototypes of complex hardware designs with an effective production cost.
Lopes et al. [ 19 ] implemented an adaptive neurofuzzy inference mechanism (ANFIS) in a system of coupled tanks to simulate a production unit that used the level of the tanks for production and concentration control (process specifications). This inference system was proved efficient in aiding and replacing human workers for establishing relationships between process specifications and controlled variables (levels). Pires and Nascimento Júnior [ 20 ] simulated a control system of a robotic arm using a neurofuzzy system as a feedforward controller. The proposed control scheme was suitable to the robotic arm adjustment, obtaining a trajectory very close to the reference.
Hussain [ 21 ] selected in his review twenty four works concerning online application of neural networks. Many of them showed a better performance of the neural controller over the conventional one. Other studies proved the effectiveness of neural networks in modeling nonlinear processes, such as fermentation and polymerization processes [ 22 , 23 ].
In this context, this paper describes the design methodology and a comparative study on the performance of fuzzy, neurofuzzy, and neural network intelligent controllers. They were implemented in order to maintain the temperature of the bromelain precipitation process from aqueous extract of pineapple wastes. The digital control was carried out by means of a Foundation Fieldbus communication system. To assess the performance of the digital controllers, the following parameters were used: overshoot, ITAE (integral of Time multiplied by Absolute Error), response time, enzymatic activity of the product and electric power consumption of the cooling system. The novelty of this paper is that, currently, there are no experimental studies about automation and process control in the production of bromelain, despite the growing number of scientific papers related to this enzyme. | 2. Material and Methods
2.1. Precipitation Plant Description
Digital controllers were developed, implemented in the computer, and experimentally tested in a pilot plant of the precipitation process, outlined in Figure 1 , located in the Automation and Process Control Laboratory at the School of Chemical Engineering , at the University of Campinas (UNICAMP).
In Figure 2 , it could be seen that the experimental system consisted of a stainless steel stirred tank with nominal capacity of 1000 mL (no. 1); a storage tank of the precipitating agent (ethanol 99.5 GL); a variable speed pump (1000 L/h maximum capacity), to enable the flow of the cooling fluid (50% v/v water/propylene glycol solution) through the tank jacket; a micropump that continuously fed the ethanol into the precipitation tank; four Pt-100 resistance thermometer detectors (no. 2)—TE 301, TE 302, TE 303, and TE 304 represented in Figure 1 —to monitor the temperature in the precipitation tank, ethanol storage, the coolant outlet and inlet, respectively; two temperature transmitters (TT 302 device, Smar) (no. 3), with digital output signal, connected to the Pt100; a Fieldbus-current converter (no. 4) (FI302 device, Smar), coupled to a frequency converter; a level transmitter (no. 5)—LT 301, in Figure 1 —which consists of a differential pressure transmitter (LD 302 device, Smar); a current-Fieldbus converter (no. 6) (IF302 device, Smar) to transmit signal of the power consumed by the coolant pump; a peristaltic pump (Masterflex Pump) for continuous feeding of ethanol in the precipitation tank (no. 7); a mechanical stirrer (no. 8). An electrical control panel ( Figure 2(b) ) was built, where it can be observed (a) the frequency converter (Danfoss VLT 2800), that drives the coolant pump speed (manipulated variable) (b) an electrical source for powering the field devices, (c) circuit breakers, and (d) fuses and (e) connectors, to allow corrective maintenance of equipments.
2.2. Operating Conditions of the Pilot Plant
The stem and rind of “Pérola” pineapples were ground and mixed to a uniform suspension in distilled water, at a dilution rate of 1 : 1 v/v. Solids were filtered from the mixture through a 0.45 μ m paper filter. The filtrate, called pineapple extract, contained the bromelain enzyme. Aliquots of 150 mL of pineapple extract were frozen at −18°C [ 24 ] until they were defrosted and individually used in the experiments.
Samples of 150 mL of pineapple aqueous extract were fed into the tank. A micropump was employed to feed the ethanol into the tank continuously at room temperature (approximately 23°C), at a fixed rate of 2.16 L/h, until the liquid volume reached 750 mL, corresponding to a 1 : 4 v/v ratio between extract and ethanol—the optimal condition for protein precipitation [ 24 ]. In order to avoid protein denaturation, the coolant flow rate was manipulated by means of a variable-speed pump, to maintain the temperature constant into the tank.
A design of experiments was proposed to find the nominal conditions for the following process variables: the ethanol micropump flow rate, the coolant pump flow rate (manipulated variable), and the coolant inlet temperature. Those nominal conditions were determined by maximizing the temperature deviation from set point and minimizing the rise time of temperature, which turns the process a challenging problem of control. The best values found were, respectively, 2.16 L/h, 374 L/h, and 0°C. The stirring rate was kept constant (150 rpm) during tests. The batch operation took about 17 minutes (time of contact between solvent and proteins).
2.3. Characterization of Bromelain
The temperature control has great influence on the final quality of the recovered bromelain enzyme (EC 3.4.22.4). Therefore, the determination of enzymatic activity was used as an important index of performance to compare the efficiency of the automatic controllers tested in this work.
The activity assay was based on the biuret colorimetric method. Under alkaline conditions, substances containing two or more peptide bonds form a purple complex with the copper salts in the reagent. The intensity of the color produced in the biuret reaction is proportional to the number of peptide bonds participating in the reaction. In this method, a unit (U) of enzyme activity is defined as the amount of enzyme able to change the absorbance reading at 540 nm by one unit in 10 min, at 37°C. The specific enzymatic activity, AE (U/g), is the relationship between the number of units of enzyme activity (U/mL) and the amount of protein (g/mL) in the sample.
2.4. The Digital Control System
In order to provide communication between the process and the local area network, a Fieldbus interface was used. An OPC server was responsible for providing the control program (OPCclient) with current values of measured variables and for communicating the control action to the frequency converter.
The digital control was carried out through a Foundation Fieldbus communication system, as shown in Figure 3 .
The field devices composing the Fieldbus network, used to monitor and control the precipitation tank, were as follows
Distributed Fieldbus interface (DFI302 device, Smar): it is manager of communication which controls the actions related to the Fieldbus system. It performs most functions required by the control system and connects the network of field devices to an Ethernet network.
Current-Fieldbus converter (IF302): its allows the interconnection of instruments with analogue output 4 to 20 mA to a Foundation Fieldbus network. It has three independent channels.
Fieldbus-current converter (FI302): it converts digital signals to analogue signals (4–20 mA). This device was connected to the frequency converter, taking information from the controller and changing the flow of coolant.
Temperature transmitters (TT302): these devices have two channels that transform analog signals from temperature detectors to Fieldbus protocol.
The Software SYSCON 7.0 was used to configure Fieldbus network devices. This software allowed to make changes, maintenance, and operations on line. The main functional blocks of SYSCON software—resource, transducer, display, analog input, analog output, and signal characterizer—have been properly configured to achieve the desired functionality of each device. The software INDUSOFT Web Studio 6.1 was used for system management and data acquisition.
2.5. Controller Design
2.5.1. Fuzzy PI Controller
This work was based on fuzzy logic concepts, wherein the knowledge accumulated by the process specialist was translated in a qualitative manner into a set of linguistic rules. To create a knowledge expert on the process, necessary for the development of fuzzy controller, experimental tests were performed to observe the behavior of the system.
In order to obtain a process reaction curve, a pseudosteady state condition was experimentally simulated by adding and withdrawing pineapple extract at the same rate (input and output) in the precipitation tank, without ethanol feeding. When the initial bulk temperature was close to the set point, ethanol began to be added at a fixed rate. At this exact moment, a disturbance of 30% of the maximum propylene-glycol pump capacity was applied. In this experiment, the pineapple extract was diluted with ethanol (1 : 1), in order to maintain the same precipitation conditions as during initial stages of the batch process. The system was monitored until the bulk temperature reached a new steady state.
The influence of the variation of the tank volume on the precipitation process was evaluated. Samples containing extract and ethanol in different proportions (from 1 : 1 to 1 : 3 v/v) were used in the pseudosteady state operation. Positive and negative disturbances were then applied (±30%) to the initial conditions of the speed of the coolant pump (manipulated variable). As in the fed-batch operation, a proportion of 1 : 1 corresponded to 300 mL, of 1 : 2 to 450 mL and of 1 : 3 to 600 mL. The process reaction curves obtained from this step contributed to the development of the Fuzzy controller.
The error ( ε ) and the change of error (Δ ε ) of bulk temperature, given by ( 1 ) and ( 2 ), respectively, were used as input linguistic variables. The speed variation of the coolant pump (Δ U ), given by ( 3 ), was used as the output linguistic variable, resulting in a fuzzy PI incremental controller type. The structure of this controller is shown in Figure 4 and it was implemented using the Fuzzy Toolbox of the MATLAB 7.0.1 computer package.
The linguistic variables were defined by
The rule base was composed of typical Mamdani-type rules for the inference engine in the fuzzy PI controller, employing the model proposed by Li and Gatland [ 25 ] and Li [ 26 ]. The original 49 rule proposals, from seven triangular membership functions (MF) for each variable, are presented in Table 1 . The triangular membership functions were used in the fuzzification procedure because of their wide application in the literature and ease of implementation. This procedure comprises the process of transforming crisp values into grades of membership for linguistic terms of fuzzy sets. The linguistic expressions for the magnitudes of the linguistic variables contain the following seven adjectives: negative large (NL), negative medium (NM), negative small (NS), zero (ZR), positive small (PS), positive medium (PM), and positive large (PL).
Using the rule base of Table 1 and the triangular membership functions for the three linguistic variables, several rules are activated simultaneously, showing different membership degrees, or areas, for the output. In order to find a unique crisp value for the fuzzy PI output (Δ U ), a defuzzification method was used based on the determination of the center of gravity of the combined areas: where, μ OUT ( u i ) is the membership function area resulting from fuzzy PI inference operation min (i.e., among the different areas obtained for Δ U calculation from simultaneous rules, the smallest one is chosen), u i is the center of area of each output membership function area, and u * is the center of area below the combined output membership function areas found from the intersection operator application. Indeed, u * gives the controller action to be implemented in the process (Δ U ).
2.5.2. Neurofuzzy Controller
The database obtained from the application of Mamdani-type fuzzy PI structure ( Section 2.5.1 ) was used for training, validation and tests of the neurofuzzy inference system (ANFIS). For the training set, the odd samples were used, for the validation the even samples, and for the tests, all the data. The ANFIS Toolbox of MATLAB software was used. Using the anfisedit command, the training and validation database were loaded, the Takagi-Sugeno fuzzy inference system (FIS) was created, and the training procedure was performed with 100 epochs. Following this training procedure, the ANFIS output was compared to the test outputs to check the controller performance.
The flowchart for designing the neurofuzzy controller is provided in Figure 5 . Using Simulink/MATLAB and OLE for process control protocol (OPC), the controller was implemented online. The Simulink diagram of the neurofuzzy control system is provided in Figure 6 .
2.5.3. Model Predictive Controller (MPC) Based on Artificial Neural Networks (ANN)
Firstly, the process was modeled using the Neural Network Toolbox of MATLAB. In the input layer, the operating variables, measured every sample time k , were used. In the hidden layer, an activation function was applied to twice the number of nodes of the input layer. Another activation function was applied to the output layer, which predicts the one-step-ahead controlled variable (PV k +1 ).
Open-loop runs were used to train the multilayered feedforward network, employing the Levenberg-Marquardt algorithm ( trainbr command in MATLAB). The open-loop data set was obtained by gathering a wide range of values of the input layer variables, including the whole network action domain.
The process dynamic was initially observed from the open-loop run with the manipulated variable at a fixed point. From there, the step disturbances in the manipulated variable, U , were planned so that the controlled variable behavior could be monitored from several runs and this database was employed to train the neural network. The database was split in two sets: 75% and 25% for training and tests, respectively. Furthermore, closed-loop runs with a fuzzy PI controller were used for the tests as well, since the neural model was expected to have good response in closed-loop situations with the implementation of the MPC optimizer for the process control. The neural model performance was assessed through dispersion plots of the testing runs (network output versus target vector), with a suitable result being proved by a slope coefficient of the linear fitting of the dispersion plots close to the unity and the linear coefficient around zero. The flow chart for designing this alternative control system is provided in Figure 7 .
The optimized weights and biases of the trained network were inserted in an electronic worksheet to reproduce the algebraic equations of a neural model. The calculated output generates a quadratic error (relative to the set point), which was defined as the objective function to be minimized by the solver ( 5 ). The solution was found from the quasi-Newton method of generalized reduced gradient, available in Excel software, by changing the manipulated variable value that should be implemented in the plant ( U ). The MPC diagram of the overall control system is provided in Figure 8 : subject to the following constraints: | 3. Results and Discussion
3.1. Determination of Set Point
To avoid denaturation of the protein, the temperature set point was determined from the literature and also from experimental testing of enzymatic activity to examine the quality of the enzyme. The results showed an activity of 0.8739 U/mL at 5°C, 0.7478 U/mL at 10°C and 0.7204 at 20°C ( Figure 9 ), which shows that at lower temperatures the effect of denaturation is attenuated. Those results were very close to those obtained by Cesar et al. [ 24 ], which do not recommend setting the temperature below 5°C. Thus, the set point selected for the precipitation temperature was 5°C.
3.2. Controller Design
3.2.1. Fuzzy PI Controller
According to the procedure described in Section 2.5.1 , the reaction curves were obtained and are shown in Figure 10 . The initial conditions of the tests were kept in 40% of rotation of the pump and 5 ° C for the temperature inside the tank.
By observing the reaction curves ( Figure 10 ), it was clear that the absolute value of the process gain ( K p ) increased as tank volume rose up, for positive disturbances (high speed of coolant pump). On the other hand, the absolute gain decreased as the volume increased, for negative disturbances (low speed). Thus, the temperature responses to positive and negative disturbances, of the same intensity, were asymmetrical. Table 2 summarizes the values of static gain from the Figure 10 .
Two main factors determine these transient and nonlinear features of the process.
Thermal exchange: at the beginning of the fed-batch process the heat exchange is deficient, as a consequence of the small heat transfer area. As the volume rises up, this area grows and heat exchange becomes more efficient. In the final condition of volume of 600 mL (1 : 3 curve), when applying a positive disturbance (high speed), there was a greater decrease in temperature inside the tank.
Heat of dissolution of the alcohol: an effect that increases the nonlinearity of the process is the heat of dissolution of ethanol in aqueous solution. The amount of heat released is greater at the beginning of the process and this effect decreases continuously during precipitation. In the condition of volume of 600 mL (1 : 3 curve), the increase in temperature caused by the dissolution of ethanol in the solution was negligible.
Due to the transient behavior and nonlinearities, discussed above, the precipitation system is characterized by having different sensitivities to the control actions, which emphasizes the limitation of the use of conventional controllers in this system. To minimize the consequences, the procedure of tuning the fuzzy PI controller involved changes in the universe of discourse, rule base, and support sets of membership functions, based on the reaction curves analysis.
The original rules of Mamdani, shown in Table 1 , were modified based on tests performed experimentally to provide better tuning of fuzzy PI controller. Adjustments are in bold in Table 3 and explained in the text above.
When the error was large positive (PL)—temperature was above the set point—and the change of error was NL, NM, or NS—the temperature was decreasing—there was not a need for great action in the coolant pump, as suggested by the original rules ( Table 1 ). The heat exchange was becoming more efficient due to the enlargement of the volume and also due to the lower impact of dissolution of ethanol on the solution. Thus, these three actions were modified to NS, smoothing the control action that reached upper limit without saturation.
When the temperature was slightly above the set point (error PS) and still increasing Δ ε = PL, there was a need for positive action on the pump speed. However, as the error was still small, it was observed that a medium positive action (PM) met the same goal as large action (PL), reducing temperature oscillation around set point.
When the temperature was slightly below the set point (error NS), and increasing rapidly (Δ ε = PL), it was observed that a small positive (PS) increase in pump speed would be enough to stop this temperature increase, since the gain of the process was large at the final conditions of batch. The rule medium positive (PM) originally suggested in Table 1 could provide a significant reduction in temperature and might cause undesirable oscillations. For the same reasons as outlined above, when error was negative medium (NM) and change of error was large positive (PL), the rule was changed to ZR, because maintaining the coolant flow rate showed to be sufficient to prevent any rise in temperature.
The triangular membership functions, and their corresponding labels of error ( ε ), change of error (Δ ε ), and the incremental control action (Δ U ), were presented in Figure 11 .
It may be noted in Figure 11 that the universe of discourse of error has a narrow range (from −1 to 1), since it was observed that the beginning of the process was the most critical stage, demanding a fast response of the manipulated variable, even for small errors ( ε ) in the controlled variable. Thus, even for small variations in the controlled variable, an intense response of the manipulated variable is produced.
Based on the analysis of the dynamic behavior of the process, a wide range (from −20 to 13) was set for the output variable (Δ U ). In terms of absolute magnitudes, the lower bound (−20) was greater than the upper bound (13), owing to the need to reduce the pump speed more quickly when the process reaches maximum error. In this region, lower coolant flow rates were able to handle the temperature rise caused by the release of heat of dissolution of the alcohol during the precipitation process.
The control surface related to the fuzzy PI controller is represented in Figure 12 , where nonlinearities incorporated in this control system are clearly observed.
3.2.2. Neurofuzzy Controller
The ANFIS controller design consisted in finding a Takagi-Sugeno (TS) fuzzy structure, which was able to describe an incremental fuzzy PI controller action, by choosing as linguistic variables ε , Δ ε , and Δ U (( 1 ), ( 2 ), and ( 3 )). The FIS file was made up of three triangular membership functions (ZR: approximately zero, PS: positive small, and PL: positive large) for the error ( ε ), seven triangular membership functions (NL: negative large, NM: negative medium, NS: negative small, ZR: approximately zero, PS: positive small, PM: positive medium, and PL: positive large) for the change of error (Δ ε ), and twenty-one linear functions for the pump speed variation (Δ U ). Another FIS file was created, for comparison of performances, with four triangular membership functions for the error ( ε ) (including PM: positive medium). The universes of discourses were determined according to subjective knowledge of the process and they were similar to that used in the fuzzy PI Mamdani controller ( Figure 11 ).
After the training procedure, the ANFIS controller outputs (FIS TS output) were tested with the samples obtained from the fuzzy PI Mamdani structure (FIS output), which allowed the observation of the high training performance. In Figure 13 , the FIS TS outputs are plotted versus the test samples (index). It could be noted that the points are coincident, even for the validation set that uses unseen data.
3.2.3. Model Predictive Controller
The operating variables for the ANN input layer, measured every four seconds (sample time), were chosen as follows: ethanol temperature ( T alc, k ), which exerts great influence in the precipitation temperature—the higher the ethanol temperature, the higher the overshoot obtained; coolant inlet ( T in, k ) and outlet ( T out, k ) temperatures, which provide information on the heat exchange in the tank jacket; coolant pump speed ( U k ), that is, the manipulated variable, so that its value will determine the controlled variable response; the liquid level ( L k ), represented by the liquid volume, provides the ANN with information on the run time, thus distinguishing equal input vectors that correspond to different output vectors; pump speed variation (Δ U k ) that indicates to the ANN which step disturbance in pump speed caused the given output; bulk temperature ( T bulk, k ), which works as a reference for the ANN prediction of the one-step-ahead bulk temperature ( T bulk, k +1 ), that was chosen as the output variable of the ANN. In the hidden layer a hyperbolic tangent activation function was applied to fourteen nodes.
Figure 14 shows the results for the open-loop and the closed-loop offline tests of the neural model. These tests, with unseen data, proved that the ANN successfully predicted the tank temperature, as it can be seen by the agreement between actual (target) and predicted values of temperature. Both linear fits presented slope coefficients close to the unity and linear coefficients around zero, approaching the diagonal line.
The optimized weights and biases of the trained network were then inserted in an electronic worksheet to reproduce the algebraic equations of the neural model. Simultaneously to the experiment, the neural model was able to predict on line the one-step-ahead bulk temperature. The results showed that the ANN was capable of learning the inherent nonlinearities and also successfully predicted the bulk temperature, thus being considered suitable for the MPC application.
3.3. Closed-loop Experimental Tests
The inlet ethanol flow rate at room temperature, the volume, and the heat transfer area variations were inherent disturbances, which drove the process away from the set point. All experimental tests were carried out under the same experimental conditions.
Figure 15 shows the temperature deviation under a well-tuned fuzzy PI Mamdani controller. Final settings were presented in Figure 11 and Table 3 .
Since the neurofuzzy tests showed the effectiveness of the training ( Figure 13 ), the ANFIS controllers were applied in the plant. The results obtained are shown in Figure 16 .
In Figure 16 , it can be noted a suitable performance from both ANFIS controllers implementation, with three and four membership functions. By adding one membership function, the overshoot and the rise time were shortened.
The neural model coupled with the Microsoft Excel solver was used as a MPC temperature controller, and the experimental results are shown in Figure 17 . The manipulated variable, to be changed by the solver, was subject to the following constraints ( 5 ): range of 0 ( U min ) to 100% ( U max ) of speed variation; manipulated variable action was smoothed by restricting its step value up to 35%. To prevent the controlled variable from leaving the training operating range, an additional constraint was added: the pump was turned off when the bulk temperature reached 4.9°C.
Figure 17 shows that the developed MPC controller was able to maintain the controlled variable around set point (5°C), with small rise time. As discussed in Section 3.2 , the overshoot observed in the first 200 seconds is due to the low liquid level in the tank, which caused the heat exchange area to be at a minimum. The dissolution heat produced during the ethanol addition can explain it as well.
Table 4 summarizes some important indexes in order to compare the performance of the implemented controllers. The best controller, in terms of overshoot and rise time, was the fuzzy PI Mamdani one.
The early stage of ethanol addition is critical. In order to keep the overshoot to a minimum, intense controller response is required, causing pump saturation, which was noted under all control strategies ( Table 4 ). However, the saturation effect was far less noticeable when the fuzzy PI controller was used, favoring conservation of the equipment.
Some other quantitative and qualitative analysis on the performance of the best implemented controller is summarized in Table 5 . It could be observed very low power consumption under fuzzy PI control.
The enzymatic activity analysis proved that fuzzy PI control outperformed the other implemented controllers because the final product (bromelain enzyme) showed high activity. From Figure 15 , it was observed the stable maintenance of bulk temperature, resulting in high quality of the product. | 3. Results and Discussion
3.1. Determination of Set Point
To avoid denaturation of the protein, the temperature set point was determined from the literature and also from experimental testing of enzymatic activity to examine the quality of the enzyme. The results showed an activity of 0.8739 U/mL at 5°C, 0.7478 U/mL at 10°C and 0.7204 at 20°C ( Figure 9 ), which shows that at lower temperatures the effect of denaturation is attenuated. Those results were very close to those obtained by Cesar et al. [ 24 ], which do not recommend setting the temperature below 5°C. Thus, the set point selected for the precipitation temperature was 5°C.
3.2. Controller Design
3.2.1. Fuzzy PI Controller
According to the procedure described in Section 2.5.1 , the reaction curves were obtained and are shown in Figure 10 . The initial conditions of the tests were kept in 40% of rotation of the pump and 5 ° C for the temperature inside the tank.
By observing the reaction curves ( Figure 10 ), it was clear that the absolute value of the process gain ( K p ) increased as tank volume rose up, for positive disturbances (high speed of coolant pump). On the other hand, the absolute gain decreased as the volume increased, for negative disturbances (low speed). Thus, the temperature responses to positive and negative disturbances, of the same intensity, were asymmetrical. Table 2 summarizes the values of static gain from the Figure 10 .
Two main factors determine these transient and nonlinear features of the process.
Thermal exchange: at the beginning of the fed-batch process the heat exchange is deficient, as a consequence of the small heat transfer area. As the volume rises up, this area grows and heat exchange becomes more efficient. In the final condition of volume of 600 mL (1 : 3 curve), when applying a positive disturbance (high speed), there was a greater decrease in temperature inside the tank.
Heat of dissolution of the alcohol: an effect that increases the nonlinearity of the process is the heat of dissolution of ethanol in aqueous solution. The amount of heat released is greater at the beginning of the process and this effect decreases continuously during precipitation. In the condition of volume of 600 mL (1 : 3 curve), the increase in temperature caused by the dissolution of ethanol in the solution was negligible.
Due to the transient behavior and nonlinearities, discussed above, the precipitation system is characterized by having different sensitivities to the control actions, which emphasizes the limitation of the use of conventional controllers in this system. To minimize the consequences, the procedure of tuning the fuzzy PI controller involved changes in the universe of discourse, rule base, and support sets of membership functions, based on the reaction curves analysis.
The original rules of Mamdani, shown in Table 1 , were modified based on tests performed experimentally to provide better tuning of fuzzy PI controller. Adjustments are in bold in Table 3 and explained in the text above.
When the error was large positive (PL)—temperature was above the set point—and the change of error was NL, NM, or NS—the temperature was decreasing—there was not a need for great action in the coolant pump, as suggested by the original rules ( Table 1 ). The heat exchange was becoming more efficient due to the enlargement of the volume and also due to the lower impact of dissolution of ethanol on the solution. Thus, these three actions were modified to NS, smoothing the control action that reached upper limit without saturation.
When the temperature was slightly above the set point (error PS) and still increasing Δ ε = PL, there was a need for positive action on the pump speed. However, as the error was still small, it was observed that a medium positive action (PM) met the same goal as large action (PL), reducing temperature oscillation around set point.
When the temperature was slightly below the set point (error NS), and increasing rapidly (Δ ε = PL), it was observed that a small positive (PS) increase in pump speed would be enough to stop this temperature increase, since the gain of the process was large at the final conditions of batch. The rule medium positive (PM) originally suggested in Table 1 could provide a significant reduction in temperature and might cause undesirable oscillations. For the same reasons as outlined above, when error was negative medium (NM) and change of error was large positive (PL), the rule was changed to ZR, because maintaining the coolant flow rate showed to be sufficient to prevent any rise in temperature.
The triangular membership functions, and their corresponding labels of error ( ε ), change of error (Δ ε ), and the incremental control action (Δ U ), were presented in Figure 11 .
It may be noted in Figure 11 that the universe of discourse of error has a narrow range (from −1 to 1), since it was observed that the beginning of the process was the most critical stage, demanding a fast response of the manipulated variable, even for small errors ( ε ) in the controlled variable. Thus, even for small variations in the controlled variable, an intense response of the manipulated variable is produced.
Based on the analysis of the dynamic behavior of the process, a wide range (from −20 to 13) was set for the output variable (Δ U ). In terms of absolute magnitudes, the lower bound (−20) was greater than the upper bound (13), owing to the need to reduce the pump speed more quickly when the process reaches maximum error. In this region, lower coolant flow rates were able to handle the temperature rise caused by the release of heat of dissolution of the alcohol during the precipitation process.
The control surface related to the fuzzy PI controller is represented in Figure 12 , where nonlinearities incorporated in this control system are clearly observed.
3.2.2. Neurofuzzy Controller
The ANFIS controller design consisted in finding a Takagi-Sugeno (TS) fuzzy structure, which was able to describe an incremental fuzzy PI controller action, by choosing as linguistic variables ε , Δ ε , and Δ U (( 1 ), ( 2 ), and ( 3 )). The FIS file was made up of three triangular membership functions (ZR: approximately zero, PS: positive small, and PL: positive large) for the error ( ε ), seven triangular membership functions (NL: negative large, NM: negative medium, NS: negative small, ZR: approximately zero, PS: positive small, PM: positive medium, and PL: positive large) for the change of error (Δ ε ), and twenty-one linear functions for the pump speed variation (Δ U ). Another FIS file was created, for comparison of performances, with four triangular membership functions for the error ( ε ) (including PM: positive medium). The universes of discourses were determined according to subjective knowledge of the process and they were similar to that used in the fuzzy PI Mamdani controller ( Figure 11 ).
After the training procedure, the ANFIS controller outputs (FIS TS output) were tested with the samples obtained from the fuzzy PI Mamdani structure (FIS output), which allowed the observation of the high training performance. In Figure 13 , the FIS TS outputs are plotted versus the test samples (index). It could be noted that the points are coincident, even for the validation set that uses unseen data.
3.2.3. Model Predictive Controller
The operating variables for the ANN input layer, measured every four seconds (sample time), were chosen as follows: ethanol temperature ( T alc, k ), which exerts great influence in the precipitation temperature—the higher the ethanol temperature, the higher the overshoot obtained; coolant inlet ( T in, k ) and outlet ( T out, k ) temperatures, which provide information on the heat exchange in the tank jacket; coolant pump speed ( U k ), that is, the manipulated variable, so that its value will determine the controlled variable response; the liquid level ( L k ), represented by the liquid volume, provides the ANN with information on the run time, thus distinguishing equal input vectors that correspond to different output vectors; pump speed variation (Δ U k ) that indicates to the ANN which step disturbance in pump speed caused the given output; bulk temperature ( T bulk, k ), which works as a reference for the ANN prediction of the one-step-ahead bulk temperature ( T bulk, k +1 ), that was chosen as the output variable of the ANN. In the hidden layer a hyperbolic tangent activation function was applied to fourteen nodes.
Figure 14 shows the results for the open-loop and the closed-loop offline tests of the neural model. These tests, with unseen data, proved that the ANN successfully predicted the tank temperature, as it can be seen by the agreement between actual (target) and predicted values of temperature. Both linear fits presented slope coefficients close to the unity and linear coefficients around zero, approaching the diagonal line.
The optimized weights and biases of the trained network were then inserted in an electronic worksheet to reproduce the algebraic equations of the neural model. Simultaneously to the experiment, the neural model was able to predict on line the one-step-ahead bulk temperature. The results showed that the ANN was capable of learning the inherent nonlinearities and also successfully predicted the bulk temperature, thus being considered suitable for the MPC application.
3.3. Closed-loop Experimental Tests
The inlet ethanol flow rate at room temperature, the volume, and the heat transfer area variations were inherent disturbances, which drove the process away from the set point. All experimental tests were carried out under the same experimental conditions.
Figure 15 shows the temperature deviation under a well-tuned fuzzy PI Mamdani controller. Final settings were presented in Figure 11 and Table 3 .
Since the neurofuzzy tests showed the effectiveness of the training ( Figure 13 ), the ANFIS controllers were applied in the plant. The results obtained are shown in Figure 16 .
In Figure 16 , it can be noted a suitable performance from both ANFIS controllers implementation, with three and four membership functions. By adding one membership function, the overshoot and the rise time were shortened.
The neural model coupled with the Microsoft Excel solver was used as a MPC temperature controller, and the experimental results are shown in Figure 17 . The manipulated variable, to be changed by the solver, was subject to the following constraints ( 5 ): range of 0 ( U min ) to 100% ( U max ) of speed variation; manipulated variable action was smoothed by restricting its step value up to 35%. To prevent the controlled variable from leaving the training operating range, an additional constraint was added: the pump was turned off when the bulk temperature reached 4.9°C.
Figure 17 shows that the developed MPC controller was able to maintain the controlled variable around set point (5°C), with small rise time. As discussed in Section 3.2 , the overshoot observed in the first 200 seconds is due to the low liquid level in the tank, which caused the heat exchange area to be at a minimum. The dissolution heat produced during the ethanol addition can explain it as well.
Table 4 summarizes some important indexes in order to compare the performance of the implemented controllers. The best controller, in terms of overshoot and rise time, was the fuzzy PI Mamdani one.
The early stage of ethanol addition is critical. In order to keep the overshoot to a minimum, intense controller response is required, causing pump saturation, which was noted under all control strategies ( Table 4 ). However, the saturation effect was far less noticeable when the fuzzy PI controller was used, favoring conservation of the equipment.
Some other quantitative and qualitative analysis on the performance of the best implemented controller is summarized in Table 5 . It could be observed very low power consumption under fuzzy PI control.
The enzymatic activity analysis proved that fuzzy PI control outperformed the other implemented controllers because the final product (bromelain enzyme) showed high activity. From Figure 15 , it was observed the stable maintenance of bulk temperature, resulting in high quality of the product. | 4. Conclusions
We have described the design and experimental testing of intelligent control algorithms for temperature control during the precipitation of bromelain with solvent. The temperature was monitored and controlled in order to minimize bromelain denaturation during the precipitation process.
Tuning the controllers proved to be a difficult task in this fed-batch nonlinear process. Fuzzy tuning was hindered by the simultaneous multiple adjustments. Nevertheless, the procedure based on the analysis of the process reaction curves proved to be an attractive strategy to provide a suitable nonlinear controller design for transient processes.
From the results, it was concluded that all proposed controllers were suitable for the precipitation tank temperature control. The fuzzy PI Mamdani controller showed better global performance criteria: small ITAE, short response time and pump saturation time, and higher enzyme activity in the product. This fuzzy PI controller also presented lower power consumption, providing a significant reduction of operating costs. This high performance of the fuzzy PI controller can be attributed to its ability to adapt to the nonlinearities.
The real-time data exchange between the softwares MATLAB and Indusoft and also between the softwares and the field devices showed to be reliable and fast through the implemented Fieldbus architecture.
The developed controllers gathered the benefits of the artificial intelligence in affording nonlinearities, making this methodology a promising new way to face complex process control problems, without spending efforts unnecessarily in rigorous mathematical modeling. | Academic Editor: Leszek Kleczkowski
This paper focuses on the development of intelligent controllers for use in a process of enzyme recovery from pineapple rind. The proteolytic enzyme bromelain (EC 3.4.22.4) is precipitated with alcohol at low temperature in a fed-batch jacketed tank. Temperature control is crucial to avoid irreversible protein denaturation. Fuzzy or neural controllers offer a way of implementing solutions that cover dynamic and nonlinear processes. The design methodology and a comparative study on the performance of fuzzy-PI, neurofuzzy, and neural network intelligent controllers are presented. To tune the fuzzy PI Mamdani controller, various universes of discourse, rule bases, and membership function support sets were tested. A neurofuzzy inference system (ANFIS), based on Takagi-Sugeno rules, and a model predictive controller, based on neural modeling, were developed and tested as well. Using a Fieldbus network architecture, a coolant variable speed pump was driven by the controllers. The experimental results show the effectiveness of fuzzy controllers in comparison to the neural predictive control. The fuzzy PI controller exhibited a reduced error parameter (ITAE), lower power consumption, and better recovery of enzyme activity. | Acknowledgment
The authors are grateful to CAPES, CNPq (Grant no. 477195/2007-5) and FAPESP (Grant no. 2008/10710-7) for financially supporting this study.
Nomenclature
Static process gain
Bias
Temperature error
Change of error
Change in control action
MV: control action
PV: plant output
μ i : Membership function (activation level)
Integral of the time-weighted absolute error = ∫ 0 ∞ t || e ( t )|| dt
Set point
Defuzzified action value
Minimum operator
OLE for process control
Index of discrete points
Negative membership function
Positive membership function. | CC BY | no | 2022-01-13 02:52:24 | Enzyme Res. 2010 Dec 27; 2010:250843 | oa_package/40/bc/PMC3014679.tar.gz |
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PMC3014680 | 21234107 | 1. Introduction
Research shows that depression is relatively rare in children but becomes more prevalent in adolescence. At the same time, the sex ratio also changes considerably; whereas there is little evidence of gender differences in children, depression during adolescence is significantly more common among girls than boys [ 1 , 2 ]. Another problem behavior that develops approximately at the same time is deliberate self-harm (henceforth referred to as self-harm), here defined as self-inflicted damage to the surface of one's own body. (It should be noted that the definition used in this study does not include behaviors like taking an overdose or self-poisoning. In this way, it is similar to the proposed diagnosis of “nonsuicidal self-injury” in the DSM-V [ 3 ], although our definition differs from this notion by not explicitly excluding suicidal intentions.). The mean age of onset of this kind of self-harm is reported to be around 12–15 years [ 4 , 5 ], and self-harm is quite frequently reported among young adolescents [ 6 – 12 ]. This raises the question how depressive symptoms and self-harm are associated, both concurrently and prospectively.
It is commonly assumed that self-harm develops as a symptom of high emotional distress (anxiety, depression, self-hate, etc.), as a way of expressing or regulating this distress [ 13 – 16 ]. This would mean that distressful emotions (e.g., depressive feelings) represent a risk factor for the development of self-harm. A risk factor is generally defined [ 17 ] as a measurable variable that must precede a negative outcome and be associated with a higher risk for this outcome, which means that risk factors can only be identified by means of prospective studies. So far, there are only a few prospective studies of emotional risk factors for self-harm. In one study [ 18 ], self-reported depressive symptoms at the age of 8 were found to predict acts of self-harm 10 years later in a community-based sample of 2,348 boys. In another study [ 19 ], predictors at the age of 12 for acts of self-harm at the age of 15 were studied; the results showed that self-reported internalizing problems and somatic complaints, as well as parent-reported externalizing problems and aggressiveness, independently predicted self-reported acts of self-harm 3 years later.
In a recent study, however, we [ 20 ] failed to find support for emotional symptoms among 13–15-year-old adolescents as a risk factor for increased self-harm. In this study, emotional symptoms were measured by Goodman's [ 21 ] Strengths and Difficulties Questionnaire (SDQ). One problem with the SDQ, however, is that its 5-item subscale for measuring emotional symptoms contains only one depression-related item (whereas it contains three items related to nervousness, fear and worry, and one somatic item). It is likely that depressive feelings of guilt, shame, worthlessness, self-disgust, self-contempt, or self-hatred are more of a risk factor for the development of self-harm than are feelings of fear, worry, or nervousness. The SDQ emotional symptoms subscale may therefore be less optimal for detecting any existing prospective association between emotional problems and self-harm. It is quite possible that, although emotional symptoms in general do not serve as a risk factor for the development of self-harm, a subset of emotional symptoms do so. One purpose of the present study was to test the hypothesis that depressive symptoms represent such a subset of emotional symptoms.
Another purpose was to analyze subcategories of depressive symptoms and investigate if certain subsets of depressive symptoms, or patterns of such symptoms, are especially associated with self-harm. For example, because self-harm implies an attack towards one's own body, it may be assumed to be more associated with feelings of self-hatred, self-contempt, self-disgust, and so forth than with feelings of sadness, lack of energy, or difficulties in concentration.
Finally, we also wanted to test the possibility that self-harm may serve as a risk factor for increased depressive symptoms. In a previous study [ 20 ], we found evidence for a bidirectional relationship between self-harm and psychological difficulties in general; that is, overall psychological difficulties predicted an increase in self-harm one year later and self-harm predicted increased psychological difficulties one year later. Our hypothesis was that such a bidirectional relationship would apply also to the association over time between depressive symptoms and self-harm. There are several reasons to expect that self-harm may be a risk factor for increased depression. First, although self-harm may have an emotion-regulating function and therefore lead to decreased emotional distress as an immediate consequence [ 14 ], it may have the opposite effect in a longer time perspective by setting the stage for depression-related processes like rumination, shame, guilt, and regret. Second, when it comes to the attention of others (parents, friends, etc.) that an adolescent deliberately harms herself, these others may respond strongly negatively, thereby causing a deterioration and even disruption of interpersonal relationships in a way that may lead to depressive symptoms.
If there is a bidirectional prospective relationship between depression and self-harm, this would mean that depressive symptoms and self-harm may enter into a self-generating “vicious cycle” where increases in the one variable lead to increases in the other and vice versa. This can be described in terms of a dynamic system, where internal feedback processes lead to the emergence and stabilization of pathological patterns that include both depression and self-harm. Because evidence suggests that there may be gender differences in both depression and self-harm, we studied this question separately in girls and boys.
To summarize, the present study was carried out (1) to study if there is a bidirectional relationship between depressive symptoms and self-harm in young adolescents, in the sense that depressive symptoms serve as a risk factor for self-harm and vice versa; (2) to investigate different patterns of depressive symptoms, how frequent they are among young adolescents, and how they are related to self-harm. Methodologically, we combined two different approaches. First, we used a variable-oriented approach to study both concurrent and prospective associations between symptoms of depression and self-harm, with the hypothesis that we would find evidence of a bidirectional relation between depression and self-harm. Second, we used a person-oriented approach [ 22 ] in accordance with an advanced analytical procedure developed by Bergman [ 23 ] to identify different subgroups of adolescents with different patterns of depressive symptoms, and then compared these subgroups to see if they differed on self-harm. We hypothesized that the analysis would identify at least one clear depression-related cluster and that depression-related clusters would contain an over-representation of girls and be associated with significantly more self-harm than the other clusters. We also expected that adolescents characterized by some categories of depressive symptoms (e.g., negative self-related feelings) would show more self-harm than others (e.g., those characterized by sadness and lack of energy). | 2. Materials and Methods
2.1. Participants
The participants were a community sample from a municipality in the south of Sweden which is fairly representative for the rest of Sweden in terms of the proportions of children living with both of their parents, and their ethnic backgrounds, but slightly more rural than Sweden as a whole, and with a slightly lower income level and educational level [ 24 ]. At Time 1, there were 532 students in Grade 7 (mean age 13.7 years) and 520 students in Grade 8 (mean age 14.7 years) in the schools of this municipality (excluding three special schools with place for around 25 individuals with severe school difficulties); 992 of these 1052 students (94%) participated. One year later, at Time 2, 984 students in Grade 8 and 9 participated. Ten individuals were excluded as multivariate outliers with stereotypic response patterns. In total, there were available longitudinal data for 879 participants (450 girls and 429 boys), who represented 83.6% of all students that were available for inclusion at Time 1.
2.2. Instruments
The participants filled out an 11-page questionnaire, which was tailor made for young adolescents and tested in a pilot study with around 200 participants [ 6 , 24 ]. The questionnaire included a number of self-assessment instruments. The present study used data from four of these instruments, plus some separate questions. As the measure of deliberate self-harm, we used a short version of Gratz' [ 25 ] Deliberate Self-Harm Inventory. To measure depressive symptoms, a Depression Index was constructed on the basis of depression-related items from three separate instruments: the Strengths and Difficulties Questionnaire (SDQ [ 21 ]), a modified version of the Emotional Tone Index (ETI [ 26 , 27 ]), and the Appearance subscale of the Body Esteem Scale for Adolescents and Adults, (BEAA [ 28 ]), with the addition of separate questionnaire items concerning sleep, alertness, self-rated health, and views on the personal future.
Deliberate Self-Harm Inventory: 9-Item Version Revised (DSHI-9r) This is a shortened and modified version of the Deliberate Self-Harm Inventory which was originally constructed and validated by Gratz [ 25 ] and then translated into Swedish and adapted to adolescents [ 6 , 8 ]. In this 9-item version of the DSHI, respondents are asked if they have deliberately engaged in any of nine different kinds of direct physical self-harm (cutting wrists, arms, or body areas; minor cutting causing bleeding; carving words, pictures, and so forth into the skin; burning oneself with cigarette, lighter, or match; severe scratching, causing bleeding; sticking sharp objects into the skin; biting oneself so that the skin is broken; punching oneself or banging one's head, thereby causing a bruise; preventing wounds from healing) during the past 6 months. Respondents are instructed to rate the number of times they have conducted these behaviours on a scale from 0 to 6, where 0 is “never” and 6 is defined as “more than five times”. A total score (from 0 to 54) on the DSHI-9r can thus be calculated by summarizing the number of times a person reports having engaged in these self-harming behaviours. The internal consistency of the DSHI-9r in the present study was α = .90. All nine items of the DSHI-9r correlated with mental health problems as measured by SDQ Total Difficulties ( r s ranging from .23 to .33).
Depression Index Depression-relevant items were selected from the 11-page questionnaire, according to their correspondence with items from standard measures of depression and the DSM-IV criteria for major depression [ 29 ]. Because the items came from different instruments with different response formats, the scores on each item were transformed to z -scores. Items referring to positive feelings were reverse scored. The items were then subjected to a principal components analysis with varimax rotation, which identified eight components with eigenvalues >1; on the basis of converging results from both a scree plot and parallel analysis, however, the number of components was reduced to 6. On the basis of these, six subscales were constructed (see the appendix): Dysphoric relations to parents (10 items of which 4 referred to positive feelings, alpha = .85), Negative self-image (6 items of which 3 involved positive statements about the self, alpha = .85), Dysphoric relations to friends (6 items which all referred to positive feelings, alpha = .73), Fatigue/somatic complaints (5 items, alpha = .70), Sadness/loneliness (4 items, alpha = .67), and Difficulties in concentration (4 items, alpha = .65). Test-retest correlations between Time 1 and Time 2 were r = .71 for the entire Depression index, r = .60 for Dysphoric relations to parents, r = .68 for Negative self-image, r = .51 for Dysphoric relations to friends, r = .61 for Fatigue/somatic complaints, r = .48 for Sadness/loneliness, and r = .61 for Difficulties in concentration.
2.3. Procedure
This research was conducted after approval by the Regional Ethics Committee at Lund University. Contact was established with school managements via head-masters who gave consent to their schools' participation in the study. Information about the form and purpose of the study was sent by mail to the parents, who were asked to contact the school teachers or the researchers if they did not want their child to participate. Parents as well as children were informed that this was a research project on the situation of young people today, in terms of how they feel, and how they perceive themselves, their feelings, relations, and life situation. The participants were also informed that their participation was voluntary, that they were free to withdraw at any time and without having to give a reason, that their answers were treated confidentially, and that no school personnel would have access to their answers. Contacts were established with representatives from school healthcare in the municipality to facilitate procedures if serious psychological problems or other circumstances related to participants would warrant an intervention. The procedure was considered ethically appropriate on the basis of previous research [ 30 , 31 ].
The 11-page questionnaire was filled out in school, as part of a separate lecture hour, and was administered by research assistants from Lund University. A teacher was present, but did not participate in the data collection. In order to guarantee the students privacy, their school desks were separated as much as possible. The students were instructed to answer all questions as best they could, but not to think too much about any answer. They were instructed not to write their names anywhere on the questionnaire. After the completion of the questionnaire, it was sealed in an envelope by the student.
2.4. Statistical Analysis
The distribution of total DSHI-9r scores and the scores on two of the depression subscales (Dysphoric relations to parents and Sadness/loneliness) were highly positively skewed and leptokurtic at both Time 1 and Time 2; logarithmic transformations were therefore conducted on these three indexes, resulting in acceptable normal distributions.
To test the hypothesis that depressive symptoms would be a risk factor for self-harm, we used both logistic regression (with incidence of new cases of repeated self-harm, at Time 2 as the dependent variable) and hierarchical linear regression (with DSH-9r scores at Time 2 as the dependent variable). To test the hypothesis that self-harm would be a risk factor for depressive symptoms at Time 2, we only used hierarchical linear regression.
Cluster analysis was used to group all participants on the basis of their different profiles of scores on the six depression scales, according to the LICUR procedure [ 23 ]. This was done in four steps. First, multivariate outliers were identified by means of the residue procedure in the statistical package for pattern-oriented analyses SLEIPNER 2.1 [ 32 ] and removed from further analysis. Second, Ward's hierarchical clustering method was applied. Four criteria presented by Bergman [ 23 ] were used to decide on the optimal cluster solution: (a) theoretical meaningfulness of the cluster solution; (b) if there is a distinct drop in the explained error sum of squares (EESS) when a cluster solution is extracted, this suggests that two insufficiently similar clusters were merged to a nonoptimal cluster solution; (c) the number of clusters should not be more than 15 and should not be expected to be less than five; (d) the size of the EESS for the chosen cluster solution should preferably not be less than 67% and at the very least exceed 50%. In addition, the homogeneity coefficient of each cluster should preferably be <1. Third, a data simulation was undertaken to verify that the explained ESS was higher than what could be expected on a random data set with the same general properties as the data set used in the real analysis. Fourth, a nonhierarchical relocation procedure was carried out in order to improve the homogeneity of the clusters and to increase the variance explained by the cluster solution. | 3. Results
As expected, girls showed more evidence of depression than boys; this did not, however, apply to all depression subscales. Gender comparisons by independent samples t -test showed that girls scored higher than boys on the total Depression index and on the subscales Negative self-image, Sadness/loneliness, and Fatigue/somatic complaints (all P < .001). On the other hand, the boys scored higher on Dysphoric relations to friends ( P < .001), and there were no significant differences ( P > .05) on Dysphoric relations to parents or on Difficulties in concentration.
More girls than boys reported self-harm. At Time 1, 45.1% of the girls and 37.9% of the boys ( χ 2 = 5.1, P < .01) reported that they had harmed themselves deliberately at least once during the past 6 months. Repeated self-harm (defined as at least 5 instances of self-reported self-harm) was reported by 20.7% of the girls and by 15.9% of the boys. The stability of DSHI-9r scores from Time 1 to Time 2 was higher for girls ( r = .57) than for boys ( r = .35).
Correlational analyses showed that total depression scores and self-harm were moderately to highly associated both in girls ( r = .58 at Time 1 and r = .55 at Time 2) and boys ( r = .39 at Time 1 and r = .46 at Time 2).
3.1. Prediction of New Cases of Repeated Self-Harm
Depressive symptoms at Time 1 predicted the incidence of new cases of repeated self-harm at Time 2 in both girls and boys. Incidence here refers to participants who reported repeated self-harm (defined as at least five instances of self-harm) at Time 2 but had reported no instance of self-harm at Time 1. The incidence rate of repeated self-harm was 10.4% (26 of 251) among the girls and 8% (21 of 254) among the boys. To test the hypothesis that depressive symptoms would predict the incidence of new cases of repeated self-harm, logistic regressions were carried out separately for girls and boys. As seen in Table 1 , the model was significant in both genders ( χ 2 (1) = 9.13, P = .003 in girls, and χ 2 (1) = 5.56, P = .018 in boys), explaining 7.3% of the variance in girls ( Nagelkerke R 2 = 0.073) and 4.8% in boys ( Nagelkerke R 2 = 0.048).
3.2. Bidirectional Associations between Self-Harm and Depression
The hypothesis of a bidirectional relationship over time between self-harm and depressive symptoms was confirmed in girls but not in boys. As seen in Table 2 , hierarchical regression analyses among girls showed that, when controlling for self-harm at Time 1, depressive symptoms at Time 1 predicted self-harm at Time 2, and, conversely, when controlling for depressive symptoms at Time 1, self-harm at Time 1 predicted depressive symptoms at Time 2. In boys, however, there was only evidence for a unidirectional relationship; although depressive symptoms predicted self-harm, the reverse was not the case.
3.3. Cluster Analysis
In total, 977 participants had full data on the depression index at Time 1 and were entered into the cluster analysis. Of these, 24 adolescents were identified as multivariate outliers and excluded by the residue procedure. Second, the application of Ward's hierarchical clustering method, together with Bergman's [ 23 ] criteria, resulted in the choice of a ten-cluster solution, explaining 58.1% of the total error sum of squares (ESS). Third, a data simulation showed that the explained ESS of the cluster solution was significantly higher than expected by chance ( P < .0001). Fourth, a non-hierarchical relocation procedure to improve the homogeneity of the clusters resulted in a ten-cluster solution that was found to explain 62.5% of the variance. The homogeneity coefficients of the clusters also were quite good, with most clusters having a homogeneity coefficient of <.40 and no cluster having a higher homogeneity coefficient than .82 (low coefficients mean high homogeneity).
Figures 1 – 4 show the profiles of z -scores for each cluster. As seen in Figure 1 , the analysis identified two depression-related clusters: one small cluster (called the Depression cluster, n = 27), which was characterized by high scores on all depression indexes ( z > 1 on all scales except on Difficulties in concentration), and one larger cluster (referred to as Mild/moderate depression, n = 81), which showed high scores ( z > 1) on Sadness/loneliness and moderately high scores on all other subscales. As seen in Table 3 , the Depression cluster showed a highly elevated depression score ( z = 1.37), whereas the Mild/moderate depression cluster showed a moderately elevated score ( z = 0.68).
In addition, there were a number of clusters that showed elevated scores on some depression indexes, without scoring high on total depression. As seen in Figure 2 , there was a three-problem cluster that combined high scores on Fatigue/somatic problems, Dysphoric relations to parents, and Difficulties in concentration (called the “Fatigue and problems with parents” cluster, n = 53) and a two-problem cluster that combined high scores on Fatigue/somatic problems and Dysphoric relations to friends (called the “Fatigue and problems with friends” cluster, n = 65). As seen in Table 3 , however, none of these clusters scored especially high on the total depression score.
As seen in Figure 3 , three “one-problem clusters” were also identified: one with a highly negative self-image but normal scores on all other indexes (“Negative self-cluster,” n = 71), another with high scores only on Sadness/loneliness (“Sadness/loneliness cluster,” n = 90), and a third with high scores on Difficulties in concentration but relatively normal scores on the other indexes (“Concentration difficulties cluster,” n = 97).
Finally, the analysis identified three relatively large “healthy clusters” which together comprised 49.2% of the total sample: one cluster with low scores ( z < −0.50) on all depression subscales (“Happy and healthy,” n = 189), another with relatively low scores on all indexes except Negative self-image where they scored close to average (the “No problems cluster,” n = 154), and a third with close to average scores on the indexes (the “Average problems cluster,” n = 126) (see Figure 4 ).
A one-way ANOVA with the ten-cluster categorization as the independent variable and the total depression index score as the dependent variable showed that the clusters differed on depression, F (9,943) = 641.1, P < .0001. Tukey post-hoc tests showed a categorization of the clusters into eight subsets, which differed significantly in the following order: Depression cluster > Mild/moderate depression cluster > “Fatigue and problems with parents” cluster > Negative self-cluster, “Fatigue and problems with friends” cluster > Sadness/loneliness cluster, and Average problems > Concentration difficulties cluster > No problems > Happy and healthy.
3.4. Gender Comparison between the Clusters
As expected, the girls were overrepresented in the depression-related clusters. The gender distribution in the ten clusters is shown in Table 3 . To test the hypothesis that girls would be overrepresented in the depression-related clusters, the observed frequency was compared with the frequency that should be expected by chance alone, and one-tailed probabilities were computed according to the fixed-margins model using EXACON [ 33 ]. The results showed that the girls were overrepresented in both the Depression cluster (observed frequency 20, expected frequency 13.5, χ 2 = 3.13, P = .009) and in the Mild/moderate depression cluster (observed frequency 53, expected frequency 40.5, χ 2 = 3.86, P = .003). Explorative comparisons of the gender distributions in the eight remaining clusters, with the Bonferroni correction ( P < .05/8 = .006), showed only one significant effect: boys were overrepresented in the Average problems cluster (observed frequency 89, expected frequency 63, χ 2 = 10.73, P < .0001, two tailed).
3.5. Comparison between the Clusters on Self-Harm
As expected, the depression-related clusters were associated with high levels of self-harm, but this was also the case for the “Fatigue and problems with parents” cluster and the Negative self-cluster. Table 3 shows the mean scores on the DSHI-9r for all the ten clusters. A one-way ANOVA with the ten-cluster categorization as the independent variable and the DSHI-9r as dependent variable showed that the clusters differed on self-harm, F (9,936) = 38.9, P < .0001. Tukey post-hoc tests showed that the Depression cluster scored higher than all the other clusters. Further, the “Fatigue and problems with parents” cluster scored higher than six of the remaining clusters, the Mild/moderate depression cluster scored higher than five of the remaining clusters, and the Negative self-cluster scored higher than the three clusters with lowest DSHI-9r scores.
To study the stability of these results, a similar one-way ANOVA was carried out with the DSHI-9r at Time 2 as the dependent variable. As seen in Table 3 , the results were highly similar, showing that the Time 1 clusters differed significantly also on Time 2 self-harm, F (9,857) = 14.6, P < .0001. Again, Tukey post-hoc tests showed that the Depression cluster scored higher than all the other clusters and that the “Fatigue and problems with parents” cluster scored higher than six of the remaining clusters. The Mild/moderate depression cluster and the Negative self-cluster also scored higher than the five clusters with the lowest self-reported frequencies of self-harm. | 4. Discussion
There are two main findings of the present study. First, there was support for the hypothesis of a bidirectional relationship between depressive symptoms and self-harm in the girls but not in the boys (where there was only support for a unidirectional relationship, depressive symptoms being a predictor of increased self-harm one year later). Second, and in line with expectations, among the ten profiles of depressive symptoms identified in the cluster analysis there were two depression-related clusters with an overrepresentation of girls and significantly increased levels of self-harm and also a single-problem cluster characterized by negative self-image and significantly higher levels of self-harm.
Importantly, the demonstration of a bidirectional prospective relationship between depression and self-harm in girls means that higher levels of one of these variables at a given time are associated with increasing levels in the other variable over time . Whereas the concurrent analyses showed that levels of depression are strongly associated with levels of self-harm, the prospective analyses showed that higher levels of depression predispose to increased levels of self-harm within the next year and conversely that higher levels of self-harm predispose to increased depression within the next year. Although the effects were not strong, they are of a more dynamic order than the concurrent associations and suggest the possibility that depression and self-harm may enter into a self-generating “vicious cycle” where increases in the one variable lead to increases in the other, and vice versa. This can be described in terms of a dynamic system, where internal feedback processes lead to the emergence and stabilization of pathological patterns of depressive symptoms and self-harm. The absence of evidence for a bidirectional relationship in boys suggests that different developmental dynamics may be involved in girls and boys—or, in other words, that self-harm has a different meaning and function in girls than in boys.
In a previous study [ 20 ], we found no support for the hypothesis of emotional problems being a risk factor for the development of self-harm. In that study, emotional problems were measured by the Emotional symptoms subscale of the Strengths and Difficulties Questionnaire (SDQ). The present results corroborate the assumption that the SDQ Emotional symptoms scale is not sufficiently sensitive to capture the role of depressive symptoms for the development of self-harm, as it contains only one depression-related item.
There are several interesting results from the cluster analytic part of the present study. First, two depression-related clusters were identified, suggesting that 2-3% of the adolescents (27 of 953) in this community sample suffered from depression and that an additional 8-9% (81 of 953) suffered from something that is reminiscent of at least “minor depression.” Both of these clusters were characterized by an overrepresentation of girls (74% and 65%, resp.) and were also characterized by significantly higher frequencies of self-harm than most other clusters. The individuals in the depression cluster showed particularly high frequencies of self-harm.
Second, the analysis identified three one-problem clusters of adolescents (the Negative self-cluster, the Sadness/loneliness cluster, and the Concentration difficulties cluster), characterized by high scores on one of the subscales without any elevated scores on the total depression index. In line with expectations, the Negative self-cluster was characterized by significantly increased levels of self-harm, whereas the other two were not. This is consistent with the hypothesis that self-harm is more associated with negative self-related emotions than with feelings of sadness. This finding also suggests the need for more specific theoretical models concerning the emotion-regulating role of self-harm. It is well established that a primary function of deliberate self-harm is the regulation of negative emotions [ 14 ]. But are all kinds of negative feelings (fear, sadness, shame, guilt, self-hate, etc.) equivalent in this respect, or are some emotional experiences more likely than others to be handled by self-harm? Because deliberate self-harm involves a direct physical attack on one's own body, and attack is more associated with anger or aggression than with fear or sadness, it may be hypothesized that self-harm is used primarily to regulate feelings of self-directed anger or aggression. It may be a task for future research to develop more specific theoretical models in this area and to develop instruments for testing these models. It should be noted that the items in the negative self-image subscale of the Depression Index in the present study were not constructed for this purpose and actually deal more with the absence of positive self-feelings than with the presence of negative self-feelings (see the appendix). The fact that this relatively weak index of a negative self-image was still able to produce results in line with the hypothesis suggests that this line of research may be worth pursuing.
A third finding from the cluster analysis is that high scores on fatigue/somatic complaints combined with dysphoric relations to parents and dysphoric relations to friends, respectively, into two separate problem clusters. For both of these, high scores on one of the dysphoric relations factors were combined with completely normal scores on the other. More specifically, the adolescents in the “Fatigue and problems with parents cluster” scored even slightly below average ( z < 0) on dysphoric relations to friends. And, conversely, the adolescents in the “Fatigue and problems with friends cluster” scored slightly below average ( z < 0) on dysphoric relations to parents. Whereas the “Fatigue and problems with parents” cluster had elevated scores on self-harm, this was not the case for the “Fatigue and problems with friends” cluster. This suggests that self-harm is more associated with negative relations to parents than with negative relations to friends.
Fourth, the results from the cluster analysis gain further strength by their stability; the clusters defined at Time 1 showed highly similar results on self-harm also at Time 2. Still, it may be asked if the clusters with more circumscribed problems represent stable profiles or if the individuals in these clusters constitute risk groups for developing more depression later on. This could be studied by an analysis of the stability of these clusters from Time 1 to Time 2.
A general comment is that the identification of subgroups like these probably cannot be done by variable-oriented approaches that rely only on the analysis of linear correlations; here, person-oriented methods like cluster analysis may represent an important complement, which make it possible to discover aspects of the data that are hard to detect otherwise.
The present study has several strengths: it uses a large representative sample of adolescents, and there were longitudinal data for over 83% of all adolescents. The study, however, also suffers from several limitations. For example, the study used only two measure points, and it is possible that other results on risk factors would have been obtained if the first measure point had been earlier or if more measure points had been added. Another limitation is that the study relied entirely on self-assessment instruments; a multimethod approach might have made it possible to draw stronger conclusions. Further, the study did not use any established measures of depression but a specially constructed index based on items selected from different parts of a large questionnaire. A content analysis of the items (see the appendix) indicates that most of the criteria of major depression, as defined by the DSM-IV [ 29 ], are represented among the items. Two exceptions, however, are the DSM-IV criteria of weight loss or weight gain, and recurrent thoughts of death and suicide. This means that the depression index used in the present study does not do full justice to the psychiatric notion of major depression. On the other hand, the use of the present kind of depression index produced some interesting findings of potential interest to the understanding of adolescent depression, which would probably not have been seen if an established measure of depression had been used. For example, the present results identified dysphoric relations to parents and dysphoric relations to friends as two separate factors, with at least partly different meaning. One limitation with the dysphoric relations to parents subscale, however, is that all the items in this subscale refer to “parents” as the unit and do not allow for the possibility that some adolescents may feel differently towards their mother and father and hence be somewhat confused as to what to respond.
Finally, a possible risk with collecting data in school settings is that insufficient privacy may impact on levels of disclosure and thus threaten the validity and reliability of the results. An alternative possibility would have been to let the participants fill out the questionnaire at home; however, this would enter other ethical concerns and possible threats to response rates, as well as to validity and reliability. Also, the reports from the research assistants who administered the questionnaires gave no reason for concern about negative effects of lack of privacy but indicated that the students in general were very focused on the questionnaire during the lecture hour that was set off for filling it out. Further, the last page of the 11-page questionnaire included a question with a four-response format asking “how interesting and meaningful” the participants thought it had been to answer the questionnaire; the fact that over 80% of the students responded “very” or “fairly” to this question corroborates the impression that at least a large majority of the students were indeed engaged in the task. | 5. Conclusion
To summarize, the present study contributes to the literature in at least two ways. First, it shows evidence of a bidirectional relationship over time between depressive symptoms and self-harm in young girls, although there was only evidence for a unidirectional relationship from depressive symptoms to self-harm among young boys. This suggests the hypothesis that depressive symptoms and self-harm in young girls may form a dynamic system, where feedback processes lead to the emergence and stabilization of self-generating “vicious cycles” of depressive symptoms and repeated self-harm.
Second, the present study uses cluster analysis in a way that gives a partly new perspective on aspects of adolescent depression and depression-related problem patterns. Apart from the identification of two depression-related clusters which were both characterized by an overrepresentation of girls and by elevated levels of self-harm, the analysis also identified a number of clusters with more circumscribed problems, and with different associations with self-harm. For example, the results also suggest that dysphoric relations to parents and to friends represent two separate dimensions that form part of different problem profiles and may be important to differentiate in order to understand the nature of depressive experiences in adolescence. Finally, the results indicate that negative self-image, sadness/loneliness, and difficulties in concentration exist as significant one-problem patterns in relatively large subgroups of adolescents, who do not show evidence of depression; of these, only the Negative self-cluster was associated with elevated levels of self-harm. The latter finding suggests that self-harm may serve to regulate negative self-related emotions rather than feelings of sadness; more research, however, is required on the kind of emotions that are associated with self-harm. | Academic Editor: Rory O'Connor
The associations between depressive symptoms and deliberate self-harm were studied by means of a 2-wave longitudinal design in a community sample of 1052 young adolescents, with longitudinal data for 83.6% of the sample. Evidence was found for a bidirectional relationship in girls, with depressive symptoms being a risk factor for increased self-harm one year later and self-harm a risk factor for increased depressive symptoms. Cluster analysis of profiles of depressive symptoms led to the identification of two clusters with clear depressive profiles (one severe, the other mild/moderate) which were both characterized by an overrepresentation of girls and elevated levels of self-harm. Clusters with more circumscribed problems were also identified; of these, significantly increased levels of self-harm were found in a cluster characterized by negative self-image and in a cluster characterized by dysphoric relations to parents. It is suggested that self-harm serves more to regulate negative self-related feelings than sadness. | Acknowledgments
The present paper was financed by the Swedish Council for Working Life and Social Research (FAS). We also wish to thank Kajsa Pettersson, Ylva Kurtén, and a number of other research assistants for invaluable help with the data collection. Finally, we want to thank two anonymous reviewers for helpful comments on an earlier draft of the paper.
Appendix
Items in the Depression Index and Its Subscales (with Response Format)
Dysphoric Relations to Parents When I am with my parents (or think about them), I feel: sad, disappointed, depressed (Never/Seldom/Often/Very often) bored (Never/Seldom/Often/Very often) angry, irritated (Never/Seldom/Often/Very often) rejected, ignored, badly treated (Never/Seldom/Often/Very often) lonely, left out (Never/Seldom/Often/Very often) uneasy, restless (Never/Seldom/Often/Very often) calm, relaxed (Never/Seldom/Often/Very often) REV happy, joyous, glad (Never/Seldom/Often/Very often) REV a sense of belongingness (Never/Seldom/Often/Very often) REV liked, loved, cared for (Never/Seldom/Often/Very often) REV.
Negative Self-Image When I am with my parents (or think about them), I feel proud of myself (Never/Seldom/Often/Very often) REV I am ashamed of my looks (Never/Seldom/Often/Very often) I am rather satisfied with my appearance (Never/Seldom/Often/Very often) REV I wish I looked better (Never/Seldom/Often/Very often) I am proud of my body (Never/Seldom/Often/Very often) REV How do you think your life will be? (Very good/Rather good/Acceptable/Not very good/Not at all good).
Dysphoric Relations to Friends Other people my age generally like me (Not true/Somewhat true/Certainly true) REV. When I am with my closest friends (or think about them), I feel: happy, joyous, glad (Never/Seldom/Often/Very often) REV proud and sure of myself (Never/Seldom/Often/Very often) REV a sense of belongingness (Never/Seldom/Often/Very often) REV eager and full of energy (Never/Seldom/Often/Very often) REV calm and relaxed (Never/Seldom/Often/Very often) REV.
Fatigue/Somatic Complaints Do you feel alert and energetic during the day? (Always/Most often/Sometimes/Seldom/Never) Do you sleep well? (Always/Most often/Sometimes/Seldom/Never) In general, how would you say your health is? (Very good/Fair/Poor) I get a lot of headaches, stomach aches, or sickness (Not true/Somewhat true/Certainly true) When I am with my parents (or think about them), I feel eager and full of energy (Never/Seldom/Often/Very often) REV.
Sadness/Loneliness I am often unhappy, down hearted, or tearful (Not true/Somewhat true/Certainly true). When I am with my closest friends (or think about them), I feel: sad, disappointed, depressed (Never/Seldom/Often/Very often) angry, irritated (Never/Seldom/Often/Very often) lonely, left out (Never/Seldom/Often/Very often).
Difficulties in Concentration I am restless, I cannot stay still for long (Not true/Somewhat true/Certainly true) I am easily distracted, I find it difficult to concentrate (Not true/Somewhat true/Certainly true) I finish the work I am doing. My attention is good (Not true/Somewhat true/Certainly true) REV When I am with my closest friends (or think about them), I feel uneasy, restless (Never/Seldom/Often/Very often). | CC BY | no | 2022-01-13 02:18:31 | Depress Res Treat. 2011 Dec 27; 2011:935871 | oa_package/f0/55/PMC3014680.tar.gz |
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PMC3014681 | 21234108 | 1. Introduction
Skin ulcers due to micro/macrovascular disease and peripheral neuropathy represent a common complication in diabetes. Patients suffering from chronic wounds have a diminished quality of life, require frequent hospitalization, and experience increased morbidity and mortality, causing great societal and economical costs [ 1 ]. Current treatments, which include relief of pressure at the wound site, aggressive surgical debridement, control of infection, and arterial reconstruction, are limited in effectiveness and often not sufficient to guarantee adequate healing [ 2 ]. In fact, a significant number of patients do not respond to conventional therapies, and recurrence of symptoms is frequent. Short protein half-life and inefficient delivery to target cells hamper some nonconventional treatments, including topical application of recombinant growth factors to promote tissue regeneration [ 3 ].
Given the increased incidence of the disease, it is necessary to develop improved therapies to treat diabetic ulcers to reduce patient discomfort and lower societal burden. Recently, stem cell application has been suggested as a possible novel therapeutic option to promote chronic wound healing [ 4 ]. In particular, mesenchymal stem cells isolated from fetal liver [ 5 ] and bone marrow [ 6 , 7 ] can enhance wound repair through differentiation and angiogenesis promotion. Mesenchymal cells sharing similar characteristics of the ones isolated from bone marrow also can be derived from adipose tissue [ 8 ]. Adipose tissue-derived stromal cells (AT-SCs), also known as stromal vascular fraction cells (AT-SVFs), are collected from adipose tissue by collagenase digestion and differential centrifugation [ 9 ]. Since liposuction involves only local anaesthesia, cells may be obtained from the patient with a repeatable, nondebilitating operation and autologously transplanted at the site of tissue regeneration. The procedure can be performed in a large-scale, reproducible manner according to GMP regulations, allowing AT-SCs to be used in preclinical studies and experimental clinical trials [ 10 ].
Adipose tissue-derived multipotential precursor cells are able to differentiate in several cells types of both mesodermal and nonmesodermal origins, including adipocytes, chondrocytes, osteocytes, myocytes, hepatocytes, endocrine pancreatic cells, neurons, and endothelial cells (for review see [ 9 ]).
AT-SCs secrete angiogenic molecules, including HGF, VEGF, PlGF, IGF, and KGF [ 11 , 12 ]. Moreover, transcriptional profiling has demonstrated the expression of proangiogenic genes [ 13 ]. Transplantation of AT-SCs has been shown to promote therapeutic angiogenesis in hind limb ischemia in mice [ 14 – 16 ]. This may be due to integration of AT-SCs into vessels in vivo [ 17 ] and to the contribution to neovascularisation mediated by a paracrine effect [ 18 ]. Moreover, AT-SCs can promote human dermal fibroblast proliferation [ 12 ] and wound healing in a mouse model [ 19 , 20 ] both via direct cell-to-cell contact and by a paracrine effect. In addition, a protective effect of AT-SCs and their secretory factors during oxidative injury has been described [ 21 ]. Improved wound healing of adipose tissue extracts was determined in a porcine experimental model [ 6 ], while AT-SCs extract did not affect murine wound repair [ 19 ].
Taken together, these studies support the development of approaches using AT-SCs administration to promote wound healing. Nonetheless, we know very little about how transplanted cells behave in vivo , as well as their ability of engraftment, persistence, differentiation, and biodistribution after in vivo administration. In vivo studies in animal models have the potential to demonstrate the clinical potential of stem cell therapy by elucidating cell biology and physiology of the transplanted cells and their progeny. Current methods of studying stem cell fate after administration mainly rely on histochemical evaluation of samples obtained from sacrificed animals. This approach is time consuming, laborious, and expensive, requiring sectioning analysis of multiple samples deriving from a large number of experimental animals. To better understand stem cell activity in vivo , rapid, affordable, and noninvasive imaging techniques are needed.
In order to evaluate engraftment of AT-SCs in a murine model of diabetic impaired wound healing, we used bioluminescent imaging to track transplanted cells. Furthermore, we were interested in determining whether concomitant overexpression of SDF-1 may further promote wound healing and cell engraftment. In fact, in the diabetic condition, impaired healing is at least in part due to SDF-1 downregulation [ 22 ], which affects migration and homing at the wound site of circulating endothelial progenitor cells (EPCs) [ 23 ]. On the other hand, exogenous administration at the lesion site of recombinant SDF-1 [ 24 ] or of a lentiviral vector expressing SDF-1 [ 25 ] reversed the impaired EPC homing in a murine model of diabetic wound healing. Conversely, SDF-1 inhibition in diabetic animals resulted in further impairment of wound repair [ 26 ]. Moreover, SDF-1 overexpression increased survival and growth of CXCR4-expressing mesenchymal stem cells both in vitro [ 27 ] and in vivo [ 28 , 29 ].
Here, we show that topical administration of AT-SCs promotes wound healing in diabetic mice and determine by BLI the biodistribution and the kinetic of engraftment of administered cells. | 2. Material and Methods
2.1. Experimental Animals
Mice used in the study were 6- to 8-week-old wild-type Swiss CD1 males, transgenic mice expressing ubiquitously GFP [ 30 ], or GFP and firefly luciferase [ 31 ] from colonies maintained in our institutional animal facility. All experimental procedures were performed according to the guidelines of the Italian National Institutes of Health and were approved by the Institutional Animal Care and Use Committee.
Induction of diabetes was obtained by intraperitoneal injection of 50 mg/kg streptozotocin (Sigma-Aldrich, St. Louis, MO) in 0.05 M Na citrate, pH 4.5, for 5 consecutive days. Two weeks after the last streptozotocin injection, animals were fasted for 2 hours and blood glucose level measured using an Ascensia Confirm glucometer (Bayern HealthCare, Basel, Switzerland). Mice with glycaemia above 200 mg/dl were selected for further studies. Three weeks later, diabetic animals were used for wound-healing experiments.
2.2. Cells Isolation
AT-SCs were obtained from 6-week-old mice as previously described [ 32 ]. Briefly, inguinal subcutaneous fat pads were digested for 45 minutes in a shaking water bath at 37°C in PBS containing 2% BSA and 2 mg/ml collagenase A (Roche Diagnostics, Mannheim, Germany). After tissue disaggregation, cells were filtered through a 40 μ m cell strainer (BD Falcon, Franklin Lakes, NJ) and collected by centrifugation at low speed (500 g) to remove floating mature adipocytes. Cells were then washed in PBS, counted, and used for wound healing experiments. With this procedure, we isolated approximately 0.8–1.2 × 10 6 cells from each 6-week-old mouse.
To obtain adipose-derived adherent stromal (ADAS) [ 13 ], sometimes referred to also as adipose issue-derived mesenchymal cells (AT-MSCs) [ 33 ], cells were plated in DMEM 20% FCS in tissue culture dishes. After an overnight incubation, the adherent cell population was obtained.
Skin fibroblasts were obtained from the same animals as previously described [ 34 ]. Briefly, skin samples were washed in PBS, then the subcutaneous tissues were eliminated and the epidermis removed by enzymatic digestion. After a wash in PBS, dermal samples were cut into small pieces and treated with 0.3% trypsin in PBS for 30 min in a 37°C water bath. Ice-cold complete medium (DMEM 10% FCS) was then added and the samples vigorously mixed on a vortex. The suspension was passed through a 40 μ m cell strainer, and cells were collected by centrifugation (10 min at 150 × g, 4°C). Cells were then washed in PBS and counted and either used as control for angiogenesis plug assay (see below) or plated on tissue culture dishes (0.4 × 10 5 cells/cm 2 ) in complete medium.
2.3. Construction of Viral Vectors
Recombinant first-generation E1-E3-deleted adenoviral (Ad) vectors for expression of SDF-1 and GFP were obtained using the Ad-Easy system as previously described [ 35 ]. After amplification, the adenoviral vectors were purified by CsCl 2 gradient ultracentrifugation [ 36 ] and dialyzed against a solution containing 3% sucrose, 10 mM Tris, pH 7.8, 150 mM NaCl, and 10 mM MgCl 2 . Viral titers were estimated by serial dilution of the viral stocks in 293 cells and expressed as plaque-forming units per ml (PFU/ml). Viral stocks were tested for absence of replication-competent adenovirus on A549 cells.
2.4. In Vitro Lesion Repair Assay
The assay was performed as described by Bilic et al. [ 37 ] in triplicate. Second passage mesenchymal cells derived from adipose tissue were grown to confluence in complete medium (DMEM 20% FCS). Cells were then transduced by adenoviral-mediated gene transfer for 1 hour at 37°C at multiplicity of infection of 10. Cells were washed with PBS then grown with serum-free medium supplemented with 0.1% bovine serum albumin (BSA). The day after gene transfer cell monolayer was lesioned using a 2 mm cell scraper without damaging the dish surface. Lesion areas were recorded with a digital camera at time zero and after 1 and 2 days. Analysis was performed by tracing the lesion edges and calculating the pixel area using the Image Analysis System (IAS, Delta Sistemi, Rome, Italy). In vitro lesion repair is expressed as percent of the value calculated at time zero.
2.5. In Vivo Angiogenesis Gel Plug Assay
The assay is based on the implant of Matrigel plugs [ 38 ]. Freshly isolated AT-SCs resuspended in PBS were mixed with Cultrex growth factor-reduced basement membrane extract (Trevigen Inc., Gaithersburgh, MD) maintained at 4°C (liquid state). An aliquot of 400 μ l of Cultrex containing 7 × 10 5 cells was injected subcutaneously into Swiss CD1 mice gathered in experimental groups of 5 animals each near the abdominal midline. The solution and the syringe were kept on ice before the injection to prevent gelling in the needle. A week later, mice were sacrificed and gel plugs removed. Samples were fixed in 4% formaldehyde and embedded in paraffin for sectioning and histological analysis. Sections were then processed with Masson Trichromic stain and capillary density determined.
2.6. Cell Administration at the Wound Site
Hyperglycaemic animals were sedated by intraperitoneal administration of Avertin (200 mg/kg, Sigma-Aldrich) and shaved on the dorsum, and a full-thickness wound was performed on the dorsal midline using a 3.5 mm-diameter biopsy punch (Stiefel, Offenbach am Main, Germany). Right after wound induction, freshly isolated AT-SCs were then administered site in a single dose (7.5 × 10 5 cells/mouse dissolved in 40 μ l of saline solution) topically at the lesion. Control animals received the same volume of saline solution. Wounded animals were then housed individually.
2.7. Wound Analysis
At different time points (0, 3, 5, 7, 10, and 14 days) after wounding, lesion closure was documented using a digital camera. Images were processed and analyzed by tracing the wound margin and calculating the pixel area using the Image Analysis System (IAS). Re-epithelialisation is reported as the percentage of the initial wound area and calculated as re-epithelialisation percentage = [1 − (area on day of analysis/area on day 0)] × 100 [ 39 ]. The day in which the full-thickness wound was seen to be completely closed was taken as the day of complete healing. Moreover, images of haematoxylin and eosin stained slides of each wound obtained from maximal cross-sections were digitally acquired, then the epithelial gap (distance between the two epithelial edges) was measured to assess re-epithelialization.
2.8. Ex Vivo and In Vivo Optical Bioluminescent Imaging
Ex vivo and in vivo imaging analysis was performed using the IVIS Lumina from Caliper Life Sciences (Caliper Life Sciences, Hopkinton, MA). For ex vivo imaging, AT-SCs isolated from inguinal fad pads from luciferase-expressing mice were plated into clear bottom tissue culture dishes and incubated in a solution of D-luciferin (Caliper Life Sciences) dissolved in prewarmed tissue culture medium (150 μ g/ml) before analysis. For in vivo analysis, mice were anesthetized with Avertin and D-luciferin dissolved in PBS (150 mg/kg body weight), was administered i.p. 10 minutes before analysis. Photons emitted from luciferase-expressing AT-SCs transplanted into the animals were collected with final accumulation times of 1 to 5 minutes, depending on the intensity of the bioluminescence emission. The same mice were analyzed at different time points after transplantation with the same procedure, providing longitudinal data of transplanted cells biodistribution and survival.
2.9. Histological Analysis
Biopsies were fixed in 4% formaldehyde for 48 hours, embedded in paraffin, and serially sectioned (7 μ m) perpendicularly to the wound surface. Every eighth section of each wound was haematoxylin and eosin stained to perform morphologic analyses. Immunohistochemistry on formalin fixed, paraffin-embedded tissue was performed with antibodies against GFP (Abcam Ltd., Cambridge, UK).
2.10. Statistical Analysis
Results are expressed as means ± SEM. Data analysis and comparisons between control and treated groups were done with INSTAT (GraphPad, San Diego). The significance of differences was assessed with a two-tailed Student t -test for unpaired data; statistical significance level was set at P < .05. | 3. Results and Discussion
3.1. SDF-1 Overexpression Promotes Lesion Repair in AT-SCs Monolayer Cultures In Vitro
We determine that AT-SCs have a repair potential in an in vitro lesion repair assay performed as previously described [ 37 ]. AT-SCs cultured in 20% FCS containing filled a 2 mm gap performed on the cell monolayer in less than 3 days. Non-transduced and mock-transduced AT-SCs maintained in serum-free medium were not able to repair the scratch. Non-transduced cells performed better than mock-transduced (Ad-GFP), possibly for toxicity associated with viral exposure, but the difference was not statistically significant at 48 hours after lesion induction. On the other hand, cells after adenoviral-mediated gene transfer of SDF-1 had an improved lesion repair potential after 48 hours from the lesion, compared both with non-transduced and mock-transduced AT-SCs ( Figure 1 ).
AT-SCs have been shown to express the CXCR4 alpha-chemokine receptor specific for stromal-derived factor-1, and its overexpression increases AT-SCs migration and proliferation [ 40 ]. Accordingly, these results indicate that SDF-1 may act as an autocrine factor on AT-SCs after a viral-mediated gene transfer that promotes cell proliferation and migration.
3.2. AT-SCs Promotes In Vivo Angiogenesis in a Gel Plug Assay
We found that the stromal fraction isolated from adipose tissue contained a population of cells capable of differentiating into endothelial-like structures in a Matrigel plug assay [ 14 ]. To perform this assay, we isolated AT-SCs from transgenic mice ubiquitously expressing GFP [ 30 ] and then transplanted the plugs containing the GFP-positive cells in GFP-negative animals. We were then able to detect endothelial-like structures derived from implanted GFP-positive AT-SCs ( Figure 2(b) ), while we failed to detect similar structure in control mice ( Figure 2(a) ). In this experimental condition, more than 99% of endothelial-like structures were derived from GFP-positive cells ( Figure 2(d) ). We also performed the assay using AT-SCs transduced with an adenoviral mock vector and AT-SCs transduced with an adenoviral vector expressing SDF-1. We observed that even in normoglycemic conditions, the number of endothelial-like structures formation was slightly improved (5%) by SDF-1 overexpression ( Figure 2(h) ); albeit in the experimental setting used to perform the analysis ( n = 5 per group), the difference versus the control group did not achieve statistical significance ( P -value ≥.05).
Matrigel plugs containing an equal number of skin fibroblasts obtained from the same animals used for isolation of AT-SCs were used as controls and failed to organize into endothelial-like structures (data not shown).
In addition, we also performed a Matrigel plug assay using a conditioned medium obtained from an overnight culture of AT-SCs and found that secreted soluble factors produced by AT-SCs may promote angiogenesis ( Figure 2(f) ). This is in accordance with several studies which have proved that AT-SCs secrete a panel of angiogenic molecules [ 21 ], which have the potential to be, at least in part, responsible for the therapeutic effect we and others have observed on wound healing.
Collectively these sets of data indicate that AT-SCs are able to promote vessel formation in vivo in a Matrigel assay by taking part in vessel-like structure organization and by secretion of proangiogenic factors.
3.3. AT-SCs Transplantation Promotes Wound Healing in Diabetic Animals
For in vivo studies, we used streptozotocin-induced CD1 diabetic mice receiving a full-thickness wound on the dorsal midline. We observed that topical administration at the lesion site of 7.5 × 10 5 AT-SCs significantly enhanced wound healing in diabetic mice ( Figure 3 ).
Mesenchymal cells isolated from adipose tissue represent a suitable target for cell-mediated gene therapy as they have been proven to be prone to viral-mediated gene transfer [ 41 ]. We then assessed whether overexpression of SDF-1, whose downmodulation plays a pivotal role in the pathophysiology of diabetic wounds, might result in a better therapeutic profile of the AT-SCs-mediated treatment. Indeed, we achieved further improvement by adenoviral-mediated SDF-1 gene transfer into AT-SCs before topical administration. Wound area and epithelial gap were significantly reduced in treated animals. In particular, the percentage of wound closure 3 days after injury was 24 in control mice 41 and 58%, respectively, in animals receiving AT-SCs and AT-SCs expressing SDF-1. At 5 days, values were 45, 68, and 78%, respectively. Full-thickness wounds were completely closed in 7 to 10 days in all AT-SCs-treated mice, while in controls, complete healing was achieved later than 14 days after the punch.
By gross examination and immunohistochemical analysis, we determined the presence at the site of tissue regeneration of GFP-positive administered cells several days after administration ( Figure 4 ). Further studies to determine biodistribution and persistence of administered cells were performed using in vivo bioluminescent imaging techniques (see below).
3.4. Tracking Transplanted AT-SCs by BLI
After in vivo administration of AT-SCs expressing luciferase, we monitored luciferase expression by real time in vivo imaging in the whole animal, together with a light photograph, to provide for anatomical references. The intensity of the signal detected by in vivo imaging can be precisely quantified and correlates with the presence of luciferase-expressing cells, and therefore with effective cell engraftment after administration. Since luciferin metabolism requires ATP, only living cells expressing luciferase are able to produce a signal. The ability to perform repeated analysis on the same animal at different time points allowed us to determine the kinetic of AT-SCs biodistribution and engraftment at the wound site.
AT-SCs were isolated from inguinal fad pads from luciferase-expressing mice. Figure 5 shows the BLI signal originated from 7.5 × 10 5 cells placed on a 96-well cell culture plate and of the same cells right after topical administration at the wound site. Quantification of the BLI signal did not show any statistically significant difference between non-transduced cells and AT-SCs transduced with adenoviral vectors expressing GFP and SDF-1 ( Figure 5 ) at the time of administration. Differences in BLI signal observed from cells placed in a 96 well tissue culture and from the same cells after administration to the dorsum of the mouse are dependent, at least in part, on the approximately 3-fold difference in the area of a 96-well compared to the area of the biopsy punch.
To determine biodistribution of delivered cells, some animals were sacrificed 1, 10, and 15 days after wound induction and topical administration at the lesion site of AT-SCs expressing luciferase. BLI was performed before and after sacrifice of the mouse and dissection of the wounded area. As shown in Figure 6 , cells were mainly located at the lesion site, with minimal spreading from the site of delivery. Moreover, performing BLI on the dissected tissue with the skin facing the CCD camera produced a signal that was consistently lower than the one obtained with flipping the piece ( Figure 6 ). This reflects the fact that administered cells mainly contributed to dermal and subdermal tissue regeneration and engrafted only in small part in the skin. We detected a positive signal in the region of the skin and in the corresponding subdermal area even at day 15, when the wound was entirely healed ( Figure 6(c) ). This clearly indicates engraftment and permanence of administered cells in the regenerated tissue even after complete healing.
The kinetics of engraftment of administered cells, expressed as percent of the BLI signal at day 0 is reported on Figure 7 , and it inversely correlates with the kinetics of wound healing as observed in Figure 3 . Most of the signal associated with transplanted cells was lost between day 1 and day 3 after transplantation. A value of approximately 10% of the intensity observed at day 0 was maintained from day 3 to day 10, indicating cell engraftment. Although at a lower level, BLI signal was detectable also at 14 days after cell administration, suggesting cell engraftment at the site of tissue regeneration.
Dramatic reduction of stem cell survival after administration is the main obstacle hampering clinical translation of cell therapy protocols. In fact, up to 99% of grafted cells may die within the first few days of transplantation due to the rigors of the host microenvironment they are transferred in. This is mainly characterized by short supply of oxygen or nutritive substrates and by a massive excess of free radicals [ 42 ]. Several strategies to promote donor cell survival have been evaluated [ 43 ]. In particular, preconditioning of mesenchymal stem cells by treatment with the recombinant SDF-1 resulted in enhanced survival and proliferation under anoxic conditions in vitro and in vivo [ 44 ].
The SDF-1 α /CXCR4 ligand/receptor axis modulates several pivotal biological functions, including increased cell growth, proliferation, migration, survival, antiapoptosis, and transcriptional activation. In particular, it has been shown that overexpression of CXCR4 increases migration and proliferation of human adipose tissue-derived stromal cells [ 40 ]. As shown in Figure 3 , we found that transplantation of AT-SCs overexpressing SDF-1 promotes wound healing in diabetic animals. Interestingly, we determined a statistically significant difference between BLI signals in animals receiving AT-SCs expressing SDF-1 relative to controls expressing GFP, 1 day after administration ( Figure 8 ). This indicates that SDF-1 overexpression enhances survival/proliferation of administered cells at the lesion site at this time point, resulting in beneficial effects. Nonetheless, no statistically significant differences between the two groups were observed at later time points (3, 5, 7, and 14 days), suggesting that SDF-1 adenoviral-mediated overexpression failed to improve long-term cell engraftment in our experimental model. However, SDF-1 overexpression may promote prohealing effects to dermal fibroblasts and keratinocytes surrounding the lesion site [ 12 , 21 ], resulting in improved healing observed in Figure 3 .
Our results show that AT-SCs administration at the lesion site improves wound healing in diabetic animals within days from wound induction. BLI imaging data indicate that a small fraction of administered cells engraft and participate in repairing the tissue at the lesion site. On the other hand, the amount of engrafted cells may be too low to fully explain the mechanisms of tissue regeneration. In addition, several evidences suggest that AT-SCs may exert their beneficial effects also via an autocrine effect promoting self-profileration and via a paracrine effects on dermal fibroblasts and keratonocytes surrounding the lesion site [ 21 ]. | 3. Results and Discussion
3.1. SDF-1 Overexpression Promotes Lesion Repair in AT-SCs Monolayer Cultures In Vitro
We determine that AT-SCs have a repair potential in an in vitro lesion repair assay performed as previously described [ 37 ]. AT-SCs cultured in 20% FCS containing filled a 2 mm gap performed on the cell monolayer in less than 3 days. Non-transduced and mock-transduced AT-SCs maintained in serum-free medium were not able to repair the scratch. Non-transduced cells performed better than mock-transduced (Ad-GFP), possibly for toxicity associated with viral exposure, but the difference was not statistically significant at 48 hours after lesion induction. On the other hand, cells after adenoviral-mediated gene transfer of SDF-1 had an improved lesion repair potential after 48 hours from the lesion, compared both with non-transduced and mock-transduced AT-SCs ( Figure 1 ).
AT-SCs have been shown to express the CXCR4 alpha-chemokine receptor specific for stromal-derived factor-1, and its overexpression increases AT-SCs migration and proliferation [ 40 ]. Accordingly, these results indicate that SDF-1 may act as an autocrine factor on AT-SCs after a viral-mediated gene transfer that promotes cell proliferation and migration.
3.2. AT-SCs Promotes In Vivo Angiogenesis in a Gel Plug Assay
We found that the stromal fraction isolated from adipose tissue contained a population of cells capable of differentiating into endothelial-like structures in a Matrigel plug assay [ 14 ]. To perform this assay, we isolated AT-SCs from transgenic mice ubiquitously expressing GFP [ 30 ] and then transplanted the plugs containing the GFP-positive cells in GFP-negative animals. We were then able to detect endothelial-like structures derived from implanted GFP-positive AT-SCs ( Figure 2(b) ), while we failed to detect similar structure in control mice ( Figure 2(a) ). In this experimental condition, more than 99% of endothelial-like structures were derived from GFP-positive cells ( Figure 2(d) ). We also performed the assay using AT-SCs transduced with an adenoviral mock vector and AT-SCs transduced with an adenoviral vector expressing SDF-1. We observed that even in normoglycemic conditions, the number of endothelial-like structures formation was slightly improved (5%) by SDF-1 overexpression ( Figure 2(h) ); albeit in the experimental setting used to perform the analysis ( n = 5 per group), the difference versus the control group did not achieve statistical significance ( P -value ≥.05).
Matrigel plugs containing an equal number of skin fibroblasts obtained from the same animals used for isolation of AT-SCs were used as controls and failed to organize into endothelial-like structures (data not shown).
In addition, we also performed a Matrigel plug assay using a conditioned medium obtained from an overnight culture of AT-SCs and found that secreted soluble factors produced by AT-SCs may promote angiogenesis ( Figure 2(f) ). This is in accordance with several studies which have proved that AT-SCs secrete a panel of angiogenic molecules [ 21 ], which have the potential to be, at least in part, responsible for the therapeutic effect we and others have observed on wound healing.
Collectively these sets of data indicate that AT-SCs are able to promote vessel formation in vivo in a Matrigel assay by taking part in vessel-like structure organization and by secretion of proangiogenic factors.
3.3. AT-SCs Transplantation Promotes Wound Healing in Diabetic Animals
For in vivo studies, we used streptozotocin-induced CD1 diabetic mice receiving a full-thickness wound on the dorsal midline. We observed that topical administration at the lesion site of 7.5 × 10 5 AT-SCs significantly enhanced wound healing in diabetic mice ( Figure 3 ).
Mesenchymal cells isolated from adipose tissue represent a suitable target for cell-mediated gene therapy as they have been proven to be prone to viral-mediated gene transfer [ 41 ]. We then assessed whether overexpression of SDF-1, whose downmodulation plays a pivotal role in the pathophysiology of diabetic wounds, might result in a better therapeutic profile of the AT-SCs-mediated treatment. Indeed, we achieved further improvement by adenoviral-mediated SDF-1 gene transfer into AT-SCs before topical administration. Wound area and epithelial gap were significantly reduced in treated animals. In particular, the percentage of wound closure 3 days after injury was 24 in control mice 41 and 58%, respectively, in animals receiving AT-SCs and AT-SCs expressing SDF-1. At 5 days, values were 45, 68, and 78%, respectively. Full-thickness wounds were completely closed in 7 to 10 days in all AT-SCs-treated mice, while in controls, complete healing was achieved later than 14 days after the punch.
By gross examination and immunohistochemical analysis, we determined the presence at the site of tissue regeneration of GFP-positive administered cells several days after administration ( Figure 4 ). Further studies to determine biodistribution and persistence of administered cells were performed using in vivo bioluminescent imaging techniques (see below).
3.4. Tracking Transplanted AT-SCs by BLI
After in vivo administration of AT-SCs expressing luciferase, we monitored luciferase expression by real time in vivo imaging in the whole animal, together with a light photograph, to provide for anatomical references. The intensity of the signal detected by in vivo imaging can be precisely quantified and correlates with the presence of luciferase-expressing cells, and therefore with effective cell engraftment after administration. Since luciferin metabolism requires ATP, only living cells expressing luciferase are able to produce a signal. The ability to perform repeated analysis on the same animal at different time points allowed us to determine the kinetic of AT-SCs biodistribution and engraftment at the wound site.
AT-SCs were isolated from inguinal fad pads from luciferase-expressing mice. Figure 5 shows the BLI signal originated from 7.5 × 10 5 cells placed on a 96-well cell culture plate and of the same cells right after topical administration at the wound site. Quantification of the BLI signal did not show any statistically significant difference between non-transduced cells and AT-SCs transduced with adenoviral vectors expressing GFP and SDF-1 ( Figure 5 ) at the time of administration. Differences in BLI signal observed from cells placed in a 96 well tissue culture and from the same cells after administration to the dorsum of the mouse are dependent, at least in part, on the approximately 3-fold difference in the area of a 96-well compared to the area of the biopsy punch.
To determine biodistribution of delivered cells, some animals were sacrificed 1, 10, and 15 days after wound induction and topical administration at the lesion site of AT-SCs expressing luciferase. BLI was performed before and after sacrifice of the mouse and dissection of the wounded area. As shown in Figure 6 , cells were mainly located at the lesion site, with minimal spreading from the site of delivery. Moreover, performing BLI on the dissected tissue with the skin facing the CCD camera produced a signal that was consistently lower than the one obtained with flipping the piece ( Figure 6 ). This reflects the fact that administered cells mainly contributed to dermal and subdermal tissue regeneration and engrafted only in small part in the skin. We detected a positive signal in the region of the skin and in the corresponding subdermal area even at day 15, when the wound was entirely healed ( Figure 6(c) ). This clearly indicates engraftment and permanence of administered cells in the regenerated tissue even after complete healing.
The kinetics of engraftment of administered cells, expressed as percent of the BLI signal at day 0 is reported on Figure 7 , and it inversely correlates with the kinetics of wound healing as observed in Figure 3 . Most of the signal associated with transplanted cells was lost between day 1 and day 3 after transplantation. A value of approximately 10% of the intensity observed at day 0 was maintained from day 3 to day 10, indicating cell engraftment. Although at a lower level, BLI signal was detectable also at 14 days after cell administration, suggesting cell engraftment at the site of tissue regeneration.
Dramatic reduction of stem cell survival after administration is the main obstacle hampering clinical translation of cell therapy protocols. In fact, up to 99% of grafted cells may die within the first few days of transplantation due to the rigors of the host microenvironment they are transferred in. This is mainly characterized by short supply of oxygen or nutritive substrates and by a massive excess of free radicals [ 42 ]. Several strategies to promote donor cell survival have been evaluated [ 43 ]. In particular, preconditioning of mesenchymal stem cells by treatment with the recombinant SDF-1 resulted in enhanced survival and proliferation under anoxic conditions in vitro and in vivo [ 44 ].
The SDF-1 α /CXCR4 ligand/receptor axis modulates several pivotal biological functions, including increased cell growth, proliferation, migration, survival, antiapoptosis, and transcriptional activation. In particular, it has been shown that overexpression of CXCR4 increases migration and proliferation of human adipose tissue-derived stromal cells [ 40 ]. As shown in Figure 3 , we found that transplantation of AT-SCs overexpressing SDF-1 promotes wound healing in diabetic animals. Interestingly, we determined a statistically significant difference between BLI signals in animals receiving AT-SCs expressing SDF-1 relative to controls expressing GFP, 1 day after administration ( Figure 8 ). This indicates that SDF-1 overexpression enhances survival/proliferation of administered cells at the lesion site at this time point, resulting in beneficial effects. Nonetheless, no statistically significant differences between the two groups were observed at later time points (3, 5, 7, and 14 days), suggesting that SDF-1 adenoviral-mediated overexpression failed to improve long-term cell engraftment in our experimental model. However, SDF-1 overexpression may promote prohealing effects to dermal fibroblasts and keratinocytes surrounding the lesion site [ 12 , 21 ], resulting in improved healing observed in Figure 3 .
Our results show that AT-SCs administration at the lesion site improves wound healing in diabetic animals within days from wound induction. BLI imaging data indicate that a small fraction of administered cells engraft and participate in repairing the tissue at the lesion site. On the other hand, the amount of engrafted cells may be too low to fully explain the mechanisms of tissue regeneration. In addition, several evidences suggest that AT-SCs may exert their beneficial effects also via an autocrine effect promoting self-profileration and via a paracrine effects on dermal fibroblasts and keratonocytes surrounding the lesion site [ 21 ]. | 4. Conclusions
We have demonstrated that topical administration of AT-SCs improves impaired wound healing in diabetic mice. Moreover, using BLI, we were able to follow biodistribution and the kinetics of engraftment, survival, and proliferation of administered cells, which proved relatively permanent in the regenerated tissue even after complete healing. In addition, our data suggest that concomitant genetic manipulation of transplanted AT-SCs designed to promote overexpression of SDF-1 may further improve diabetic impaired wound healing. | Academic Editor: Zongjin Li
Chronic ulcers represent a major health problem in diabetic patients resulting in pain and discomfort. Conventional therapy does not guarantee adequate wound repair. In diabetes, impaired healing is partly due to poor endothelial progenitor cells mobilisation and homing, with altered levels of the chemokine stromal-derived factor-1 (SDF-1) at the wound site. Adipose tissue-associated stromal cells (AT-SCs) can provide an accessible source of progenitor cells secreting proangiogenic factors and differentiating into endothelial-like cells. We demonstrated that topical administration of AT-SCs genetically modified ex vivo to overexpress SDF-1, promotes wound healing into diabetic mice. In particular, by in vivo bioluminescent imaging analysis, we monitored biodistribution and survival after transplantation of luciferase-expressing cells. In conclusion, this study indicates the therapeutic potential of AT-SCs administration in wound healing, through cell differentiation, enhanced cellular recruitment at the wound site, and paracrine effects associated with local growth-factors production. | Acknowledgments
This work was supported by EU FP6 STREP LSHB-CT-2005-512102 and by the Italian Ministry of Health (no. RF 351467). J.C.W. was supported by the NIH grants nos. HL099117 and EB009689. The authors thank S. Artuso, I. Manni, G. Piaggio, and S. Straino for assistance. | CC BY | no | 2022-01-13 03:18:30 | Stem Cells Int. 2010 Dec 26; 2011:304562 | oa_package/e8/d2/PMC3014681.tar.gz |
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PMC3014684 | 21234109 | 1. Introduction
Investigation of a human syndrome known as Mendelian susceptibility to mycobacterial diseases (MSMD) (OMIM 209950) has, in the past 15 years, led to the identification of a series of genetic defects in the IL-12/IFN- γ axis. These include defects in three autosomal genes controlling the response to IFN- γ : IFNGR1 , encoding the ligand binding, first chain of the IFN- γ receptor; IFNGR2 , encoding the signaling, second chain of the IFN- γ receptor; STAT1 , encoding the signal transducer and activator of transcription 1 downstream from IFN- γ receptor. They also include defects in two other autosomal genes controlling the production of IFN- γ : IL12B , encoding IL-12p40 shared by IL-12 and IL-23; IL12RB1 , encoding the first chain of the IL-12 and IL-23 receptor (IL-12R β 1). In addition, there is one X-linked gene that encodes nuclear factor-kB essential modulator ( NEMO ) [ 1 – 3 ].
IL-12R β 1 defect was first described in 1998 [ 4 ] and is the most common among the known genetic disorders that predispose to mycobacterial infections. The spectrum of infections reported in these patients is, however, surprisingly narrow. These patients display selective susceptibility to weakly virulent Mycobacteria , such as environmental mycobacteria (EM) and live bacille Calmette-Guérin (BCG) vaccine (an attenuated substrain of Mycobacterium bovis ), and Salmonella . They showed almost no increased susceptibility to other pathogens including other bacteria and ubiquitously distributed viruses and fungi [ 5 ]. Evidence suggests that the immune system is redundant in its response to many intracellular microorganisms. For other organisms like varicella zoster virus, Th1 activation and IFN- γ production was found to be stimulated by IFN- α not IL-12 [ 6 ].
In a very recent report, that included the largest cohort of patients with IL-12R β 1 deficiency (141 patients) [ 7 ], the mean age of onset of the first infection was 2.4 years. The severity of the disease varied significantly from subjects who were asymptomatic until adulthood to patients who died in early childhood from complications of the disease. The mortality rate was 32% among symptomatic subjects and the mean age of death was 7.5 years, mostly secondary to BCGosis or EM disease.
In patients with IL-12R β 1 deficiency who do not respond to prolonged therapy with multiple anti-mycobacterial drugs, the mycobacteria frequently develop resistance to some of these medications. The management of those patients subsequently becomes very difficult and frequently they will not recover using antimycobacterials alone. Since the most important reason of those patients' failure to control mycobacterial infections is the inability of their T cells and NK cells to produce IFN- γ in response to IL-12 stimulation, IFN- γ is an attractive candidate therapy that can be tried in those patients [ 8 ]. However, the optimal dose and duration of using IFN- γ are not clear. | 2. Subject and Methods
2.1. Study Patient
Our patient was referred to us at 6 months of age with left axillary lymphadenitis that grew the vaccine strain of Mycobacterium bovis (BCG), a routine vaccine given in day one of life in Saudi Arabia. There was no granuloma formation on the lymph node biopsy. She was otherwise well apart from intermittent fever and night sweats. Her parents were not consanguineous but from the same tribe. She had a 5-years-old brother who had left axillary BCGitis in infancy that resolved promptly after treatment with isoniazid alone for 6 months. She also had a 7-years-old sister who has been completely healthy. She did not respond to isoniazide and rifampicin. Sensitivity results then showed that the organism was resistant to both, so she was started on ethambutol 300 mg OD, cycloserine 250 mg OD, and moxifloxacin 200 mg OD for which the organism was sensitive. Meanwhile, immunological investigations revealed that our patient's lymphocytes have no IFN- γ production in response to IL-12+BCG as compared to controls (those investigations were performed in Dr. Casanova's lab in Paris. Her parents were travel controls). Her brother had a similar cellular phenotype (i.e., no IFN- γ production after BCG and BCG+IL-12 stimulation in whole-blood assay) ( Table 1 ). In addition, no cell surface-expressed IL12RB1 could be detected from their PHA-T cell blast (data not shown). Genetic testing confirmed that she and her brother have homozygous 1336delC mutation in the IL12RB1 gene, leading to complete IL12R β 1 deficiency. Her sister was functionally and genetically normal. To our knowledge this is the only family with this specific type of mutation to be reported [ 7 ].
2.2. Study Plan
After parental consent, baseline and followup investigations were obtained including CBC, ESR, liver enzymes, lymphocytes subsets, and tuberculin skin test (TST) every 3 months. Immunoglobulin levels, serum mycobactericidal activity, and lymphocytes proliferation in vitro were performed every 6 months.
The patient was seen in the clinic every 6 weeks. IFN- γ -1b (Imukin, Boehringer Ingelheim) was started at a dose of 50 μ g/m 2 subcutaneously 3 times/week. If there is no significant response within 3 months the dose will be doubled to 100 mcg/m 2 , at the same frequency.
2.3. Cellular Studies
Blood samples were taken from the patient before starting treatment with IFN γ (S1), 6 months (S2) and 12 months (S3) after starting treatment. PBMCs were isolated from whole blood by density centrifugation (Lymphoprep, Nycomed, Oslo, Norway) and assayed for in vitro proliferative responses by the thymidine incorporation method against phytohemagglutinin (PHA) (Sigma, St. Louis, Mo.) and IL2 (R&D System, Abington, UK).
The proliferation results are expressed as mean count per minute of triplicate cultures for the antigen concentration giving maximum response minus the mean count-per-minute values for 12 wells without antigen (medium only).
2.4. Serum Mycobactericidal Assay
Two specimens of the patient's serum were collected at 6 months and 12 months of the study period, 2 hours after anti-mycobacterial drug administration, to determine the serum inhibitory and bactericidal titers against the patient's mycobacterial isolate as well as mycobacterium bovis BCG vaccine strain (Statens Serum Institute, Copenhagen S, Denmark) and mycobacterium tuberculosis H37RV reference strain (ATCC, Atlanta, GA, USA), as previously described [ 9 ]. | 3. Results
Our patient was started on IFN- γ at a dose of 50 μ g/m 2 three times a week in addition to her anti-mycobacterial medications as above ( Figure 1 ). Three months later, the patient's condition was slightly worse, so ethionamide 250 mg OD was added to her drug regimen and IFN- γ dose was upgraded to 100 μ g/m 2 three times a week. Six months from the beginning of the study her clinical condition continued to worsen and she developed a left chest wall abscess. At that time amikacin was added to her drug regimen for 6 weeks only. She was continued on the same dose of IFN- γ . Nine months from the beginning of the study there was no clinical improvement. She was on four anti-mycobacterial medications as mentioned above. At this point the dose was upgraded to 200 μ g/m 2 . One month later the enlarged lymph nodes and the chest wall abscess started to discharge pus that was positive for acid-fast bacilli on ZN stain and the culture grew Mycobacterium bovis . The patient felt better and the abscesses and the discharging lymph nodes healed. One month later she developed right pleural effusion, but remained clinically stable. Cultures from the effusion were negative. After 12 months from the beginning of the study we ran out of IFN- γ and the patient continued on her usual anti-mycobacterial medications. She then started to deteriorate gradually. Three months later she developed massive pneumonia and died. The only adverse effect from IFN- γ therapy that was noted throughout the study at the different doses was fever (up to 39°C) and lethargy up to 8 hours from giving the injection. Her CBC, liver enzymes, lymphocyte subsets, and immunoglobulin levels were not significantly changed and her ESR fluctuated between 80–107 mm/hr throughout the study period.
The spectrum of proliferative responses of T cell following stimulation with PHA and IL-2 is shown in Figure 2 . The percent increase of T cell proliferation in response to PHA (1 : 10 dilution) rose from 17% at baseline to 26% at 6 months of the study, reaching 32% at 12 months ( P = .02) after increasing the dose of IFN- γ to 200 μ g/m 2 . There was, however, no change after IL-2 stimulation, where percent increase of T cell proliferation was 20%, 18%, and 17% at baseline, 6 months, and 12 months, respectively.
There was no significant difference between the serum mycobactericidal activity at 6 months and at 12 months. | 4. Discussion
Our patient had a severe phenotype of IL-12R β 1 deficiency. In addition, the organism was resistant to anti-BCG antibiotics. Therefore, it was more stringent to try IFN- γ as a therapeutic agent. IFN- γ was tried initially at a dose of 50 μ g/m 2 3 times a week based on the experience of using it in other conditions like chronic granulomatous disease (CGD) [ 10 ]. However, it was not surprising that it did not work at this concentration with our patient since, unlike patients with CGD, she is not able to produce IFN- γ and the pathophysiology of the disease is completely different. The clinical effect noted after using the high-dose IFN- γ (200 μ g/m 2 ) may be attributed to this medication. This could be supported by the deterioration in the patient's condition after cessation of IFN- γ . Since there was no difference in the mycobactericidal activity between 6 months and 12 months of the study period, the changes in the patient's clinical condition are unlikely to be due to the anti-mycobacterial medications.
The increase in the lymphocyte proliferation in response to PHA at 12 months as compared to 0 and 6 months may be secondary to indirect stimulation of the patient's lymphocytes with the high-dose IFN- γ . However, the persistently negative tuberculin skin test suggests that IFN- γ therapy did not improve specific DTH to mycobacterial PPD antigens. Since there were no additional adverse effects with the 200 μ g/m 2 dose of IFN- γ , our experience with this patient may encourage physicians to start directly with this dose especially when there is no good response to maximum anti-mycobacterial therapy after few months of treatment.
Achieving clinical resolution is very important in the management of patients with IL-12R β 1 deficiency and BCGosis. Patients with BCG disease rarely have recurrence or develop EM disease, if they responded to multidrug antibiotic treatment [ 7 ]. On the other hand, patients frequently develop recurrence of salmonellosis, sometimes involving the same serotype. Interestingly, more than a quarter of the genetically affected siblings remain asymptomatic. This strongly suggests that the IL-12 pathway is redundant for the primary immune response against Mycobacteria and Salmonella in a substantial proportion of patients.
IFN- γ was tried in some patients with IL-12R β 1 deficiency and BCGosis. There are only two reports of three patients where IFN- γ was tried. The first report includes 2 patients with BCGosis treated with IFN- γ for 18 months in addition to antimycobacterials [ 11 ]. One patient survived and the other died. The patient who responded had maintained a normal number of Th cells during treatment and had good proliferative response to mycobacterial antigens in vitro compared to the other patient. The second report included one patient who had BCGosis for 13 years and failed multiple first- and second-line antimycobacterials. She was having chronic diarrhea and her serum showed no in vitro bactericidal nor bacteriostatic activity against BCG. She fully recovered when IFN- γ was used for one year plus the addition of intravenous anti-mycobacterial treatment to her regimen [ 9 ]. Two other patients with similar presentation were treated similarly by the same group and did well (direct communication with Dr. Rosenzweig).
In many countries around the world BCG vaccine is given at birth or few months afterword. In Saudi Arabia, BCG vaccine is given in day 1 of life. This was mainly because of high incidence (243/100,000 in 1978) and problems with compliance with vaccination schedule. Subsequently, the incidence dropped to 90/100,000 in 1990 and now 11/1000,000 (Ministry of Health statistics). Recent studies showed that giving BCG vaccine at birth induces significant mycobacterial immune response as early as 2 months of life. When this response was tested at about 9 months of age it was found to be maintained and comparable to infants who received the vaccine at 2 or 4.5 months of life [ 12 , 13 ].
In conclusion, in one patient with IL-12R β 1 deficiency, IFN- γ at a dose of 200 μ g/m 2 , but not at lower dosages, was found to have a positive clinical effect with no additional adverse effects. This medication holds promise in the management of such patients especially if used early in the course of disease. Multicenter studies are needed to establish the effectiveness, dose, and duration of IFN- γ treatment on a large number of patients. | Academic Editor: Nathalie Winter
IFN- γ has been used in the treatment of IL-12R β 1 deficiency patients with disseminated BCG infection (BCGosis), but the optimal dose to reach efficacy is not clear. We used IFN- γ in the treatment of a 2.7-year-old patient with IL-12R β 1 deficiency and refractory BCG-osis. IFN γ was started at a dose of 50 μ g/m 2 3 times per week. The dose was upgraded to 100 mcg/m 2 after 3 months, then to 200 mcg/m 2 6 months afterwards. Serum mycobactericidal activity and lymphocytes number and function were evaluated throughout the study. There was no clinical response to IFN- γ with 50 or 100 μ g/m 2 doses. However, there was some response to the 200 μ g/m 2 dose with no additional adverse effects. The serum mycobactericidal activity was not significantly different during the whole treatment period. Lymphocytes proliferation in response to PHA was significantly higher after 3 months of using the highest dose as compared to the lowest dose. The tuberculin skin test reaction remained persistently negative. We conclude that in a patient with IL-12R β 1 deficiency, IFN- γ at a dose of 200 μ g/m 2 , but not at lower dosages, was found to have a noticeable clinical effect with no additional adverse effects. | Conflict of Interest
The authors declare no conflict of interest. | Acknowledgments
The authors thank Norberto Símboli for providing the mycobactericidal assay protocol. The authors also thank Jacqueline Feinberg and Lucille Janière for their help with the diagnostic workup and Mohammad Sarwar for technical support. This work was supported by a grant from the Research Center, College of Medicine, King Saud University. | CC BY | no | 2022-01-13 01:53:39 | Clin Dev Immunol. 2011 Dec 22; 2011:691956 | oa_package/7d/cd/PMC3014684.tar.gz |
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PMC3014685 | 21234394 | 1. Introduction
Mycotic diseases in delphinoids sometimes cause fatal outcomes or difficulties for the cares of animals [ 1 – 3 ]. Lobomysosis caused by Lacazia loboi (formerly Loboa loboi ) is listed as the most famous mycosis in dolphins and zoonotic mycosis [ 1 – 12 ]. Apart from lobomycosis, Aspergillus spp. [ 1 – 3 , 12 – 15 ], Candida albicans and other Candida spp. [ 16 – 20 ], Chladosporium sp. [ 21 ] and Chladophialophora bantiana [ 3 ], Cryptococcus neoformans [ 18 – 20 , 22 – 24 ], Fusarium spp. [ 25 ], Sporothrix schenkii [ 26 ], Trichophyton sp. [ 27 ], Trichosporn sp. [ 1 – 3 ], and zygomycetes, [ 28 – 30 ] which are common to human fungal infections, have also been documented as causative agents for pulmonary, disseminated and cutaneous fungal infections in the animals [ 1 – 3 ]. Highly pathogenic mycoses caused by Coccidioides immitis [ 31 ], Histoplasma capsulatum [ 32 ], and Blastomyces dermatitidis [ 33 ] have also been reported.
Besides being highly pathogenic, the above fungal species were isolated from exhalation, although the findings do not support correlations between mycoses—fungal pneumonia and/or disseminations and these organisms [ 3 , 34 ]. In fact, even in healthy dolphins, pathogenic fungal species were isolated from exhalation [ 35 ]. Most species of pathogenic fungi isolated from exhalation were environmental contaminants while C. albicans and other Candida spp. existed as normal fungal residents of mucous membranes [ 3 , 35 ]. Furthermore, these human pathogenic yeast species were isolated from more than 70% of captive dolphins and environmental water samples [ 19 ].
According to Buck [ 19 ] and Dunn et al. [ 17 ], there is no transmission or diffusion of Candida spp. between dolphins and environments. However, there has been no investigation of the correlation between human pathogenic yeast isolates from dolphins and their keeping conditions, including captive pools and staff members, based on molecular biological studies.
The present study aims to investigate the fungal flora of pathogenic yeast species from the exhalation of dolphins, captive pools, staff members, and air in front of the dolphin show stage at the Churaumi Aquarium, Okinawa, Japan, to clarify correlations among the isolates from dolphins, captive environments and staff members using genotypes of multiple drug resistance 1 gene ( MDR1 ) having adequate sites of diversity for strain identification [ 36 ] and the internal transcribed spacer (ITS) 1 5.8 s-ITS 2 regions of ribosomal RNA gene ( ITS rDNA ) sequences recommended as the bar-coding gene of pathogenic fungi for differentiation of species [ 37 ]. | 2. Materials and Methods
2.1. Dolphins
The fungal flora of exhalation in dolphins captive at the Churaumi Aquarium, Okinawa, Japan, were investigated. The investigations were carried out on 20 individuals in August 2006 as sample collections in the summer time, and February 2007 in the winter. The sampled dolphins were as follows: two bottlenose dolphins ( Trusiops truncatus ), six (five in 2007 because of death) Indo-Pacific bottlenose dolphins ( Tursiops aduncus ), one Pacific bottlenose dolphin ( Trusiops aduncus ), three dolphins of F1 offspring between bottlenose dolphins and Pacific bottlenose dolphins, two Pacific white-sided dolphins ( Lagenorhynchus obliquiens ), six false killer whales ( Pseudorca crassidens ), and one rough-toothed dolphin ( Steno bredanensis ). The sex, age or estimated age, and housing periods in the aquarium are shown in Table 1 . The survey was performed with the permission of Churaumi Aquarium, Okinawa, Japan, with a perspective for animal welfare.
2.2. Isolation and Identification of Pathogenic Yeasts from Dolphins
Four exhalations from each animal were collected. Two potato dextrose agar plates supplemented with 100 mg/L of chloramphenicol (CPDA) and two CHROMagar Candida plates (Kanto Chemical Co. Ltd., Tokyo, Japan) were placed approximately 40 cm above the blowhole. One plate per an exhalation was used. The plates were cultured at 25°C for 1 week, and the sprouted yeast colonies were counted. Colonies were collected according to slight differences in color on CHROMagar Candida plates and in size on CPDA. The collected yeast colonies were cultured on potato dextrose agar slants at 35°C for 48 hours. Colonies having growth ability at 35°C were identified on the basis of color on the CHROMagar Candida plates and species-specific polymerase chain reaction (PCR) for detecting topoisomerase II gene ( Top II PCR) [ 43 ]. When plural isolates from one animal having identical genotype based on MDR1 and ITS rDNA sequences have existed, the isolate was treated as one isolate. Mycelial colonies that grew on the plates were ignored at the present study.
2.3. Captive Pools and Discharged Water to the Sea
Samples were collected from eight pools of the aquarium for dolphins, two for manatees ( Trichechus manatus ), and at a discharging point of all pools to the sea. The relationship among pools and the water system is shown in Table 2 .
Pools 1, 2, 3, and 8 communicated with each another and are supplied, seawater directly; this is indicated as water group A. Pools 4, 5, 6, and 7 indicated as water group B, also communicate with each another, and are supplied by sea water, and overflowing water from a fish aquarium. Pools 9 and 10 are supplied by salty well water, indicated as water group C. Seawater in discharge point is indicated as water D.
Water for captive pools is taken 300 m from the shore and at a depth of 20 m. Water exchange by an overflow system works at 8, 4, and 24 times of the volume of water per day at water systems A, B, and C, respectively. A complete change of water is provided 2 or 3 times a week, and this is aided by a scrubbing brush and by using 12% concentration of hydrochloride solution.
Pools 1, 2, and 3 are used for the dolphin show at least 4 times a day, and sometimes the splash sprays on the audience. A special exhibition of training of a dolphin with an artificial tail fin is held at pools 4 and 5 at least twice a day, and the touching of dolphins by registered visitors is allowed every weekend and on holidays. Pools 9 and 10 also have opportunities for registered visitors to feed the manatees.
The inhabiting dolphin members are not fixed. They are placed depending on the programs of dolphin shows, health conditions, and affinities. In contrast, the housing of the manatees is fixed depending on the sex. The dolphins and manatees are nursed and treated by the same staff members. Foot-bathing tubs with hydrochloride are placed at each pool entrance.
2.4. Isolation of Pathogenic Yeasts from Water Samples
Five hundreds milliliter of water samples taken from the surface of the captive pools were filtered with a 0.22 μ m pore-sized filter. The filters used for filtration were washed with 5 mL of sterile saline. One milliliter of the saline was put on CPDA and CHROMagar Candida plates and cultured at 25°C for 7 days. Yeast-form colonies were picked up and maintained on PDA slants at room temperature and identified by Top II PCR [ 43 ]. Mycelial colonies that grew on the plates were ignored at the present study.
2.5. Oral Pathogenic Yeast Flora of Staff Members
Twenty-four staff members (11 men, 13 women, 20–50 years old) were studied under personal agreement with informed consent. The survey was performed with the permission of Churaumi Aquarium, Okinawa, Japan. The ethic committee at the Chiba University judged that the present study had no infringement.
A sterile cotton tip was placed on the center of the tongue for 1 minute with rolling movements. The cotton tip was soaked in 2 mL of sterile-distilled water and then stirred vigorously for 10 seconds. One hundred microliters of the water was spread on a CHROMagar Candida plate cultured at 35°C for up to 7 days in duplicate. The sprouted yeast colonies were picked up and maintained on PDA slants at room temperature and identified by Top II PCR [ 43 ].
2.6. Airborne Fungi during Dolphin Show
One hundred liters and/or 500 L air was collected with an air sampler (Gunze, Tokyo, Japan) using an agar strip containing CPDA during the 4 dolphin shows at 11:00, 13:30, 15:00, and 16:00 in February 2007. The agar strips were cultured at 25°C for 7 days. The yeast colonies were picked up and cultured on PDA slants at 35°C for 2 and 7 days if the colonies were identified.
2.7. DNA Extraction
Fungal DNA was extracted with a DEXPAT Kit (TaKaRa, Ohtsu, Japan), following the manufacturer's protocol with slight modification, from cultures incubated on PDA slants at 25°C for 48 to 96 hours. Approximately 100 μ L of fungal mass was transferred to a sterilized microtube (1.5 mL) and homogenized with 0.5 mL of DEXPAT solution with a plastic pestle. The mixture was incubated at 100°C for 10 minutes and centrifuged at 12,000 pm (13, 201 g) for 10 min. The supernatant was used as the DNA sample.
2.8. Multiple Drug Resistant Gene 1 (MDR 1) Sequencing
The DNA was amplified with primers described by Tavanti et al. [ 36 ]. The primer sets for C. albicans were MDR1_CAF (5′-TGT TGT GTT TCA CTT TAC CT-3′) and MDR1_CAR (5′-AGG AGC ACC AAA TAA TGG GA-3′), and those for C. tropicalis were MDR1_CTF (5′-TGT TGG CAT TCA CCC TTC CT-3′) and MDR1_CTR (5′-TGG AGC ACC AAA CAA TGG GA-3′). DNA extract at 2.5 μ L, a piece of Ready-to-Go beads (Amersham Pharmacia, Tokyo, Japan), 2.5 μ L of 10 pM of the above primers, and 17.5 μ L of distilled water were mixed. Amplification was performed for an initial denaturing step of 7 min at 94°C, 30 cycles of 1 min at 94°C for DNA denaturation, 90 seconds at 55°C for primer annealing, 90 sec at 72°C for primer extension, a final extension of 10 min at 72°C, and a 4°C soak. The amplified PCR product was confirmed by electrophoresis on 1.0% agarose in 1x/TBE buffer (0.04 M Tris-boric acid, 0.001 M EDTA [pH 8.0]) and ethidium bromide staining. The amplified product was purified with a PCR purification kit (QIAquick PCR Purification Kit, QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Cycle sequencing was performed with BigDye fluorescent-labeled DyeDeoxy protocols (BigDye Terminator ver. 1.01; Amersham Pharmacia, Piscataway, NJ, USA) as follows: 15 sec at 96°C, 30 sec at 55°C, and 4 min at 60°C for 25 cycles, followed by a 4°C soak. All sequencing reactions were run on an automated DNA sequencer (3100, Applied Biosystems, Foster City, CA, USA) after purification by ethanol precipitation. DNA sequences were aligned by GENETEX-MAC genetic information processing software (Software Development Co., Ltd. Tokyo, Japan).
2.9. ITS rDNA Sequencing
We used the same reaction system as that used for MDR1 detection with a piece of Ready-to-Go beads and the primer set ITS-5 (5′-GGA AGT AAA AGT CGT AAC AAG G-3′), and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) [ 38 ]. Amplification was performed for an initial denaturing step of 4 minutes at 95°C, 30 cycles of 1 minute at 94°C for DNA denaturation, 90 seconds at 50–53°C for primer annealing depending on samples, 2 min at 72°C for primer extension, a final extension of 10 min at 72°C, and a 4°C soak. After confirming the amplified DNA by electrophoresis, being purified using a kit, it was labeled with the primers ITS-5, ITS-4, ITS-2 (5′-GCT GCG TTC TTC ATC GAT GC-3′) and ITS3 (5′-GCA TCG ATG AAG AAC GCA GC-3′) [ 38 ] as follows: 15 sec at 96°C, 30 sec at 50 to 55°C, and 4 min at 60°C for 25 cycles, followed by a 4°C soak, and sequenced.
Because yeast is a diploid organism, analysis of MDR 1 and ITS rDNA sequences from both C. albicans and C. tropicalis was based on the data set of only the variable bases or the apparently predominant peak used previously with C. albicans multilocus sequence typing. When showing almost equal peaks, the base was determined as a wobble base using a universal code; K: G+T, M: A+C, R: A+G, S: C+G, W: A+T, and Y: C+T [ 36 , 39 , 40 ].
2.10. Genotypings
The MDR 1 and ITS rDNA sequences from both C. albicans and C. tropicalis were deposited in the GenBank via DDBJ (DNA database of Japan, Mishima, Shizuoka, Japan). The combined sequences of MDR 1 and ITS rDNA sequences were analyzed by the Unweighted Pair Group Method with Arithmetic mean (UPGMA) using GENETYX-MAC ver. 13.0 (GENETYX Corporation, Tokyo, Japan) genetic information processing software and given a serial number of genotype.
2.11. Susceptibility Testing
Susceptibility tests were performed according to the broth microdilution-modified method of the CLSI M27-A3 standard [ 41 , 42 ] accepted standard using RPMI 1640 medium (Sigma, Poole, UK) buffered to pH 7.0 with MOPS (Sigma) and serial concentrations of amphotericin B (AMPH-B), flucytosine (5-FC), itraconazole (ITZ), fluconazole (FLCZ), miconazole (MCZ), and micafungin (MCFG). The latter three antifungals were included even though the method was originally described for use with AMPH-B, 5-FC, and ITZ. The test was performed in 96-well round-bottomed plastic plates using 100 mL of RPMI 1640 medium with fungal cells and antifungal substances (Dry plate koubo you, Eiken Co. Ltd., Tokyo, Japan). Data were obtained from duplicate trials. The mean or the lower data were taken. Reading results and the evaluation of the susceptibility categorized as susceptible, doze-dependent susceptible, intermediate, resistant and nonsusceptible on 5-FC, ITZ, FLCZ, and MCFG were followed to CLSI guideline [ 41 , 42 ]. The category for susceptibility extracted from the CLSI guideline was shown in Table 1 .
Twelve references isolates each for C. albicans and C. tropicalis stored in our center were added as references for molecular biological studies and the susceptibility test to antifungal substances. | 3. Results
3.1. Isolates from Dolphins
The pathogenic yeasts isolated from exhalation of dolphins were C. albicans , C. tropicalis , and C. glabrata . The total numbers of colonies and identified species are shown in Table 1 . Fourteen out of 20 dolphins, corresponding to 70% of the animals, had some kinds of pathogenic yeast species. The holding rates of C. albicans , C. tropicalis, and C. glabrata were 40%, 30%, and 5%, respectively.
Except for dolphin number 1, the rest of the fungal-positive animals had the same species of Candida in investigations of both August 2006 and February 2007 ( Table 2 ). The genotypes and susceptibility to antifungal drugs of the C. glabrata isolate are not shown in the present study because there was only one isolate throughout the study.
Mycelial fungal species were also obtained from dolphin samples although the number of colonies was 1 or 2 per animal. The filamentous fungal species isolated from the exhalation of dolphins were shown in Table 3 .
3.2. Isolates from Water, Staff Members, and Air
Collected water samples from 8 out of 11 sites from the captive pools and draining place had C. albicans and/or C. tropicalis during the summer investigation while the winter investigation resulted in 4 of the 11 sites showing only C. albicans isolates. The total number of colonies was less than 5 regardless of the collecting time or place. Except for Pool No. 10 nursing the male manatees, all water sources were positive for pathogenic yeasts for at least one collection period corresponding to 90.9% ( Table 4 ).
Candida albicans from 1 man and 2 women, C. dubliniensis from 1 woman, C. parapsilosis from 1 man, and Cryptococcus albidus from 1 man and 1 woman were isolated from the oral samples of staff members, corresponding to 29.2% (7 out of 24 individuals). The number of colonies per plate was more than 100, regardless of the sample. Except for C. albicans , genotypes and susceptibility to antifungal drugs were not shown in the present study.
Although mycelial fungal species were Aspergillus niger , A. ochraceus , Cladosporium sp., Fusarium sp., Penicillium sp., and Trichoderma sp. identified in the basis of morphology, there was no pathogenic yeast from the air samples collected in the front of the dolphin shows.
3.3. Consistent Fungal Species between Dolphins and Environments
The consistent fungal species through the dolphins, the water samples, and staff members were C. albicans and C. tropicalis, as shown in Tables 5 and 6 , respectively. The plural isolates from an animal or a site at the same collection period were due to the size of the colony on CPDA and/or CHROMagar Candida, the color on CHROMagar Candida, and the genotype based on the combination of MDR1 and ITS rDNA .
3.4. MDR 1 and ITS rDNA Sequences in C. albicans
The sequences of MDR 1 in C. albicans , consisting of 645 bases, were divided into 16 genotypes with at least 98.8% identity, and those in ITS rDNA , consisting of at least of 447 bases, were divided into 12 genotypes with at least 99.5% identity. Twenty-four genotypes based on combined sequences of MDR 1 and ITS rDNA among C. albicans isolates showed more than 99.2% identity. Although we tried to determine the ITS sequences on isolates IFM 55378, 55281, and 55298 many times, these sequences were impossible to complete, because of extremely overlapping sequence. The accession numbers of the genes and the genotypes were shown in Table 5 .
Except for C. albicans isolates derived from the dolphin No. 11 having 2 different genotypes of C. albicans collected at the 2006 summer, there was no dolphin having different genotype simultaneously at the same collecting time. Candida albicans isolates from dolphin numbers 3, 4, and 13 showed different genotypes depending on the collecting seasons. Isolates IFM 55378 derived from the dolphin No. 9 and IFM 55281 from the dolphin No. 16 were treated as exceptions for genotyping analysis, because of lacking ITS rDNA sequences. Candida albicans isolates from the captive pools; numbers 1, 2, and 4 of the same collecting period showed different genotypes, except for the isolate IFM 55298 lacked the genotype of ITS rDNA .
The genotypes A, B, G, and H are common among C. albicans isolates from dolphins and environment. The genotype C is identical between a dolphin and a staff. There was no common genotype between the isolates from the aquarium and the references in C. albicans . There was no common genotype between the isolates from the aquarium and the references in C. albicans .
3.5. MDR 1 and ITS rDNA Sequences in C. tropicalis
The sequences of MDR 1 in C. tropicalis , consisting of 645 bases, were divided into 11 genotypes with at least 97.5% identity, and those in ITS rDNA , consisted of at least 435 bases, were divided into 5 genotypes with at least 94.5% identity. Thirteen genotypes based on combined sequences of MDR 1 and ITS rDNA among C. tropicalis isolates had more than 97.2% identity. In addition, the ITS rDNA sequence of isolate IFM 55379 derived from dolphin No. 10 was not determined and treated as an exception for genotype analysis because of extremely overlapping signals. The accession numbers of the genes and the genotypes were shown in Table 4 .
Candida tropicalis isolates from dolphin No. 7 collected in the summer had 2 genotypes and in the winter had independent one. Isolates from dolphin No. 2 were identical irrespective of the collecting time while dolphin No. 14 had different clones depending on the collecting time.
The genotypes A and B are common among C. tropicalis isolates from dolphins and environment. In addition, the genotype A was detected not only in isolates at the aquarium but also in the reference ones.
3.6. Antifungal Susceptibility
The susceptibilities to antifungal agents were shown in Tables 5 and 6 . No isolate showed resistance to AMPB among the C. albicans and C. tropicalis isolates from dolphins, environments, and reference.
Three of 15 (20%) from dolphins, 1 of 12 (8.3%) from the environments, and 1 of 12 (8.3%) from the references in C. albicans isolates showed resistance to 5-FC while none of C. tropicalis isolates regardless of origins showed resistance to 5-FC.
Thirteen of 15 (86.7%) from dolphins, 7 of 12 (58.3%) from the environments, and 1 of 3 (33.3%) from the staffs showed resistance or dose-dependent susceptibilities to FLCZ; however there was no isolate that showed resistance to the compound in the reference C. albicans isolates. Eight of 10 (80%) from dolphins, 1 of 6 (16.7%) from the environments, and 3 of 12 (25%) from the reference C. tropicalis isolates showed resistance or dose-dependent susceptibilities to FLCZ.
Twelve of 15 (80.0%) from dolphins, 10 of 12 (83.3%) from the environments, and 1 of 3 (33.3%) from the staffs showed resistance susceptibilities to ITZ; however no isolate showed resistance to the compound in the reference C. albicans isolates. Eight of 10 (80%) from dolphins, 3 of 6 (50%) from the environments, and 6 of 12 (50.0%) from the reference C. tropicalis isolates showed resistance or dose-dependent susceptibilities to ITZ.
There was no correlation between resistance or dose-dependent susceptibilities to antifungal agents and the genotype of MDR1 or ITS rDNA in either C. albicans or C. tropicalis isolates. In addition, one isolate derived from the captive pool no. 5 collected at the winter 2007 showed extremely resistant to MCFG as 16 μ g/mL. The susceptibilities to MCZ were listed as reference data. | 4. Discussion
4.1. Isolating Rates for Pathogenic Yeasts from Dolphins
According to Buck et al., the holding rates of Candida spp. in free-ranging dolphins were as follows: C. albicans , 7.0%; C. tropicalis , 14.3%; and Candida sp., 14.3% [ 35 ]. At the present study, the holding rates of Candida spp. in dolphins and captive-pools were 70% and 90.9%, respectively, which were higher than those of free-ranging dolphins. Similarly, anther report by Buck et al. [ 35 ] demonstrated that the captive environments of dolphins showed a higher incidence, over 70%, in feces and pool waters of captive bottlenosed dolphins ( Tursiops truncatus ). It suggested that the data from various aquariums or institutions might vary depending on the nursing conditions and the climates of the aquarium. Further studies will confirm the average data of the holding ratio of pathogenic yeast species in captive dolphins and their nursing environments with considerations of age, sex, and physiological data.
4.2. Relationship between Fungal Exhalation and Health
The relationship between fungal exhalation phenomena from blowholes and health condition has not been confirmed [ 3 , 19 ], although many veterinarians, animal-keepers, and nurses in aquariums in Japan consider the isolations of Candida spp. from exhalation as being indicative of illness or weakness in dolphins [ 34 ]. We agree that a small numbers of total colonies in C. albicans , C. glabrata, and C. tropicalis isolates might be attached as normal fungal residents of mucous membranes, as described by Buck in 1980 [ 19 ], however, we had a doubt on the negative correlation between large numbers of Candida spp. colonies and a predictive sign of weakened health or preillness. There were 4 dolphins that dead after August 2006; for example dolphin No. 5 died in August 21, 2006 by pneumonia and colitis, No. 18 in December 24, 2007, No. 14 in January 15, 2008, and No. 13 in April 3, 2008 by pneumonia with long-term treatments. Two out of 4 dolphins showed a large numbers of Candida spp. in the breath when sampled indicating the correlation between large numbers of Candida spp. colonies and a predictive sign of weakened health or preillness. On the other hand, dolphin No. 7 had 407 C. tropicalis colonies in the summer and none in the winter, dolphin No. 10 had 601 colonies in the summer and 428 colonies in the winter did not die, and the majority of live dolphins had low Candida spp. rates. We could not confirm the relationship between the number of C. tropicalis colonies and the health condition of dolphins from these findings.
Although some pathogenic mycelial fungal species such as Aspergillus niger , Aspergillus spp., Phoma sp., Curvularia lunata , Aspergillus sp. , Schizophyllum commune , Aureobasidium pullulans , and Fusarium sp. were isolated from exhalation from blowholes, there was no correlation on the health of dolphins. We should wait for the accumulation of data on the fungal flora from exhalation and body conditions including blood and other physiological examinations, for judging the existence of the correlation.
4.3. Seasonal Characteristics on the Isolates
Interestingly, water source-derived C. tropicalis isolates disappeared in the winter, suggesting that C. tropicalis might have some difficulty surviving in winter conditions, and even in subtropical areas. The average temperatures of the environment and captive pools in February were 19.2°C and 22.2°C, while those in August were 31.4°C and 28.8°C, respectively. It is considered that the differences in water temperature might be one of the factors for the existence of C. tropicalis . Future investigations may confirm this phenomenon.
4.4. Genotypes
Genotypes based on MDR 1 seemed to be suitable for molecular epidemiological study in a confined area due to adequate sites of diversity [ 36 , 39 ]. On the other hand, genotypes of ITS rDNA could be useful for the identification of some intraspecies diversity and strain differentiations [ 44 ], and could show correlations to geographic, regional, and/or host-dependent genotypes of pathogenic fungi determined by multiple gene analysis [ 45 ]. Furthermore, the combination of 2 genes; MDR 1 and ITS rDNA, could indicate more detailed diversity of the genotypes of Candida spp. than those by MDR 1 or ITS rDNA alone. Then we discussed on the distributions of genotypes for C. albicans and C. tropicalis isolated in the aquarium based on the combined genotypes of MDR 1 and ITS rDNA . In the basis of these combinations, we could demonstrate the existences of coincident pathogenic yeast species and their genotypes in both C. albicans and C. tropicalis between or among dolphins, captive-pools and a staff member although Buck have denied the possibility that pathogenic yeasts are dispersed to other dolphins and environments [ 19 ]. Especially, the common genotype of C. albicans to both a dolphin and a staff member might be exchanged between them since the animal has been receiving medicine and surgical treatments from the staff member working as a veterinarian.
Interestingly, the genotype of C. albicans isolated from dolphin No. 5 which suddenly died of bacterial colitis and pneumonia was detected in the isolate from dolphin No. 3 and captive pools numbers 1, 2, and 4. Those of C. tropicalis isolated from the same dolphin No. 5 have also been detected in the isolate from dolphin No. 10, from captive pools No. 5 and 6, and from drained seawater. The animal might be a dispersal source for both C. albicans and C. tropicalis to other animals as well as to the environment.
The source of pathogenic yeasts might be related to the environmental water since common genotypes among the dolphins and pool water samples were found in C. albicans and C. tropicalis isolates. Isolates from captive pools free of dolphins had the same genotypes of Candida spp. isolates as the dolphins. Nevertheless, any systematic relationship of the water supply could not be found between pathogenic yeast species and captive pools or seawater. Exchanges of dolphins, common staff members for dolphins and manatees, and/or the influence of the audience might play roles in the dispersing, exchanging and introducing pathogenic yeasts. Further studies and detailed molecular profiles of the isolates may confirm the principle sources of the pathogenic yeasts.
The coincidence of genotypes in dolphins and in environmental isolates to the reference isolates of C. tropicalis suggested that such genotypes might be introduced from audiences or from sea water, and/or be very common in the world.
4.5. Attention for Sample Collection
Attention to plural isolates from the same animal at the same sampling period might be important. Although the differences in the colonies were slight with regard to size on CPDA or color on CHROMagar Candida, the clones showed different genotypes and/or susceptibilities to antifungal agents as detected in the C. albicans isolates IFM 55273 and IFM 55274 derived from dolphin No.11 isolated at the August 2006. Therefore, at least 2 or more colonies, depending on size and/or color, should be selected for identification, molecular biological analysis, and susceptibilities to antifungal drugs.
4.6. Risk to Be Audience
The fishy smell in the auditorium of the dolphin show indicates a possible spread of the breath, including pathogenic yeasts, to the audience, in spite of the fact that such pathogenic yeast isolates from air samples collected in the front of the dolphin shows were not detected. This suggests that the possibility of inhaling or being exposed to pathogenic Candida spp. from the exhalation of dolphins is relatively low. Nevertheless, it seems dangerous to approach the blowholes to a distance closer than 40 cm. For example, activities such as kissing or touching dolphins and, for pregnant women, swimming with dolphins should be approached with caution. The exact distance from the blowholes of dolphins from where it would be free of yeast-blow needs to be measured. Although there was no record of fungal infection caused by inhalation of the exhalation of dolphins, an immunocompromised person should be strongly urged to avoid such close contact with dolphins.
4.7. Characteristics on the Susceptibilities to Antifungal Drugs
The high ratio in isolations of pathogenic yeasts derived from the dolphins compared with the reference strains was the same as that in human oral fungal flora with HIV-infected or immunocompromised hosts [ 46 , 47 ]. Frequent administrations of antibiotics and steroids might be one of the explanations, but data regarding the parameters concerning stress and immunosupression, defense mechanisms against microorganisms, and drug metabolisms in the animals, even in normal immune data or blood chemical profiles, are not yet sufficient for meaningful discussion, although the correlation between the occurrence of lobomycosis and immune status of the dolphins was reported in Floridan bottlenose dolphins [ 10 , 48 – 51 ].
Furthermore, the higher incidences of resistance to azole-related antifungal agents in the isolates from dolphins and environments might be related to the sodium chloride in sea water, a speculation drawn from the correlations among resistance to chemical compounds, pathogenicity, and sodium chloride [ 52 ].
4.8. Correlation between the Genotype and Resistance to Antifungal Agents
According to Tavanti et al., MDR 1 alone cannot define the relationship between genotypes and profiles of antifungal agents [ 36 ]. In the present study, no correlation was found between resistance or dose-dependents susceptibilities to azole-related antifungal agents and the genotypes of MDR1 and/or ITS rDNA in either C. albicans or C. tropicalis , since an identical genotype based on MDR1 and ITS rDNA sequences has shown different profiles of susceptibilities to azole-related antifungal agents, regardless of origin. The reference isolates, giving a higher ratio of resistant isolates, could not help in determining the specific genotypes based on the combination of MDR1 and ITS rDNA sequences. | 5. Conclusions
The detection of common genotypes on Candida spp. among dolphins, environments, and staff members pointed to the dissemination of pathogens at the aquarium. Thus, it seemed to be important to consider the effects on audiences from dolphins and the reverse relations for controls of zoonotic infections. In addition, the sequence of MDR 1 showed adequate numbers of variations, indicating that the gene might be useful for molecular epidemiological studies. | Academic Editor: Pedro J. Ginel
Genotypes of Candida spp. isolated from exhalation of 20 dolphins, 11 water samples from captive pools, and 24 oral cavities of staff members in an aquarium using a combination of multiple drug resistance 1 gene ( MDR1 ) and the internal transcribed spacer (ITS) 1 5.8s-ITS 2 regions of ribosomal RNA gene ( ITS rDNA ) sequences were studied. The holding ratios of the dolphins, captive pools, and staff members were 70, 90, and 29%, respectively. Isolated pathogenic yeast species common to the dolphins and environments were Candida albicans and C. tropicalis . Identical genotypes in both Candida spp. based on the combination of MDR1 and ITSrDNA were found in some dolphins, between a dolphin and a staff, among dolphins and environments, and among environments. The results indicated the diffusion and exchange of pathogenic yeasts at the aquarium among dolphins and environments. The isolates at the aquarium showed higher rates of resistance to azole antifungals compared to reference isolates. | Acknowledgments
This study was supported by the Special Research Fund for Emerging and Re-emerging Infections of the Ministry of Health, Welfare, and Labor, Japan, and in part by the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology, Japan, cooperated by Drs. Katsuhiko Kamei and Ayako Sano. | CC BY | no | 2022-01-13 07:44:14 | Vet Med Int. 2010 Dec 22; 2010:349364 | oa_package/a3/0f/PMC3014685.tar.gz |
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PMC3014686 | 21234395 | 1. Introduction
We live in an era of surgical innovation that has seen the development and expansion of various types of laparoscopic surgery in which the incisions made are increasingly small. It is well established that laparoscopic surgery, in comparison with more traditional methods, results in fewer post-operative complications and leads to earlier patient mobility and recovery of the normal activities of daily life [ 3 , 4 ]. The safety of laparoscopic cholecystectomy (LC) for the elderly has also been confirmed in many studies as an acceptable procedure and is now the preferred method for cholecystectomy [ 1 , 3 – 11 ].
Care-related outcomes such as mortality and post-operative complications have been studied extensively, but to the best of our knowledge, there have been no studies measuring functional changes in elderly patients undergoing cholecystectomies [ 1 , 7 – 11 ]. The effects of age differences in elderly patients on the return to a good quality of life or on resource use also had not been previously or sufficiently investigated. Older patients are likely to have many chronic conditions that could impair their physical and functional recovery; it is important to monitor post-operative outcomes such as changes in activities of daily life [ 4 , 8 ]. To complete the less-invasive LC procedure without conversion to open cholecystectomy (OC) in older patients, many surgeons will routinely use pre- or intraoperative bile duct interventions (BDIs), including endoscopic retrograde cholangiopancreatography (ERCP), intraoperative cholangiography (IOC), percutaneous gallbladder or common bile duct (CBD) drainage, endoscopic dilatation or sphincterotomy of the ampulla of Vater, and stone extraction from the CBD [ 12 – 16 ]. When compared with cases where LC was used without any interventional techniques, the use of added interventions can cause more stress to older patients and delay their recovery or worsen their physical condition; any evaluation of the safety of LC must include an evaluation of the type of intervention used. A few studies have demonstrated the safety of LC for older patients, and these were conducted in single centers or on a limited number of cases [ 1 , 7 , 9 , 10 ]. They did not, however, measure the variation in patients' functional changes during hospitalization [ 9 ]. OC was reported to be used more than LC in seriously ill patients, regardless of age. If studies comparing the safety of LC to OC in older patients are to be done, they need to be randomized to eliminate any selection bias [ 7 , 8 , 11 , 17 ].
Using a Japanese administrative database from fiscal year (FY) 2004 to 2008, we examined variation in the use of OC in patients ≥65 years of age. We analyzed the advantages of using LC instead of OC by propensity scoring in which we concurrently estimated the effects of age and OC on hospital resource use, postoperative complications and functional changes in recovering patients. | 2. Methods
This retrospective study used both a Japanese administrative database and claim data that were incorporated into the Ministry of Health, Labour and Welfare (MHLW) database as well as our own research project that was designed to develop a Japanese case-mix classification system. Eighty-two academic and 1,346 community hospitals were enrolled in 2008. Anonymous health insurance claims data with detailed clinical information had been collected annually for this database for 4–6 months beginning July 1, 2002, and the information was provided to our research team. The database contained the date and quantity of care provided during hospitalization; therefore, it was used to assess hospital performance and payments [ 18 ].
Our database included a total of 8,010,361 possible patients from the 1,006 hospitals that have participated voluntarily in our research project from 2004 to 2008. In this group, we identified 13,709 cholecystectomy patients (11,677 LC and 2,032 OC) who were treated for benign gallbladder diseases in 122 hospitals participating in our project for five consecutive years. From the group of 13,709 cholecystectomy patients, 4916 were aged ≥ 65 and were enrolled in this study. Our project was approved by the ethical committee of the University of Occupational and Environmental Health, Fukuoka, Japan. | 5. Results
Out of a total of 4,916 cholecystectomy patients, there were 3,692 LC patients from 122 hospitals and 1,224 OC patients from 117 hospitals. Of the LC patients, 1,071 (29.0%) were treated in 35 hospitals and 295 (24.1%) OC patients were treated in 34 academic hospitals. Twenty-one patients (two LC and 19 OC) were deceased and excluded. Older patients and those with greater CCI or acute gallbladder inflammation underwent OC more frequently. Preoperative ERCP or BDIs were performed more often in OC. Operating room time, LOS, TC, BI improvement score and complications were higher in OC, whereas BI scores at admission and discharge were lower ( Table 1 , Figure 1 ).
Advanced age (≥75 years), male sex, transport by ambulance to the hospital, presence of inflammation, and CCI of ≥2 were significant indicators for OC, but higher BI score at admission and surgery at an academic hospital was associated with less indication for OC ( Table 2 ).
In the propensity score-paired matching cohorts, longer operating room time, longer LOS, and higher TCs were observed for OC, but the BI improvement score did not differ significantly between OC and LC for these parameters ( Table 3 , Figure 2 ).
Patients ≥75 years of age had longer LOS, and those ≥85 years of age had higher TCs. Patients between 75 and 84 years of age had a lower BI improvement score. OC was significantly associated with longer operating room time and LOS, and higher TCs, but not with BI improvement score. Complications were associated with greater LOS, TCs and less BI improvement scores ( Table 4 ).
A risk of complications was observed in OC [odds ratio (OR) 1.285; 95% confidence interval (CI): 0.927–1.782] and patients with CCI ≥ 3 [OR: 1.894; 95% CI: 1.001–3.583]. The risk of complications was not related to age or BI at admission. Age, CCI ≥ 3, and complications were associated with BI deterioration: 75–84 years [OR: 2.908; 95% CI: 1.369–6.173], ≥85 years [OR: 3.998; 95% CI: 1.261–12.678], CCI ≥ 3 [OR: 3.998; 95% CI: 1.364–11.717], and complications [OR: 3.729; 95% CI: 1.768–7.865]. OC was not an independent indicator of BI deterioration ( Table 5 ). | 6. Discussion
The present study was conducted to compare the advantages of LC versus OC in older patients in relation to changes in physical condition and ability to function. OC was employed more often in patients ≥75 years of age and those with greater CCI or the presence of gallbladder inflammation. Preoperative ERCP or BDIs were required more often in OC. Multivariate analysis of the propensity score matching cohorts revealed that LC had the advantage of fewer complications, shorter LOS, and lower TCs compared with OC. Operating room time and resource use were greater in OC. Neither cholecystectomy procedure caused significant variations in BI improvement scores or BI deterioration. Age did not determine the complications, but advanced age and complications were independent indicators of the functional recovery.
We observed an age disparity in the use of OC and LC in the older patients. Compared with the 75–84 year-old group, OC was used more frequently than LC for patients ≥85 years of age. This corresponds to the findings of a Swedish community study of cholecystectomies by Rosenmüller et al. [ 8 ]. We found that acute admission and perioperative use of ERCP were indicated more in OC; these results also agree with the findings of Rosenmüller et al. [ 8 ]. Generally, OC is considered for seriously ill and older patients as indicated in this study in which it was reported that BI at admission was lower in OC [ 8 , 11 ]. These findings might cause a selection bias for OC that could exaggerate the benefits of laparoscopic surgery over conventional surgery.
Previous studies have confirmed the advantage of LC only in terms of mortality and complications; our study adds new and additional information concerning post-operative recoveries. In an aging population, major care-related outcomes related to the change in activity of daily life should also be considered. Kugler et al. estimated the functional recovery by measuring the change in BI score combined with information from the Hessian Stroke Data Bank [ 2 ]. In this study, however, the BI score was originally analyzed as an ordinal variable as opposed to a continuous one. The difference between ages 55 and 65 was not equivalent to that between ages 25 and 35; therefore, logistic regression needed to be applied to measure the association of OC with deterioration in BI score to correct for this discrepancy.
Since a randomized study for the elderly might be difficult to perform and unethical in that it would also depend on patient comorbidities, we constructed the propensity score matching cohorts using the administrative database. This kind of study might help surgeons make the right decision as to which factors are associated with functional recovery as well as which procedure is best for the elderly patient in actual clinical situations. Knowledge of functional changes would also contribute to the determination of healthcare policies for the elderly in the medically advanced G7 countries, where an ever-increasing number of aging patients require expensive surgical innovations [ 23 ]. The use of laparoscopic surgery should be favored because older patients are expected to benefit from fewer post-operative complications and/or earlier functional recovery.
The extra costs of surgical innovations that promote earlier functional recovery would be offset by the overall benefits to the healthcare system derived from reducing LOS. Those costs should incorporate additional procedures such as the study BDIs that allow for the completion of laparoscopic surgery without conversion to OC. We attempted to address the concerns about treatment options for elderly patients by overcoming any selection bias as comprehensively as possible. Preoperative BDI, which is an indicator of disease severity, does not always appear to influence post-operative functional status, but the occurrence of post-operative complications appears to be a factor influencing functional recovery. As complications were not necessarily correlated with age, the key to achieving favorable outcomes in elderly patients is to manage post-operative complications [ 10 ]. OC and acute cholecystitis were also observed to be independent predictors of complications. Surgeons should choose the most appropriate surgical method to encourage the best possible post-operative recoveries for elderly patients. Clinical experts should also develop an educational program or model for teaching LC operating skills for cases in which more complicated gall bladder problems such as gallbladder inflammation are diagnosed [ 24 ].
Several limitations to our study should be discussed. The study period was limited to 4 months which might diminish our ability to generalize our results. However, our larger sample size and use of propensity scoring appeared to improve the validity of this study. Because the MHLW extended the study period to 12 months from FY 2010, use of this extensive database will expand the number of cases analyzed and overcome the initial limitation. A second point is that the duration of hospitalization in Japan is generally 2-3 times longer than that in Western countries [ 23 ]. Japanese hospitals generally provide wound management and nursing home services in addition to acute medical care; costs would reflect this type of care [ 25 ]. A third point that requires discussion is that some clinical information about conversion from LC to OC and the timing of the cholecystectomy were not included. OC cases in our study included those whose surgeries had been converted from LC to OC because the Japanese procedure codes do not define this conversion. However, it has been reported by Wolf et al. that the complications and LOS are similar between straightforward OC cases and those whose surgeries were converted from LC; our results therefore would not have been significantly distorted [ 11 ]. The last point to discuss is that the frequency of the procedure might have an effect on the quality of cholecystectomy care, physical condition, and functional status with emphasis on the residual respiratory function in fragile older patients [ 26 ]. Our administrative database should provide answers to these concerns because it has the quantity and date of use of every medical care item entered into it. | 7. Conclusions
Our study investigated the quality of cholecystectomy care in patients ≥65 years of age using an administrative database and propensity score-paired matching analysis. The mix of patient cases explained the variation in use of cholecystectomy. After correcting for selection bias and relevant covariates, the LC method remained advantageous over OC in terms of fewer complications and lower resource use. Patients' functional status was not influenced by the type of cholecystectomy, but was affected by advanced age and complications. Surgeons should use LC in the elderly as much and as prudently as possible by planning the necessary preoperative treatment strategy and obtaining the necessary skills to complete LC without conversion to OC. | Academic Editor: Stanley Ashley
The safety of laparoscopic cholecystectomy (LC) in patients ≥65 years of age requires further investigation of postoperative outcomes before it becomes more widely accepted as a safe technique. The advantages of using LC versus open cholecystectomy (OC) in elderly patients were analyzed using propensity score matching. The demographics, cholecystitis severity, comorbidities, complications, and admission and discharge Barthel Index (BI) scores of patients with benign gallbladder diseases were analyzed. Outcomes were analyzed by age, length of stay (LOS), total charges (TCs), BI improvement, and postoperative complications. OC, which was indicated in severe disease cases, increased hospital resource use and caused more complications than LC, but did not improve BI. Advanced age and OC resulted in greater LOS and TCs and was the best indicator of BI deterioration. Whenever possible, surgeons should use LC in elderly patients to minimize postoperative complications and allow them to regain a good quality of life. | 3. Variable Definitions
Study variables were as follows: age, sex, use of an ambulance (in an emergency situation), discharge outcome, discharge destination (to their home or other facility), presence of inflammation (as an indicator of the principal diagnosis), comorbidities, physical condition, and functional status at admission and discharge expressed by the Barthel Index (BI) score, biliary or procedure-related complications, use of IOC, pre- and/or postoperative ERCP or BDI, and hospital teaching status (community or academic hospital).
Study patients were stratified into three age groups: 65–74, 75–84, and ≥85 years. Diagnoses were classified according to the International Classification of Disease 10th version (ICD code). A maximum of four comorbid conditions or four complications per patient were recorded in the database. To assess the severity of pre-existing comorbid conditions, we used the Charlson Comorbidity Index (CCI) [ 19 ]. Patients were divided into four groups and assigned a CCI of 0, 1, 2, ≥3. Using the ICD codes related to benign biliary disease (D135, K80–82), we categorized gallbladder status as follows: acute inflammation (K800, K803, K810 and K822) and chronic or other specified inflammation (K801, K804, K811–9, K820–1, K823, K830–2). The remaining ICDs were classified as no inflammation. Biliary or procedure-related complications included wound complications, hematoma or others (T81–T87), acute pancreatitis (K85), bowel obstruction (K560–7, K660 and K913), and peritonitis or intraabdominal abscess (K650–9) [ 20 ]. Academic hospitals were defined as university hospitals that were responsible for educating medical students and postgraduate trainees as well as carrying out clinical research.
Preoperative percutaneous gallbladder and CBD drainage, endoscopic dilatation and sphincterotomy of the ampulla of Vater, stone extraction, and stent insertion were classified as BDIs. ERCP was also examined as a factor independent of BDI. Patients who underwent conversion from LC to OC were classified as OC cases because of the lack of conversion information at this time.
We calculated the operating room time; this time included induction of general anesthesia, insertion of the epidural anesthesia where applicable, preparation for video-monitoring, and extubation of the endotracheal tube as well as the skin-to-skin time. We also measured length of stay (LOS) and total charges (TCs) billed during admission. TC is considered to be a good estimate of healthcare costs [ 21 ]. The TCs included fees for physician consultation and administration, and costs of instruments, laboratory tests and imaging. The BI improvement score, often used as a quality of life indicator for the elderly, was also recorded. It was calculated as the BI score at discharge minus the score at admission; a negative BI score indicated a deterioration in BI score [ 2 ].
4. Statistical Analysis
Frequencies and proportions for categorical data for OC and LC cases were compared by Fisher's exact test. Continuous variables were compared using analysis of variance. The variations in LOS, TC, and the operating room time between OC and LC were also indicated in the box chart. Logistic regression was used to evaluate the OC-associated study variables. To reduce possible selection bias for cases indicating OC, we defined propensity score paired-matched cohorts and compared operating room time, resource use, and BI improvement score in each of the LC and OC groups [ 22 ]. Data from deceased patients were excluded and a mixed linear regression model was used to correlate age and OC with operating room time, LOS, TCs, and BI improvement score. In this model, every study hospital was treated as a random effect to control for independent hospital preferences for the type of cholecystectomy and hospital-specific standard practices. Logistic regression was used to evaluate the association of age and OC with complications and BI deterioration. Statistical analysis was performed using SPSS version 16.0, with a two-tailed level of significance set at P < .05. | Conflicts of Interest
The authors have no competing conflicts of interest to declare.
Acknowledgment
This study was funded in part by Grants-in-Aid for Research on Policy Planning and Evaluation (Japanese Ministry of Health, Labour and Welfare, H19 Seisaku-sitei 001). | CC BY | no | 2022-01-13 02:25:54 | Gastroenterol Res Pract. 2010 Dec 22; 2010:490147 | oa_package/80/fa/PMC3014686.tar.gz |
PMC3014687 | 21234396 | 1. Introduction
The incidence of type 1 diabetes mellitus in Belgium varies between 10.9 to 15.4/100.000 [ 1 ]. In type 1 diabetic pregnancies, an increase in congenital malformations, miscarriage, macrosomia, shoulder dystocia, and other obstetric complications is described [ 2 – 19 ]. Macrosomia is defined as birth weight above the 90th percentile for gestational age. According to the modified Pederson hypothesis, maternal hyperglycemia results in transplacental diffusion of a higher amount of glucose to the fetus, resulting in a stimulation of the fetal beta cells and increased fetal insulin secretion. This finally results in fetal macrosomia. Macrosomia is related to obstetrical and neonatal complications such as asphyxia, shoulder dystocia, and neonatal trauma during delivery [ 6 – 9 ].
The aim of this study was to look for differences in birth weight in patients with diabetes type 1 as compared to the general obstetric population in the context of the actual therapy of diabetes. | 2. Methods
A retrospective analysis was performed of an existing data base. The Study Centre for Perinatal Epidemiology registers data on all hospital deliveries (this is 99% of all deliveries) and most home deliveries in Flanders, Belgium. Data are collected on both maternal and neonatal outcome. The diagnosis of type 1 diabetes was accepted as given by the treating physician. For this study, hypertension was defined as a systolic blood pressure ≥140 mmHg and/or diastolic ≥90 mmHg; no further subdivision in pre-eclampsia, gestational hypertension, and pre-existent hypertension was made. Fetal death is death before delivery, early neonatal death is death in the first 28 days of live, and perinatal death was considered the sum of fetal and early neonatal death. For every fetus, the presence of congenital anomalies was registered as eventual transfer to a neonatal intensive care unit. Caesarean or vaginal delivery and the use of forceps or vacuum are also registered. Women with type 2 diabetes or gestational diabetes were excluded from our study.
Dichotomic variables were compared using Chi squared test, significance accepted at P < .05, odds ratios, and 95% confidence intervals. To compare birthweight as a continuous variable per gestational week, a Shapiro-Wilk test was performed to test for normality; nonnormally distributed data were compared using the Mann-Whitney test, significance accepted at P < .05. Statistical analysis was done with SPSS 16.0. | 3. Results
We studied patients who gave birth between January, 1st, 2002 and December, 1st 2004; during this period 354 women with type 1 diabetes gave birth and 177.407 control women.
Table 1 gives a general overview of the obstetric data of our population. There were significantly more women in the type 1 diabetes group over age 35 years and significantly more maternal hypertension. Table 2 demonstrates the fetal-neonatal outcome. Clearly fetal and perinatal deaths are significantly higher in the type 1 diabetes group as compared to controls, and significantly more congenital anomalies were seen in neonates from diabetic mothers. This correlates with more transfers to a neonatal intensive care unit. Neonates were almost 5 times as often born by Caesarean section in case of maternal diabetes type 1. Babies of mothers with diabetes type 1 were also more often preterm.
Table 3 presents birth weight per gestational week for diabetes type 1 and the control group. Figure 1 presents a graphic view of the median birth weight per gestational week in diabetic and nondiabetic pregnancies.
For none of the gestational weeks birthweight was normally distributed (probably due to low numbers), as the number of deliveries in the type 1 diabetic group was extremely low before 33 weeks (less than 5), we only compared median birthweight from week 33 till week 41. This demonstrates, as shown in Table 3 , that until 38 weeks there is a significant difference in birth weight for the diabetic pregnancies; from 39 weeks on no significant difference is present. | 4. Discussion
Although a higher birth weight in type 1 diabetes is generally accepted, only few studies have actually described birth weight per gestational week in diabetic pregnancies as compared to nondiabetic controls. Most studies give mean birth weights, not specifying gestational age. Lauszus et al. [ 13 ] described birth weight but made a division only in term and preterm pregnancies not allowing to determine whether a difference is or is not present at different gestational ages.
Sibai et al. [ 14 ] compared birthweight in pregestational diabetes, chronic hypertension, and uncomplicated pregnancies before 35 weeks and between 35 and 37 weeks and noted a higher birthweight in pregestational diabetes. Dos Santos Silva et al. [ 19 ] made a very raw division in less than 33 weeks, between 33 and 36 weeks and more than 36 weeks but did not use a control group.
The only study comparable to our present one we were able to find was that by Jensen et al. [ 10 ] who compared mean birth weight in the diabetic group versus a control group and did not find a significant difference. Evers et al. [ 16 , 17 ] also used a control group but did not provide data per gestational week. As far as we know this is the first nationwide study presenting data on birth weight per gestational week in pregnancies with type 1 diabetes versus nondiabetic controls. Our data confirm that neonates from mothers with type 1 diabetes have a significantly increased risk for perinatal death, are more frequently born by Caesarean section, have more congenital anomalies, and more frequently need neonatal intensive care. As the number of babies born from type 1 diabetic mothers below 33 weeks was extremely low, it seemed not relevant to make a statistical comparison. When analyzing data between 33 and 41 weeks a significant difference was seen until 38 weeks. From 39 weeks on, there was no significant difference in birth weight between both groups. This can be due to chance, but another explanation might be that those pregnancies with type 1 diabetes that continue until 39 and more weeks are the ones with the least problems; all others will have been induced in the mean time.
Our study has several important limitations. We do not have any data on the quality of the diabetic control of our patients, we cannot also control for maternal weight or maternal weight gain, nor can we exclude the influence of other risk factors such as maternal smoking. | 5. Conclusion
This retrospective analysis demonstrates that birth weight for neonates from mothers with type 1 diabetes is higher than that in a control group but the difference disappears at 39 weeks, probably because only the best regulated diabetic mothers continued their pregnancy until that week. | Academic Editor: William A. Grobman
Our aim was to investigate whether birth weight in mothers with diabetes mellitus type 1 is higher as compared to nondiabetic controls. Methods . A retrospective study was performed using an existing database covering the region of Flanders, Belgium. Data included the presence of diabetes type 1, hypertension, parity, maternal age, the use artificial reproductive technology, fetal- neonatal death, congenital anomalies, admission to a neonatal intensive care unit, and delivery by Caesarean section or vaginally. Results . In the period studied, 354 women with diabetes type 1 gave birth and were compared with 177.471 controls. Women with type 1 diabetes more often had a maternal age of over 35 years (16.7% versus 12.0%, P = .008, OR 1.46; 95% CI 1.09–1.95). They more frequently suffered hypertension in pregnancy (19.5% versus 4.7%, P < .0001, OR 4.91; 95% CI 3.73–6.44). Perinatal death was significantly higher in the diabetes mellitus group (3.05% versus 0.73%, P < .0001, OR 4.28; 95% CI 2.22–8.01). Caesarean section was performed almost 5 times as frequently in the diabetes versus the control group (OR 4.57; 95% CI 3.70–5.65). Birth weight was significantly higher in diabetic pregnant women from 33 until 38 weeks included, but those reaching 39 weeks and later were not different with control groups. Conclusion . In Belgium, diabetic pregnancy still carries a high risk for fetal and maternal complications; in general birth weight is significantly higher but for those reaching term there is no significant difference in birth weight. | CC BY | no | 2022-01-13 03:04:56 | Obstet Gynecol Int. 2010 Dec 22; 2010:397623 | oa_package/d9/f2/PMC3014687.tar.gz |
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PMC3014691 | 21234398 | Rheumatoid arthritis (RA) is a debilitating autoimmune disease of global prevalence. The disease is characterized by synovial inflammation leading to cartilage and bone damage. Most of the conventional drugs used for the treatment of RA have severe adverse reactions and are quite expensive. Over the years, increasing proportion of patients with RA and other immune disorders are resorting to complementary and alternative medicine (CAM) for their health needs. Natural plant products comprise one of the most popular CAM for inflammatory and immune disorders. These herbal CAM belong to diverse traditional systems of medicine, including traditional Chinese medicine, Kampo, and Ayurvedic medicine. In this paper, we have outlined the major immunological pathways involved in the induction and regulation of autoimmune arthritis and described various herbal CAM that can effectively modulate these immune pathways. Most of the information about the mechanisms of action of herbal products in the experimental models of RA is relevant to arthritis patients as well. The study of immunological pathways coupled with the emerging application of genomics and proteomics in CAM research is likely to provide novel insights into the mechanisms of action of different CAM modalities. | 1. Herbal CAM for the Treatment of Inflammatory Autoimmune Arthritis
Conventional (allopathic) anti-inflammatory drugs are the mainstay of treatment for a variety of immune disorders, including rheumatoid arthritis (RA) [ 1 – 5 ]. The nonsteroidal anti-inflammatory drugs (NSAIDs) and biologics (e.g., antitumor necrosis factor (TNF)- α antibody and the decoy TNF- α receptor) represent a prominent group of such drugs. However, the usage of these drugs is associated with severe adverse effects, including gastrointestinal bleeding and cardiovascular complications [ 3 , 5 , 6 ]. Owing to the side effects and the high cost of conventionally used anti-inflammatory drugs, patients with arthritis are increasingly using complementary and alternative medicine (CAM) modalities of treatment [ 7 – 21 ]. Over 36% Americans used CAM products annually for different disorders and the trend is on the rise [ 11 , 22 – 25 ]. Traditional Chinese medicine, Ayurvedic medicine, Kampo, and Homeopathy are among the major contributors to the natural products consumed by patient populations. However, despite the increasing usage and popularity of CAM products in the western world [ 11 , 22 – 26 ], one of the main limitations of their use is the meager information about their mechanisms of action and objectivity in evaluating efficacy [ 27 , 28 ]. This also is one of the main reasons for skepticism about CAM in the minds of both the lay public and the professionals [ 25 , 29 – 31 ]. Thus, there is a need for continued studies on the mechanistic aspects of action of CAM products.
A diverse group of diseases is characterized by inflammation that can be triggered not only by foreign microbial antigens but also by self-antigens. The response to self-antigens results in autoimmune inflammation. Therefore, like the infectious diseases, the autoimmune diseases (such as multiple sclerosis (MS), type-1 diabetes mellitus (T1D), RA, and atherosclerosis) are also associated with inflammation. Considering that autoimmune diseases result from a dysregulated immune system [ 36 , 37 ], it is imperative to examine and unravel the immunological basis of the therapeutic activity of CAM products against autoimmune disorders as well as other conditions involving inflammation [ 27 , 28 , 38 – 42 ]. This paper is focused on the immunomodulation of autoimmune arthritis by herbal CAM products. We have described here in detail adjuvant arthritis (AA) ( Figure 1 ) as a prototypic experimental model of RA. Conceptually, the main immune effector pathways in AA are broadly representative of various other animal models of arthritis, for example, collagen-induced arthritis (CIA), streptococcal cell wall-induced arthritis (SCWIA), and proteoglycan-induced arthritis. We have elaborated upon specific immune pathways in arthritis that are modulated by a variety of herbal preparations originating from plants native to different regions of the world ( Table 1 , and Figures 2 and 3 ). These immune mechanisms include the cellular and humoral responses, the cytokine response/balance, and the cellular migration into the target organ.
The above-mentioned immunological events in the pathogenesis of arthritis also offer many promising targets for therapeutic intervention (Figures 2 and 3 ). We recommend that these and other customized immune parameters be considered for testing besides various biochemical and pharmacological parameters for the evaluation of the mechanisms of action of herbal CAM products. The herbal CAM shown in Table 1 are representative of those tested for the modulation of immunological events that contribute to their antiarthritic activity in vivo in experimental models of arthritis. Understandably, there are several other natural products that possess antiarthritic activity, but their effects on the immune system have not yet been tested. Given the scope of this paper on immune modulation, we have excluded most of those products from Table 1 .
2. Rheumatoid Arthritis
RA is prevalent (0.8%) throughout the world and affects all races [ 5 , 84 ]. Women are affected approximately three times more often than men. The age of onset is between late twenties and early fifties, but no age is immune to the disease. In children and young adults, the disease manifests as juvenile chronic arthritis (JCA). RA is a chronic multisystem disease characterized by persistent inflammatory synovitis usually involving peripheral joints in a symmetric distribution [ 5 , 85 ]. The synovial inflammation, if uncontrolled, may lead to cartilage damage, bone erosions, and ankylosis of the affected joints [ 5 ]. Twin studies and family studies indicate that there is a genetic predisposition to RA [ 86 ], and about 70% of patients have HLA-DR4 or -DR1 alleles or both. The precise target autoantigen for RA has not yet been identified. Type II collagen (CII), aggrecan, immunoglobulin binding protein (BiP), and heat-shock protein 65 (Hsp65) are among the antigens that have been implicated in the pathogenesis of RA [ 5 , 85 ]. As mentioned above, NSAIDS are the mainstay of therapy for a large proportion of patients with RA. However, because of adverse reactions, high costs, and limited efficacy of these drugs in many patients [ 3 – 5 ], the use of CAM by RA patients is becoming increasingly popular in USA and other developed countries [ 9 , 10 , 13 , 14 , 16 , 20 ].
3. Adjuvant Arthritis: An Experimental Model of RA
AA can be induced in the Lewis (RT.1 l ) rat by immunization with heat-killed Mycobacterium tuberculosis (Mtb) (H37Ra) [ 87 ]. The disease manifests as inflammation of the paws including the paw joints. The paw inflammation affects primarily the ankles, wrists, and smaller joints. The arthritic inflammation starts after 8–10 days, peaks between day 15–17, and then undergoes a spontaneous, gradual recovery in the subsequent 12–15 days ( Figure 1 ). The primary immune reaction in paw joints is the mononuclear cell infiltration of the synovial tissue, which if uncontrolled, can lead to damage to cartilage and bone [ 87 ]. Mycobacterial hsp65 (Bhsp65) has been invoked in the pathogenesis of AA. Following Mtb injection, Bhsp65 is taken up by the regional draining lymph nodes where antigen-presenting cells (APCs) process and present this antigen to naïve T cells ( Figure 3 ). The T cells bearing receptors specific for epitopes within Bhsp65 then get activated and undergo proliferation. These antigen-primed T cells then leave the lymph nodes to enter into the peripheral circulation. These T cells then migrate out from the blood vessels into the target organ, the joint, where they initiate the immune pathology ( Figure 3 ). Rat AA shares several features with human RA, and thereby, it serves as an excellent model for RA [ 87 ].
The AA model has extensively been used for studies regarding the pathogenesis of autoimmune arthritis [ 32 , 43 , 88 , 89 ] as well as for the testing of new natural [ 33 , 34 , 58 ] or synthetic antiarthritic therapeutic products. A variety of herbal CAM products have been shown to attenuate the severity of the disease in the rat AA model ( Table 1 ). These herbs modulate different immunological effector and regulatory pathways (discussed below in detail) involved in the disease process (Figures 2 and 3 ). Another model of chronic inflammation leading to bone loss has also been employed to examine the role of natural products (e.g., green tea) in limiting bone damage and bone loss, which accompanies chronic arthritis [ 90 , 91 ].
4. Heat-Shock Proteins (Hsps) Serve as the Target Antigens in Autoimmune Arthritis
Hsps have been associated with many autoimmune diseases such as RA, Crohn's disease, MS, and systemic lupus erythematosus (SLE) [ 92 ]. Hsps may also induce protection against arthritis [ 93 ]. Most hsps are acute stress reactants that insure cell survival under hostile conditions. They also are the molecular chaperones involved in protein folding and other functions for maintaining the structural integrity of other proteins [ 92 ]. A T-cell clone that was arthritogenic for the Lewis rat was found to be specific for the epitope 180–188 (p180–188) of Bhsp65 [ 88 ]. In juvenile chronic arthritis (JCA) patients, there was T-cell reactivity to p180–188 of Bhsp65, as well as to the partially homologous determinant within articular cartilage link protein [ 94 ]. The T cells of these patients also showed significant response to human hsp60 [ 95 ] and Bhsp65 [ 94 , 96 ], emphasizing the importance of Hsp65 as one of the major antigens in arthritis pathogenesis. Other hsps, including hsp70 and hsp47 have also been invoked in the pathogenesis of AA [ 97 ]. Similarities in immune response to disease-related antigens in rodent models and humans have provided useful insights into the disease pathogenesis.
It has been shown that natural plant products such as flavonoids and Celastrol can alter the cellular expression of hsps [ 98 – 100 ]. These observations are potentially of significance in view of the observed disease-protective effects of green tea flavonoids [ 33 ] and the ethanol extract of Celastrus [ 58 ]. However, the precise mechanistic link between the two sets of observations remains to be fully defined. In regard to the modulation of antigen-specific T-cell response to hsps by CAM modalities, we have shown that feeding of the Chinese herbal formula Huo Luo Xiao Ling (HLXL) dan to Lewis rats significantly reduced the T-cell proliferative response to Bhsp65 [ 34 ], and this effect was associated with suppression of arthritis. On the contrary, feeding of rats with green tea extract [ 33 ] or Celastrus [ 58 ] failed to influence T-cell proliferative response to Bhsp65 despite significant reduction in the severity of AA. However, as described below, these rats showed significant changes in cytokine response to Bhsp65 but without any change in T-cell proliferation, revealing a dichotomy of the two immune parameters. In support of our results, several other natural products (e.g., Quercetin, Resveratrol, Kaempferol, Vineatrol) have also been reported to influence lymphocyte proliferative responses, with most of them leading to inhibition of proliferative response [ 101 – 103 ]. Also reported is the dissociation between proliferation and function of lymphocytes exposed to natural products like Kaemferol [ 103 ].
5. CD4+CD25+ T-Regulatory Cells (Treg) Are Vital for Self-Tolerance and Regulation of Autoimmunity
Many types of regulatory T cells, including Th2, Th3, Treg, NKT cells, and Tr1 have been described [ 104 ]. The most recent addition to the group of regulatory T cells is the CD4+CD25+ T-regulatory cell (Treg) [ 105 – 107 ]. Treg have emerged as the central controllers of autoimmunity in a variety of experimental models of human autoimmune diseases [ 105 – 107 ]. Importantly, in animal models, CD4+CD25+ T-cell therapy via adoptive transfer of cells can effectively delay and suppress a variety of immunological diseases including diabetes, colitis, and gastritis [ 105 – 107 ]. On the contrary, the in vivo depletion of Treg leads to the early initiation and/or aggravation of autoimmune arthritis [ 106 – 109 ] as well as other autoimmune diseases.
There are two distinct types of Treg: the “natural Treg” that developed in the thymus and the “adaptive (induced) Treg” that developed in the periphery in response to antigen exposure [ 106 ]. The mechanism of action of Treg involves cell-cell contact between Treg and the responder cells, and it requires activation of Treg via the T-cell receptor (TCR) [ 105 – 107 ]. Secreted TGF- β and IL-10 have been suggested to mediate suppression by Treg in vivo.
There is evidence for a reciprocal control of the differentiation of T helper 17 (Th17) and Treg. The differentiation of the proinflammatory and pathogenic Th17 cells is induced by the simultaneous presence of TGF- β and IL-6, whereas the presence of TGF- β alone induces the generation of Treg expressing the transcription factor Foxp3 [ 110 ].
Over the last 5–10 years, the significance of determining the frequency and suppressive function of Treg for evaluating the autoimmune disease process as well as assessing the efficacy of therapeutic products for different autoimmune diseases is increasingly being realized [ 105 – 107 , 111 ]. Defects in Treg have been reported in RA [ 112 ], and this reduced activity of Treg can be restored following successful therapy, for example, with anti-TNF- α in the case of RA [ 112 ]. A recent study in the area of transplantation research has shown that dendritic cells treated with triptolide (derived from the Chinese herb Tripterygium wilfordii ) promotes the expansion of Treg in vitro [ 57 ]. It is hoped that assessment of Treg number and function would be incorporated regularly in studies aimed at defining the mechanisms of action of various CAM modalities, including natural plant products. Also, as described above, there are various other types of regulatory T cells besides Treg [ 104 ]. It is likely that different CAM modalities might have differential effect on distinct subsets of regulatory T cells, such that one product may have a more pronounced effect on Treg, while the other might instead have a major effect on Th2 or Tr1 type of regulatory cells.
6. Antibodies Contribute to the Pathogenesis of Autoimmune Arthritis
Studies in a spontaneous model of autoimmune arthritis have underscored the importance of antibodies in mediating the immune pathology in this disease; the pathology is initiated by T cells but subsequently perpetuated by antibodies [ 113 ]. Studies in the CIA model in mice have clearly demonstrated the importance of antibodies to type II collagen (CII) in the disease process [ 114 ]. However, at this time there is not much information about the physiologic role of antibodies to hsp65 in AA. The pathogenic effect of anti-Bhsp65 antibodies in AA has not been excluded formally. On the contrary, there is evidence from work done by other investigators and us [ 35 , 115 ] pointing to the protective effect against AA of anti-Bhsp65 antibodies. In one study, the AA-protective effect of anti-Bhsp65 antibodies was attributed to the production of IL-10 from mononuclear cells [ 115 ].
In the CIA model, extracts of green tea [ 59 ] pomegranate [ 60 ], and Taxol [ 63 , 64 ] have been shown to suppress arthritis, and this effect was associated with a significant decrease in anti-CII antibodies. A similar effect on the clinical disease, the proinflammatory cytokines, and the serum IgG2a was reported in another study on CIA following treatment with curcumin, a major component of turmeric [ 61 ]. Turmeric extract has also been shown to induce protection against arthritis in streptococcal cell wall-induced arthritis model of RA [ 78 ]. In one of our studies based on the AA model, we observed that feeding the polyphenolic extract of green tea to Lewis rats resulted in a significant decrease in the antibody response to Bhsp65 [ 33 ]. Similar results were obtained with a traditional Chinese medicine, HLXL, which is a mixture of 11 different herbs [ 34 ]. In both cases, the decrease in antibody response was associated with a corresponding reduction in the severity of arthritis. However, not all antiarthritic herbs tested by us caused a decrease in antibody response to Bhsp65. In another study, we observed the opposite as the feeding of Celastrus to Lewis rats led to an increased anti-Bhsp65 antibody response despite a significant suppression of clinical arthritis [ 58 ].
At present, we do not have additional information to clarify the differences in the functional attributes of the antibody subsets that are predominantly altered following feeding of different plant products. However, we propose that anti-Bhsp65 antibodies produced during the course of AA in the Lewis rats belong to two main categories, pathogenic and protective [ 35 ]. In this context, we suggest that different herbs target distinct subsets of antibodies such that reduction in clinical arthritis might involve either the suppression of pathogenic antibodies or the enhancement of protective antibodies, or both. Furthermore, studies of antibody patterns using a panel of antigens [ 40 , 41 ] targeted in arthritis and other autoimmune diseases might provide a useful readout for the effect of CAM products on the disease process.
7. Regulation of Autoimmunity via T-Helper (Th1)-/Th2-Type Cytokine Balance
Proinflammatory cytokines TNF- α , IL-1 β , and IL-6 produced by macrophages and other immune cells are of critical importance in the initiation and propagation of arthritis [ 116 , 117 ]. Among the T cell, the Th1 cells secrete IFN- γ and TNF- α , whereas the counter-regulatory Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 [ 118 ]. Th1 cells are primarily involved in the pathogenesis of certain organ-specific autoimmune diseases, whereas Th2 cells play a major role in systemic autoimmunity. The role of Th1-Th2 balance in regulation of autoimmunity has been validated through several animal model studies. The susceptibility or resistance to disease [ 119 ] and protection from disease [ 120 ], as well as improvement of the disease in RA patients [ 117 ] was associated with a change in cytokine balance to Th2 type. The change in Th1/Th2 balance could occur either by a decrease in the proinflammatory cytokine (e.g., IFN- γ ) or an increase in the anti-inflammatory cytokine (e.g., IL-4/IL-10), or both [ 42 , 118 ].
In a study on CIA, activation of the Th2 response was shown to inhibit IFN- γ production as well as reduction in the severity of arthritis [ 121 ]. Other investigators have reported the downmodulation of CIA coupled with suppression of proinflammatory cytokines (e.g., TNF- α , IL-1 β , and IL-6) by treatment of mice with extracts of green tea [ 59 ], pomegranate [ 60 ], or Plectranthus amboinicus [ 68 ]. A similar effect has been observed in vitro with Moutan cortex [ 122 ]. In 3 separate studies in AA using different natural plant products, namely, Celastrus [ 58 ], green tea [ 33 ], and HLXL [ 34 ], we observed that each of these three herbal products induced protection against AA coupled with an altered Th1/Th2 ratio. The latter effect was caused primarily by an increase in IL-10 while IFN- γ remained unchanged. Enigmatically, it has also been observed that proinflammatory Th1 cytokines such as IFN- γ and TNF- α might display dual roles as inflammatory and immunosuppressive cytokines [ 123 ]. For example, the suppression of inflammation by IFN- γ has been observed in AA [ 123 ]. Therefore, herbal CAM-induced changes in the level of certain cytokines with dual functions need to be evaluated with caution.
8. T-Helper 17 (Th17) Cells Mediate Inflammation and Tissue Damage in Arthritis
Th17 cells secrete IL-17, which has been shown to be involved in inflammatory and autoimmune diseases [ 110 , 124 ]. Th17 subset of T cells is distinct from Th1 and Th2 cells, and the differentiation of Th17 cells is induced by the concurrent exposure to TGF- β and IL-6 [ 110 ]. Retinoic acid-related orphan receptor gamma-t (ROR γ t) is the transcription factor required for the differentiation of Th17 cells. IFN- γ , IL-2, IL-4, and IL-27 have been shown to inhibit the activity of Th17 cells, whereas IL-21 and IL-23 are important for the clonal expansion and stabilization (maintenance) of Th17 cells [ 110 ]. IL-17 has been implicated in the pathogenesis of autoimmune diseases including arthritis [ 125 ]. Abundant quantities of IL-17 have been found in the synovial fluid of RA patients [ 125 ]. The in vivo blockade of IL-17 by soluble IL-17 receptors or by neutralizing anti-IL-17 antibody can significantly attenuate arthritis in rodents [ 126 ]. Furthermore, mice deficient in IL-17 [ 127 ] or IL-17 receptor [ 128 ] were found to be resistant to the induction of CIA.
In the preceding section, we have summarized the results of our earlier studies showing that a shift in Th1 to Th2 ratio induced by natural plant products was associated with reduced severity of AA in Lewis rats [ 33 , 34 , 58 ]. In two of these studies, we also tested the IL-17 response. Importantly, feeding rats with green tea [ 33 ] or HLXL [ 34 ] led to a significant reduction in IL-17 response. Thus, the concurrent changes in Th1/Th2 ratio and IL-17 response culminated into a beneficial antiarthritic activity of green tea and HLXL.
9. Chemokines and Adhesion Molecules Orchestrate the Migration of Leukocytes into the Target Organ in Arthritis
The migration of lymphocytes, macrophages, and other cells from blood into the joints is orchestrated by defined interactions mediated by chemokines and adhesion molecules [ 129 , 130 ]. Chemokines are chemoattractant cytokines that direct the migration of leukocytes from blood vessel lumen into the target site of inflammation in the periphery. The expression of chemokines and their receptors is influenced by cytokines and other inflammatory mediators. Dysregulated expression of chemokines and/or their receptors may lead to immune pathology. The blocking or neutralization of these molecules via antagonists or antibodies is being explored for the treatment of arthritis in experimental models [ 131 , 132 ] and RA patients [ 133 , 134 ]. Thus, study of the levels of expression of different chemokines and adhesion molecules, and the blockade of these biomolecules by appropriate reagents can serve as an important tool for defining the mechanisms of actions of CAM products that have antiarthritic activity.
Many herbal products have been reported to modulate the expression of specific chemokines in different tissues [ 135 – 142 ], and many of these chemokines are relevant for the trafficking of leukocytes into the joints in arthritis as well [ 129 , 130 ]. In one of our studies, we reported a simple method to study the in vivo migration of radiolabeled leukocytes in vivo [ 44 ]. We also showed a clear association between the kinetics of migration of leukocytes through the joints and the susceptibility to AA [ 44 ]. The radiolabel can be replaced by a fluorescent dye as needed for future use of such assays in CAM studies.
10. Concluding Remarks
This paper is focused on cellular and humoral immunological effector mechanisms that mediate the action of a wide variety of herbal CAM for the treatment of experimental autoimmune arthritis. However, natural products can contribute to the suppression of inflammation and arthritic processes via altering specific molecular mediators of these pathways. For example, the antiarthritic activity of various compounds (Tea polyphenols, Boswellic acid, Morin, etc.) purified from natural products has been attributed in part to their anti-oxidant activity [ 33 , 59 ] and to their action on nuclear factor-kB (NF-kB), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and matrix metalloproteinases (MMPs) (reviewed in [ 18 ]). Therefore, future studies on herbal products would benefit from including test parameters that span across pathological, immunological, biochemical, and molecular biology-related aspects of the disease process. For immunological aspects, we hope to see more CAM studies both in vitro and in vivo on the newer cytokines (e.g., the IL-17/IL-23 axis) and Treg. Furthermore, the study of genomics and proteomics of CAM [ 143 – 145 ] is representative of several modern research tools whose investment in CAM research is currently underway. This in turn would not only enhance the depth and scope of investigations into CAM research, but also provide an interface where CAM and conventional medicine could find a common ground for understanding the mechanisms of action of therapeutic products and their practical use for the ultimate benefit of the patients. It is rather difficult to predict with certainty the natural products or compounds that might end up as successful therapeutic agents for RA. Nevertheless, on the basis of the results obtained from animal models of RA as well as the delineation of multiple immunological and molecular targets of the indicated herbal products, we find Tea polyphenols, Celastrol, Triptolide, Curcumin, Boswellic acids, and HLXL as promising candidates for further preclinical and clinical trials in RA. | Acknowledgments
The authors thank Hua Yu, Ying-Hua Yang, and Steva Komeh-Nkrumah for their helpful critique and suggestions, and Siddaraju Nanjundaiah for his help with the fine artwork. This work was supported by Grants (R01AT004321, P.I.: KDM, and PO1 AT002605, P.I.: BMB) from the National Center for Complementary and Alternative Medicine, National Institutes of Health, Bethesda, MD, USA. | CC BY | no | 2022-01-13 02:20:17 | Evid Based Complement Alternat Med. 2011 Dec 22; 2011:986797 | oa_package/9f/d6/PMC3014691.tar.gz |
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PMC3014692 | 21234399 | 1. Introduction
1.1. Clinical and Economical Importance of Diabetes Mellitus
According to an estimation of the World Health Organization (WHO) in November 2009, more than 220 million people worldwide suffer from diabetes mellitus, a figure that will probably double by the year 2030. The economic and human costs of this disease are devastating. WHO estimates that in the period 2006–2015, China will lose $558 billion in foregone national income due to heart disease, stroke, and diabetes alone. The total cost of diabetes in the United States in 2002 was $132 billion. According to the statistics of the NIH in 2007, 23 million Americans had diabetes, and the estimated lifetime risk for Americans born in 2000 to develop diabetes is 1 in 3. In adults, diabetes is the most common cause of blindness, nontraumatic amputations, and end-stage renal disease as well as the sixth most common cause of death. In Germany, every 19 minutes a person with diabetes suffers a heart attack. With regard to these facts, it is of high clinical and economical importance, but also in the interest of the patients and their quality of life, to find new therapeutic options for the treatment of diabetes. Primarily, this research should be focused on the treatment of the metabolic disorder underlying diabetes to stop or slow down its progression. But also the treatment of diabetes symptoms and complications need to be addressed to improve the quality of life and prognosis of diabetic patients. Yet only limited knowledge exists about the pathogenesis, the cardiovascular consequences, and the prevention of this disease [ 1 ]. Recent data suggest that the cardiovascular complications in diabetes mellitus are associated with oxidative stress [ 2 , 3 ], as previously shown for hypercholesterolemia and arteriosclerosis [ 4 , 5 ]. The sources of these reactive oxygen and nitrogen species (RONS) have been identified to be NADPH oxidases, mitochondria, xanthine oxidase, as well as an uncoupled endothelial nitric oxide synthase (eNOS) [ 6 – 8 ]. The present review will address the question whether organic nitrates may be used for treatment of diabetic patients, and if there are differences between the organic nitrates being in clinical use at present.
1.2. Biochemical and Metabolic Consequences of Diabetes Mellitus
The relationship between diabetes and the resulting cardiovascular complications is complex, but damage to the vascular endothelium obviously plays a major role [ 9 ]. Chronic exposure of proteins from plasma and the cell membrane to hyperglycemia leads to the attachment of glucose molecules to proteins, a process known as nonenzymatic glycosylation. Subsequent slow reactions cause the formation of advanced glycation end products (AGEs) [ 10 ] known to inactivate nitric oxide (NO) and impair endothelium-dependent vasodilation [ 11 ]. However, short-term hyperglycemia leads to similar negative effects on vascular responses, suggesting that this is not the only mechanism [ 12 ]. It was shown that hyperglycemia inhibits eNOS-dependent NO formation in favour of superoxide production, leading to an impaired endothelium-dependent vasodilation [ 13 ]. Besides the formation of advanced glycation end-products and diacylglycerol (DAG), the simultaneous increase in production of reactive oxygen species (especially superoxide, O 2 •− ) contributes to inhibition and uncoupling of eNOS via depletion of BH 4 (summarized in Figure 1 ). Moreover, in diabetic patients, a higher production of vasoconstrictive compounds, such as endothelin-1 and angiotensin-II, has been observed. The increase in DAG and angiotensin-II is a direct consequence of the activation of the renin-angiotensin-aldosterone system (RAAS) in the setting of diabetes [ 14 ], which is also closely related to endothelial dysfunction [ 15 , 16 ]. Since DAG is a potent activator of the protein kinase C [ 17 , 18 ] with subsequent activation of the NADPH oxidase [ 19 ], RAAS-induced activation of this superoxide source was demonstrated by clinical [ 20 ] as well as animal experimental studies [ 21 ]. There is even some evidence that suppression of the RAAS is able to prevent the development or progression of diabetes mellitus type II in hypertensive patients [ 22 ]. The events taking place during activation of the RAAS are summarized in Figure 2 .
Leukocyte infiltration into the vascular wall was also dramatically increased. The increased concentrations of cytokines and matrix metalloproteinases lead to a decreased synthesis and increased degradation of collagen in atherosclerotic plaques reducing the stability of the fibrous cover plate, finally causing plaque rupture, thrombus formation, and myocardial infarction. In addition, the thrombocytes in diabetic individuals are larger in size, express more glycoprotein receptors on the surface, and show more pronounced aggregation in response to different stimuli (for review see [ 23 ]). Patients with diabetes mellitus have a 2- to 4-fold increased risk for infarction, and the mechanistic basis for macrovascular complications is progression of atherosclerosis. Endothelial dysfunction is a hallmark in the early stage of diabetes mellitus type 1 and 2 [ 24 ]. Oxidative protein modifications such as 3-nitrotyrosine formation [ 25 ], oxidative disruption of the zinc-sulphur-cluster, and uncoupling of eNOS [ 26 ] as well as increased levels of toxic aldehydes [ 27 ] are common features of diabetes and may contribute to the observed disorders. A broad variety of different antioxidants have been demonstrated to beneficially influence diabetic complications [ 27 – 30 ]. | 4. Conclusion and Clinical Implications
The endothelium plays a pivotal role in modulating the reactivity of vascular smooth muscle through the formation of several vasoactive substances. There is good evidence for endothelial and smooth muscle dysfunction in diabetes which is shared by the pharmacologically-induced phenomenon of nitrate tolerance. Oxidative stress plays an important role in the setting of both vascular complications and may explain the presence of nitrate resistance in diabetic vessels, a major drawback for the use of nitrates in diabetic patients. Since nitrate tolerance as well as diabetes-associated vascular dysfunction nicely respond to antioxidant treatment, this may be the key to improve the safety and efficacy of a given organic nitrate in diabetic patients. One strategy could be a combination therapy of an antioxidant (e.g., hydralazine or lipoic acid) for which a relief of nitrate tolerance has been previously demonstrated. The combination of ISDN and hydralazine improved the efficacy of the nitrate dramatically in black patients with heart failure. Another promising attempt would be the use of pentaerithrityl tetranitrate which is devoid of nitrate tolerance and oxidative stress due to induction of antioxidant pathways (e.g., HO-1 and ecSOD). It remains to be established whether these highly promising observations, that are mainly based on animal experimental data, may be translated to the clinical situation. First evidence for beneficial effects of PETN treatment in diabetic patients comes from a cost comparison analysis contrasting PETN and ISDN prescribed to diabetic patients in primary care practices in Germany [ 139 ]. PETN therapy tended to produce a saving in costs compared to ISDN therapy in diabetic patients when costs for comedication were taken into account and after adjustment for age and comorbidity. Future effort may also be directed towards the development of new organic nitrates that are devoid of tolerance and oxidative stress. A promising strategy is the synthesis of hybrid molecules by introduction of a nitrate function into established cardiovascular drugs such as AT 1 -receptor blockers or glitazones (both drug classes have been demonstrated to prevent the development of nitrate tolerance). These compounds not only could possess intrinsic antioxidant effects, but also provide synergistic antihypertensive, antihyperglycemic, and antiatherosclerotic effects. | Academic Editor: Norman Cameron
Organic nitrates represent a class of drugs which are clinically used for treatment of ischemic symptoms of angina as well as for congestive heart failure based on the idea to overcome the impaired NO bioavailability by “NO” replacement therapy. The present paper is focused on parallels between diabetes mellitus and nitrate tolerance, and aims to discuss the mechanisms underlying nitrate resistance in the setting of diabetes. Since oxidative stress was identified as an important factor in the development of tolerance to organic nitrates, but also represents a hallmark of diabetic complications, this may represent a common principle for both disorders where therapeutic intervention should start. This paper examines the evidence supporting the hypothesis that pentaerithrityl tetranitrate may represent a nitrate for treatment of ischemia in diabetic patients. This evidence is based on the considerations of parallels between diabetes mellitus and nitrate tolerance as well as on preliminary data from experimental diabetes studies. | 2. Organic Nitrates
2.1. Clinical Use of Organic Nitrates
Nitroglycerin (glyceryl trinitrate) and the long-acting nitrates (isosorbide-5-mononitrate [ISMN], isosorbide dinitrate [ISDN], and pentaerithrityl tetranitrate [PETN]) have been used in cardiovascular medicine for >100 years. Despite the fact that the potential mechanisms for relief of myocardial ischemia with nitrates are multiple and not yet fully understood [ 32 ], nitrates are widely utilized for the various anginal syndromes, and are also used in congestive heart failure and in patients with left ventricular dysfunction [ 33 , 34 ]. The nitrovasodilators are a group of drugs that result in the formation of NO or a related species within vascular smooth muscle cells [ 35 ]. Following the discovery of endothelium-derived NO [ 36 ], the idea of NO being the active principle of nitrates was very attractive to the scientific community, and it led to the speculation that they may replace a compromised endothelial NO production [ 37 ], such as in patients with coronary heart disease. Nitrates also have antiaggregatory effects, and recent evidence confirms that these drugs decrease platelet aggregation and thrombosis formation [ 38 ], although for nitroglycerin, an activation of thrombocytes was described [ 39 , 40 ]. This may play an important role in the therapy of acute unstable myocardial ischemia, including unstable angina and myocardial infarction. The hemodynamic effects of nitrates were known for a long time. They are primarily modulated by decreased myocardial work that results from smaller cardiac chambers that operate with lower systolic and diastolic pressures. These changes are secondary to a redistribution of the circulating blood volume from cardiac tissue to the venous capacitance system, with a decrease in venous return to the heart. The effects of nitrates on afterload as well as on arteries are also useful for the decrease in myocardial oxygen consumption. Considerable evidence supports a variety of mechanisms whereby nitrates lead to increased coronary blood flow, including stenosis enlargement, epicardial coronary artery dilation, improvement of endothelial dysfunction, enhanced collateral size and flow, and prevention or reversal of coronary artery vasoconstriction [ 33 ].
2.2. Bioactivation of Organic Nitrates
There is a consensus that the principle mechanism of organic nitrate-induced smooth muscle relaxation is achieved via NO signalling pathways, including activation of soluble guanylyl cyclase, increase in cyclic GMP levels and activation of cyclic GMP-dependent protein kinases, and/or cyclic nucleotide-gated ion channels which was extensively reviewed [ 32 ]. Recently, the organic nitrate-derived NO hypothesis was challenged by two independent studies using state-of-the-art-methods to detect nitrate-derived NO formation which showed a huge discrepancy between nitroglycerin-derived NO and NO-induced vasodilation [ 35 , 41 ]. This, however, questions the relevance of nitroglycerin-derived NO formation in the clinically relevant concentration range whereas NO was detected for ISMN and ISDN during vasodilation of isolated aortic rings. For nitroglycerin, several bioactivating systems have been proposed during the last decades including xanthine oxidoreductase, glutathione-S-transferases, and cytochrome P450 enzymes (for review see [ 32 , 34 ]). It should be noted that these reports on nitroglycerin-derived NO formation were based on high suprapharmacological concentrations (>10 μ M) of the nitrate. Mononitrates and dinitrates have been reported to be mainly bioactivated by cytochrome P450 enzymes. In 2002, the mitochondrial aldehyde dehydrogenase (ALDH-2) was identified as an organic nitrate bioactivating enzyme [ 42 ]. This so-called nitrate reductase activity denitrates nitroglycerin to its 1,2-glycerol dinitrate metabolite and nitrite. This reaction relies on reduced thiols at the active site of the enzyme and on the presence of reduced dithiols as the electron source. During bioconversion of nitroglycerin, but also in the presence of reactive oxygen and nitrogen species, the active site thiols of ALDH-2 are oxidized, and the enzyme loses its activity [ 43 ]. Interestingly, ALDH-2 bioactivates nitroglycerin, PETN, and its trinitrate metabolite PETriN but not ISMN and ISDN [ 44 ], which could explain the different potency of these nitrates.
2.3. Clinical Tolerance, Endothelial Dysfunction, and Differences between Organic Nitrates
Tolerance and cross-tolerance to endothelium-dependent vasodilators (endothelial dysfunction) are a major limitation for chronic nitrate treatment. Clinical tolerance and cross-tolerance may involve counterregulatory mechanisms (pseudotolerance) including release of catecholamines and activation of the renin-angiotensin-aldosterone system (RAAS) making these phenomena even more complicated [ 32 ]. The first evidence for an involvement of the renin-angiotensin-aldosterone-system (RAAS) in GTN-induced nitrate tolerance came from observations that long-term inhibition of angiotensin-converting enzyme improves nitrate tolerance in dogs [ 45 , 46 ]. Additional studies showed that AT 1 -receptor blockade and ACE inhibition positively influence the development of tolerance, and it was hypothesized that this protective effect is mediated via inhibition of NADPH oxidase-dependent ROS formation [ 45 , 47 – 53 ]. In contrast, there is a limited number of studies reporting on lack of effect of ACE inhibitors and AT 1 -receptor blockers on nitrate tolerance [ 54 – 56 ] and nitrate-induced oxidative stress [ 57 ]. Downstream to the RAAS system is the protein kinase C (PKC), which was also identified as an important constituent in the development and maintenance of GTN-induced nitrate tolerance [ 58 – 60 ]. Moreover, it was demonstrated that GTN-induced activation of PKC mediates late preconditioning, a protective effect which is probably based on initial ROS formation and mild oxidative damage [ 61 ]. The contribution of NAD(P)H oxidases to ROS production and oxidative damage in response to GTN therapy was established by a number of independent studies [ 62 – 64 ]. Nevertheless, NADPH oxidase, as a source of ROS formation in the setting of tolerance, was questioned since DPI, an unspecific inhibitor of flavin-dependent oxidoreductases, could not prevent the impaired response to GTN in tolerant vessels ex vivo [ 65 ]. In summary, the majority of previous work supports the contribution of the RAAS, PKC, and NADPH oxidase to the development of nitrate tolerance (the sequence of events is provided in Figure 2 ) although some studies neglect the importance of this pathway.
Tolerance and/or endothelial dysfunction have been observed in response to chronic treatment with nitroglycerin [ 66 , 67 ], ISMN [ 68 ], and ISDN [ 69 ] (studies before 1990 reviewed in [ 70 ]) whereas no tolerance phenomena or markers such as oxidative stress were observed for long-term administration of PETN [ 67 , 71 ]. These reports indicate that tolerance development is uniform to all organic nitrates if applied in clinically effective dosage for longer periods of time. A remarkable exception seems to be PETN [ 72 ]. Some of these effects are related to special pharmacokinetics of PETN [ 73 , 74 ], but, upon chronic administration, PETN also induces antioxidant pathways at the genomic level and cardioprotective genes (e.g., Apex1 and NFAT5) [ 75 ]. Among these induced antioxidant genes are heme oxygenase-1 (HO-1) and ferritin [ 76 ], both possessing highly protective properties. There is good experimental evidence that at least part of the beneficial profile of long-term PETN treatment is based on activation of the heme oxygenase-1/ferritin system [ 77 ] and preliminary data in heme oxygenase-1 knockout mice support these previous findings. According to recent, unpublished data from our laboratory, PETN even acts differently at the purified ALDH-2, preventing inactivation of this nitrate reductase. Since ALDH-2, beyond bioactivating organic nitrates, was recently identified as an important protective enzyme, preventing ischemic damage in experimental myocardial infarction [ 78 ], the conservation of the enzymatic activity of ALDH-2 by an organic nitrate may be of great importance for long-term treatment of patients with ischemic heart disease. Nitroglycerin not only inactivates ALDH-2 but also downregulates the enzyme at the protein level [ 79 ]. More differences between organic nitrates in clinical use were recently discussed in detail [ 80 ]. The differences between nitroglycerin and PETN are summarized in Figure 3 .
2.4. Nitrate Tolerance and the “Oxidative Stress Concept”
The “oxidative stress concept” in the setting of nitrate tolerance was established by Munzel and colleagues [ 82 ] and refined during the last couple of years [ 66 , 83 ]. In essence, the concept consists of increased superoxide formation in response to nitrate treatment which decreases NO bioavailability, leads to peroxynitrite formation, NOS uncoupling, and impairs NO/cyclic GMP signalling [ 66 ]. Moreover, oxidative inhibition of prostacyclin synthase [ 84 ] as well as mitochondrial ALDH activity [ 85 ] may present other key events in the development of nitrate tolerance. Previously, the vascular NADH oxidase was considered as the most important source of reactive oxygen species (ROS) in the setting of nitrate tolerance [ 32 ]. Today this concept has been extended to induction of mitochondrial oxidative stress by nitroglycerin treatment [ 83 , 86 ] and a crosstalk between mitochondrial ROS with NADPH oxidases in the cytoplasmic membrane [ 87 ]. Induction of oxidative stress was also described for ISMN in experimental hypertension [ 88 ], and indirect evidence was presented recently by demonstrating that the antioxidant vitamin C reverses ISMN-induced endothelial dysfunction in healthy volunteers [ 68 ]. However, until now, there is no direct proof for induction of oxidative stress by ISMN from clinical trials [ 89 ]. For ISDN, increased activation of NADPH oxidase was demonstrated in experimental MI as well as the adverse effects of the nitrate on the function of endothelial progenitor cells [ 90 ] although previous work by Keimer et al. revealed no induction of oxidative stress under ISDN or PETN therapy in healthy volunteers [ 91 ]. Indirect evidence for a role of oxidative stress in ISDN therapy comes from the highly beneficial effects of hydralazine combination therapy [ 92 ] since hydralazine is a powerful antioxidant [ 93 ] and has been demonstrated to prevent nitroglycerin-induced tolerance [ 62 ]. Related to these observations on adverse effects of nitrate therapy, Nakamura et al. presented evidence that long-term nitrate therapy increases cardiovascular mortality based on a retrospective analysis using databases from two large-scale postinfarction studies [ 94 ]. It should be noted that this analysis did not distinguish between different nitrates although there is increasing evidence that organic nitrates should not be treated as a homogenous class of compounds. Moreover, it is important to keep in mind that PETN did not share the adverse effects of the other nitrates since it was devoid of tolerance [ 67 , 71 , 74 ], induction of oxidative stress [ 71 , 90 ] and downregulation of protective genes [ 75 , 77 ]. Recent data has shown that nitroglycerin and PETN, although both considered as NO donors, exert completely opposite effects with respect to gene regulation: PETN in vivo treatment induced cardioprotective genes (e.g., Apex1 and NFAT5) whereas nitroglycerin in vivo administration induced cardiotoxic genes (e.g., Egr1, fos, junD, and MITF) [ 75 ]. Another highly important property of PETN may be the induction of the extracellular superoxide dismutase (ecSOD) [ 95 ] and glutathione peroxidase (GPx) [ 96 ], two highly protective antioxidant proteins. These experimental and clinical data on the beneficial effects of PETN were recently supported by the double-blind, placebo-controlled PENTA study conducted by Warnholtz and colleagues with the main finding that chronic PETN treatment does not induce endothelial dysfunction or nitrate tolerance, but even improves GTN potency [ 97 ]. Relative changes in mean flow volume and mean flow velocity upon ischemia increased in the PETN group versus controls.
3. Use of Organic Nitrates for Therapy of Diabetic Patients
3.1. Nitrate Resistance and Diabetes
The endothelial dysfunction present in hypercholesterolemic, type 2 diabetic, smoking, and ischemic heart disease patients reflects the reduced nitric oxide bioavailability, which can be assessed by different methods [ 98 ]. Patients with chronic heart failure did not show a significant effect in response to the NOS inhibitor N G -methyl-L-arginine suggesting that NO does not contribute to basal vascular tone in these individuals. The responses to nitroglycerin and to serotonin were impaired, suggesting that there is smooth muscle dysfunction and endothelial dysfunction in patients with chronic heart failure [ 99 ]. Nitrate tolerance can be seen as an extreme example of nitrate resistance [ 100 ], and is thought, at least in part, to be secondary to the increased oxidative stress present in the aforementioned disease states. Thus, clinical nitrate tolerance and nitrate-induced activation of the renin-angiotensin-aldosterone system (RAAS) as well as increased oxidant stress (see Section 2.3 ) may be regarded as an extension of the primary pathophysiological phenomenon of nitric oxide resistance (see Figure 2 ) [ 100 ]. Since diabetes is also associated with severe activation of the RAAS (see Section 1.2 ), the effects of diabetes and nitrate therapy could be additive and further impair endothelial function as well as nitrate potency. Hence, the potency of nitrates may be blunted by an underlying primary NO resistance to give a “primary” nitrate tolerance [ 101 ] whereas the mechanistically similar “secondary” nitrate tolerance (pseudotolerance) develops under chronic treatment [ 102 ]. These two nitrate tolerance states can even occur within the same patient. However, to date, it is not completely clarified whether pseudotolerance (secondary nitrate tolerance) contributes to endothelial dysfunction in response to an impaired NO responsiveness (NO resistance). However, evidence supporting this notion comes from a multivariate analysis of two observational coronary secondary prevention studies which showed increased mortality in chronically nitrate-treated ischemic heart disease patients following recovery from an acute cardiac event [ 94 ]. Also, a randomized controlled trial was performed and showed that continuous nitroglycerin treatment leads to endothelial dysfunction in both ischemic heart disease patients [ 103 ] and healthy volunteers [ 104 ]. In a series of experiments, impaired vasodilator responses to nitroglycerin were demonstrated in patients with diabetes mellitus when compared with age and sex matched controls [ 105 , 106 ].
These impaired responses were somewhat surprising since nitrate-induced vasodilation has been shown to be increased when the endothelium is damaged and diabetes is associated with impaired endothelial function [ 9 ]. A possible explanation may be that the biotransformation of organic nitrates requires intracellular sulfhydryl groups in order to produce vasoactive intermediates [ 107 ]. Oxidative depletion of these sulfhydryl donors will cause impaired responsiveness to organic nitrates. In diabetes antioxidant activity is decreased [ 108 ]. The increase in oxidative stress and free radical activity that occurs in diabetes probably alters the redox equilibrium of intracellular thiols leading to primary oxidation or depletion of these essential sulfhydryl donors. But also succination of thiols may contribute to decreased sulfhydryl concentration [ 109 ]. Depletion of thiols is also envisaged in rats with streptozotocin-induced experimental diabetes that have significantly decreased serum antioxidant capacity (which largely depends on serum thiol groups) (unpublished observations, Oelze and Daiber). This is one possible explanation for an impaired nitrate response observed in diabetes [ 102 , 105 ]. According to the recent development in the nitrate field, the mitochondrial nitrate reductase ALDH-2 could play a role as well. This enzyme requires dithiols (e.g., dihydrolipoic acid) in order to bioactivate nitroglycerin or PETN [ 43 ] and depletion of the pool of reduced thiols would probably lead to impairment of enzymatic activity. Although PETN also requires dithiols for bioactivation, this organic nitrate has slow pharmacokinetics preventing an overcharge of the bioactivating capacity of the organism [ 74 , 110 ]. This hypothesis is in accordance with the recent observation that ALDH-2 activity is decreased in the testis of diabetic animals [ 111 ] although this observations needs further animal experimental or clinical verification in vascular tissue. Vice versa, inactivation of ALDH-2 by nitroglycerin was demonstrated to increase the infarct area in experimental MI [ 78 ] and ALDH-2 activation is related to PKC ε [ 112 ]. Recently, Ma et al. have demonstrated that ALDH-2 knockout aggravated, and ALDH-2 overexpression improved, ischemic damage in response to experimental MI [ 113 ]. Interestingly, these authors revealed a correlation between ALDH-2 and AMP-activated kinase (AMPK), which is an important metabolic enzyme involved in the management of disease states such as diabetes [ 114 – 116 ]. Besides the loss of AMPK activity by GTN-induced inactivation of ALDH-2, diabetic complications could be further aggravated by impaired breakdown of toxic aldehydes—it may be speculated that inhibition of ALDH-2 and AMPK may contribute to increased mortality of diabetic patients. In conclusion, for patients with congestive cardiac failure, type II diabetes and acute myocardial infarction resistance to the action of organic nitrates was demonstrated. In addition, there is good evidence that a significant percentage of normal subjects have impaired organic nitrate vasodilator potency [ 117 ].
3.2. Effectiveness of Antioxidants in Diabetes- and Nitrate-Associated Vascular Complications
It was previously shown for the setting of nitrate tolerance and diabetes that there is increased formation of reactive oxygen species and/or decreased breakdown of those species leading to oxidative stress [ 2 , 3 , 66 , 82 ]. Accordingly, there is a large number of studies demonstrating that cotreatment with antioxidants ameliorates nitrate- and diabetes-induced vascular oxidative stress and dysfunction. Examples include the use of direct antioxidants such as hydralazine [ 62 , 93 ], vitamin C [ 40 ], superoxide dismutase [ 82 ], N-acetylcysteine [ 118 ], ebselen [ 84 ], and drugs with indirect antioxidant properties such as statins [ 119 ], AT 1 -receptor antagonists [ 59 ], ACE inhibitors [ 52 ], and the β -blocker carvedilol [ 120 ] (more compounds are reviewed in [ 32 ]). The antioxidant lipoic acid could improve nitrate tolerance which could be partially due to the fact that lipoic acid is the reducing cofactor of the nitrate bioactivating enzyme ALDH-2 [ 43 , 74 , 121 ]. It should be also noted that NO itself is a highly potent superoxide scavenger, although the resulting formation of peroxynitrite may limit the importance of this antioxidant property. Accordingly, organic nitrates with high turnover to NO may provide intrinsic antioxidant effects. The important role of oxidative stress for the development and progression of nitrate tolerance was further supported at a molecular level by the observation that vascular dysfunction in response to nitroglycerin was improved by genetic deletion of p47 phox , an essential subunit of the superoxide generating NADPH oxidase (Nox2) [ 87 ]. In contrast, genetic deletion of the important mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) caused an additional aggravation of nitrate-induced vascular dysfunction [ 83 ]. Interestingly, diabetes shows a very similar responsiveness to antioxidant treatment indicating that similar mechanisms as in nitrate tolerance account for vascular complications in diabetic patients and animals (see the aforementioned triggers for nitrate resistance and endothelial dysfunction). Improvement of vascular dysfunction and oxidative stress in diabetes was shown for direct antioxidants such as vitamin C [ 122 , 123 ], lipoic acid [ 28 , 124 ], ebselen [ 25 ], N-acetylcysteine [ 125 ], glutathione [ 126 ], and drugs with indirect antioxidant properties such as statins [ 127 ], AT 1 -receptor antagonists, or ACE inhibitors [ 8 , 21 , 128 ]. It should be noted that vitamin C was infused at high doses whereas oral administration did not improve endothelial dysfunction in diabetic patients [ 129 ] and even increased mortality in diabetic women [ 130 ]. Further evidence for an important role of oxidative stress for diabetic complications was provided at a molecular level by demonstrating that oxidative modifications contributing to diabetic retinopathy were prevented by overexpression of the MnSOD in a transgenic mouse model [ 131 ]. Likewise cardiomyocyte function and oxidative stress were beneficially influenced by overexpression of catalase in experimental models of type 1 and 2 diabetes [ 29 ]. Based on these data, there are striking parallels between nitrate tolerance and diabetes mellitus that converge at the level of oxidative stress as well as vascular dysfunction, which may explain nitrate resistance in the setting of diabetes. In accordance with these considerations, the clinical study of Picano and coworkers demonstrated that diabetes and nitrate therapy evoke synergistic oxidative DNA damage [ 132 ] suggesting that the pathophysiological mechanisms are similar in the setting of diabetes and nitrate tolerance. Comparable observations were made in atherosclerotic rabbits where nitroglycerin treatment synergistically increased the protein tyrosine nitration (a marker for peroxynitrite formation) in hyperlipidemic animals [ 133 ].
3.3. Is Diabetes Mellitus Another Battlefield for Pentaerithrityl Tetranitrate?
As discussed above, the use of organic nitrates in diabetic patients is associated with certain risks: besides reduced efficacy of the drugs due to nitrate resistance in these individuals, there might be synergistic damage to the vascular system since nitrate treatment and diabetes both are associated with oxidative complications. Since PETN is devoid of tolerance [ 67 ], oxidative stress [ 71 ] and even normalized vascular function in experimental atherosclerosis [ 134 ], it might be expected that this nitrate demonstrates beneficial effects in the setting of diabetes. Considering the mechanisms underlying the protective effects of PETN, mainly based on induction of HO-1 [ 77 ], ferritin [ 135 ], ecSOD [ 95 ], and other protective genes/proteins [ 75 ] as discussed afore (summarized in Figure 1 ), PETN indeed could be a suitable nitrate for treatment of ischemia in diabetic patients. Heme oxygenase-1 induction by hemin was also able to suppress nitroglycerin-induced tolerance [ 77 ]. Of note are the observations that induction heme oxygenase lead to increased expression of ecSOD [ 31 ], and pharmacological as well as genetic overexpression of heme oxygenase protects from diabetic cardiovascular complications [ 31 , 136 ] (summarized in Figure 1 ), whereas genetic deficiency in HO-1 aggravates myocardial ischemia/reperfusion injury in diabetic mice [ 137 ]. Therefore, it may be speculated that PETN-induced heme oxygenase-1 and ecSOD induction are able to improve vascular function, redox state and even prognosis in diabetic patients. According to an ongoing study in our laboratory, PETN, in contrast to ISMN, is able to improve vascular complications in experimental diabetes. PETN corrected endothelial dysfunction by approximately 50% and improved nitrate resistance almost completely whereas ISMN had only minor effects on vascular dysfunction. Vascular and cardiac mitochondrial ROS formation as well as cardiac NADPH oxidase and serum xanthine oxidase activities were significantly improved by PETN whereas ISMN even increased NADPH oxidase activity. There is also evidence that PETN improves eNOS function and normalizes endothelial dysfunction in experimental diabetes via restoration of tetrahydrobiopterin levels, probably by preventing oxidative depletion of BH 4 and upregulation of GTP-cyclohydrolase, the BH 4 synthesizing enzyme. These beneficial effects of PETN on eNOS function, NADPH oxidase activity, and oxidative stress have recently been demonstrated for experimental hypertension in angiotensin-II infused rats [ 138 ]. To our best knowledge, this was the first report on the improvement of angiotensin-II-induced hypertension by an organic nitrate.
Glossary
Clinical nitrate tolerance : impaired vasodilatory potential of a given organic nitrate in response to chronic use of the drug (may comprise of “pseudotolerance,” “NO resistance,” “endothelial dysfunction” and impaired nitrate bioactivation).
Nitrate tachyphylaxis : fast onset of in vitro or in vivo tolerance due to high-dose acute treatment with an organic nitrate (most probably comprises of impaired nitrate bioactivation).
Primary tolerance : resistance of the vasculature to endogenous and/or exogenous vasodilators encountered in certain pathophysiological states such as diabetes or coronary artery disease. This primary tolerance may be directed to organic nitrates termed “nitrate resistance” or to other NO donors as well as endogenous NO formation termed “NO resistance.”
Secondary tolerance : resistance of the vasculature to exogenous vasodilators encountered after prolonged (clinical tolerance) or high-dose (tachyphylaxis) use of these drugs. This secondary tolerance may be directed to organic nitrates termed “nitrate tolerance” or to other vasodilators generally termed “desensitization.”
Pseudotolerance: loss of vasodilatory potential of a given drug due to humoral counterregulation (e.g., activation of the RAAS). Pseudotolerance is closely correlated with the “rebound phenomenon” upon withdrawal of the drug.
NO resistance : inadequate vasodilatory response of the vasculature to exogenous or endogenous nitric oxide encountered in certain pathophysiological states such as diabetes or coronary artery disease or upon development of “secondary tolerance.”
Nitrate resistance : special form of “primary tolerance.” Inadequate vasodilatory response of the vasculature to exogenous organic nitrate therapy encountered in certain pathophysiological states such as diabetes or coronary artery disease or upon development of “secondary tolerance.”
Endothelial dysfunction : inadequate vasodilatory response of the vasculature to exogenous or endogenous endothelium-dependent vasodilators encountered in certain pathophysiological states such as diabetes or coronary artery disease or upon chronic use of organic nitrates (e.g., “cross-tolerance” to acetylcholine).
Conflict of Interests
A. Daiber received vascular research grants and lecture fees from Actavis Deutschland GmbH. | Acknowledgments
The support by research grants from the Medical Center of the Johannes Gutenberg University (MAIFOR and Forschungsfonds) is gratefully acknowledged. Moreover, the authors would like to thank Actavis Deutschland GmbH for continuous financial support of their organic nitrate research. The authors are also indebted to Thilo Weckmüller and Margot Neuser, their graphic artists, for preparing the schemes.
Abbreviations
Angiotensin-converting enzyme
Mitochondrial aldehyde dehydrogenase
Extracellular superoxide dismutase (copper, zinc-containing)
Endothelial nitric oxide synthase
Heme oxygenase-1
Isosorbide-5-mononitrate
Isosorbide dinitrate
Manganese superoxide dismutase (mitochondrial isoform)
Nitric oxide (nitrogen monoxide)
Pentaerithrityl tetranitrate
Renin-angiotensin-aldosterone system
Reactive oxygen species. | CC BY | no | 2022-01-13 02:21:51 | Exp Diabetes Res. 2010 Dec 27; 2010:213176 | oa_package/c4/5e/PMC3014692.tar.gz |
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PMC3014696 | 21234403 | Our ability to provide analgesia and sedation for children has evolved over the past several years. We have progressed from papoose boards to oral sucrose solutions to soothe babies during procedures. Many procedures that were traditionally performed in the operating room are being performed in remote settings: inpatient wards, satellite units, and emergency rooms. The delivery of pediatric sedation is no longer restricted to a limited group of specialists, but instead is delivered by specialists, and physicians as well as nonphysicians, in the field of anesthesia, hospital medicine, pediatrics, intensive care medicine, dental medicine, emergency medicine, and radiology. Some sedatives and analgesics have been introduced to market within the past decade whereas others, still in use, have existed for over a century.
The ability of infants to recognize pain was initially underappreciated. Clinical and bench research, however, have sensitized us to the newborn's capacity to feel pain and has, subsequently, laid the groundwork for ongoing research into the pathophysiology of pain and clinical tools for proper assessment [ 1 , 2 ]. Acute pain management options for children continue to evolve, encompassing all routes of delivery: oral, rectal, topical, subcutaneous, mucosal, intramuscular, parenteral and recently intranasal. Some of the recent introductions of this century include our appreciation of the analgesic and sedative benefits of oral sucrose in newborns and the use of alternative delivery routes, such as intranasal fentanyl for analgesia [ 3 , 4 ]. There has also been continued interest comparing the benefits of nonsteroidal anti-inflammatory medications to narcotics [ 5 ]. Despite the advances in our knowledge and application of analgesics, patient safety continues to be a concern, particularly as unexpected adverse events, a morphine overdose in breast milk of a mother taking codeine for example, continue to occur [ 6 ].
Analgesia and sedation practices are not uniform; guidelines, policies, and protocols differ among professional organizations, provider groups, countries, institutions and among providers within the same institution. The inability to reach a consensus on safe practice and appropriate guidelines threatens our ability to provide safe, consistent care and fuels debate and malcontent amongst and between some specialties.
The magnitude of human and financial cost of jeopardizing patient safety in sedation is large and adverse outcomes should be rare. The numerical value of rare should not be a percentage; for example, a 99.9% probability of having a given outcome or 0.1% (1 in 1000) probability of a serious adverse outcome as a result of sedation is not acceptable. An acceptable aim for pediatric sedation should be “six sigma” which will reduce adverse outcome to 3-4 errors per a million incidents [ 7 ].
Ensuring that the practice of pediatric analgesia and sedation follows the same rigorous safety monitoring at all times by all providers, and in any setting across the world is a common responsibility shared by healthcare providers caring for children.
Savithiri Ratnapalan Savithiri Ratnapalan Keira Mason Keira Mason Sharon E. Mace Sharon E. Mace | CC BY | no | 2022-01-13 02:39:08 | Int J Pediatr. 2010 Dec 26; 2010:454731 | oa_package/07/84/PMC3014696.tar.gz |
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PMC3014697 | 21234404 | 1. Introduction
The introduction of human insulin in the 1990s led to an increase in the number of sudden nocturnal deaths of young people with type I diabetes. This specific type of death in diabetes was termed the “dead in bed” syndrome and it was hypothesized that the deaths were caused by hypoglycemia [ 1 ]. The pathophysiological mechanisms behind the deaths are still not understood although circumstantial evidence suggests that they are cases of fatal cardiac arrhythmia. The proposed proarrhythmic effect of hypoglycemia is thought to be mediated by sympathoadrenal activation and hypokalaemia [ 2 ].
It has been reported that insulin-induced hypoglycemia affects repolarization of the cardiac cells in both healthy subjects [ 3 , 4 ] and people with diabetes [ 5 – 7 ]. The altered repolarization is notable on the electrocardiogram (ECG) as a flattened T wave [ 4 , 6 ] and a prolonged heart rate-corrected QT interval (QTc) [ 3 , 5 ]. A prolonged QTc is associated with an increased risk of sudden cardiac death [ 8 , 9 ], and QTc has thus been the primary variable investigated in studies of the proarrhythmic effect of hypoglycemia. The degree of QTc prolongation during clamped hypoglycemia ranges from 5 ms [ 6 ] to 60 ms [ 3 ] with one study reporting a prolongation of 156 ms [ 5 ]. Thus, in some studies the prolongation of QTc is seen to be insignificant while other studies show a significant and potentially dangerous prolongation. We hypothesize that differences in methodology when measuring the QT interval and heart rate correcting the QT interval may partly explain the discrepancies between the reported prolongations of QTc. It is known that Bazett's formula for heart rate correction is associated with both over- and undercorrection of QTc at heart rates outside a narrow range [ 10 ]. Nevertheless, Bazett's formula is still the most often used heart rate correction, when investigating QTc during hypoglycemia. The methodology when measuring the T wave offset also has an impact on the QT interval as for example the often used tangent method is more sensitive to the flattening of the T wave seen during hypoglycemia [ 11 ]. In addition, modern insulin analogues may have a different effect on the QTc than human insulin, although this has previously been shown not to be the case [ 12 ].
The “dead in bed” syndrome and the potential prolongation of QTc during hypoglycemia are still of concern to many patients and physicians. If these tragic deaths are to be avoided, an increased understanding of the phenomenon is necessary. It has been suggested that if patients with an increased risk of prolonged QT during hypoglycemia could be identified, a selective beta blocker might have a therapeutic role [ 2 ]. However, before considering preventive treatment, it is important to clarify if hypoglycemia indeed causes significant prolongation of QTc and identify the factors in study methodology that may cause the discrepancies among the reported results in the literature. Thus, in the present study, we investigate the potential bias associated with measurement technique, heart rate correction, and type of insulin when measuring the QTc during hypoglycemia. | 2. Methods
2.1. Subjects
The study population consisted of 10 subjects (6 men, 4 women; age: 32 ± 9 years) with type 1 diabetes (C-peptide negative). Subjects had HbA1c <10%, duration of diabetes of 15 ± 10 years, and none had signs of neuropathy. Each subject was studied on two weekends separated by at least 1 month. At each weekend hypoglycemia was induced two times: saturday at 2 AM and 10 PM. Subjects were randomized to use either insulin aspart (Iasp) (NovoRapid, Novo Nordisk A/S, Denmark) or human insulin (HI) (Actrapid, Novo Nordisk A/S, Denmark) the first weekend in a cross-over design so the other type of insulin was used at the next weekend. Written informed consent was obtained from all subjects, and the study protocol was approved by the Regional Ethics Committee.
2.2. Procedures
A catheter (Venflon, Viggo AB, Sweden) was inserted into an antecubital arm vein for administration of insulin and glucose. Hypoglycemia was induced by a single bolus of insulin (0.1 U/kg bodyweight) injected directly into the blood. Blood glucose was measured by a HemoCue Analyzer (HemoCue AB, Angelholm, Sweden). Measurements of blood glucose were taken 30 minutes prior to insulin injection and at least every 5 minutes following the injection. When blood glucose <2.5 mmol/L was reached, intravenous glucose (10%) was administered to restore the blood glucose.
2.3. ECG Measurements
ECG was recorded from lead II by disposable Ag/AgCl electrodes (Blue Sensor L, Ambu A/S, Denmark). ECG was sampled by a data acquisition system (Portilab 16 + 2, Twente Medical Systems International, Holland) at 400 Hz with 12-bit resolution.
Epochs of 60 seconds of ECG were analyzed 30 minutes prior to insulin injection ( t −30 ), 10 minutes prior to insulin injection ( t −10 ), at insulin injection ( t 0 ), 15 minutes after insulin injection ( t 15 ), at blood glucose nadir ( t hypo ), and 90 minutes after blood glucose nadir ( t post ) ( Figure 1 ). QT intervals were measured from each epoch using both a manual annotation (MA) and a semi-automatic “slope intersect” (SI) method.
MA measurements were carried out by independent QT experts (Spacelabs Healthcare, Washington, USA) blinded to the study design and all other information except the ECG. Each epoch was manually reviewed for artifacts and a representative beat was obtained. Distortion of the representative beat was minimized by selecting areas of the epoch with minimal artifacts to include in representative beat generation. From the representative beat QT intervals were measured from the first high-frequency deflection of the QRS complex to the offset of the T wave. Average RR interval of the QRS complexes selected for the representative beat was used to provide heart rate correction. In addition, the R peak and T peak amplitudes relative to the isoelectric line were measured. All measurements were done manually using an electronic caliper and reviewed by a cardiologist.
SI measurements were carried out using custom analysis software developed in MatLab (Version 7.8.0.347, The Mathworks, Inc., Natick, MA, USA). Templates representing the average PQRST complex in epochs were generated using manually selected areas with minimal artifacts. The end of the T wave in each template was determined automatically using the “slope intersect" method [ 13 ]. In addition, the R peak and T peak amplitudes relative to the isoelectric line were also measured. All templates and associated fiducial points were manually reviewed on-screen in random order by an observer blinded to the corresponding blood glucose and the subjects' clinical data. Templates were rejected if artifacts precluded reliable measurements but no adjustments to the QT interval were made to reduce the subjectivity of the measurements. The median RR interval in each segment was used to heart rate correcting the QT interval. In both MA and SI methods QT intervals were corrected by Bazett's formula (QTcB) [ 14 ] and Fridericia's formula (QTcF) [ 15 ]. As a measure of the T wave flatness the T peak to R peak amplitudes ratio (T/R Ratio) was calculated.
2.4. Data Analysis
Measurements at t −30 , t −10 and t 0 were averaged and collectively called t baseline to reduce the amount of statistical analyses. A linear mixed effects model was used to analyze the changes in ECG variables. Time (levels: t baseline , t 15 , t hypo , and t post ) and treatment (levels: Iasp, HI) were included as fixed effects in the model together with a time-treatment interaction term. Subjects were included in the model as a random effect on the intercept. Weekend was included as a random effect within subjects and episodes as a random effect within weekends. Separate models for each ECG variable were fitted using restricted maximum likelihood. An analysis of variance of the fitted model was used to test for significant changes in variables. With variables showing statistical significance, Dunnett's posthoc tests were used to test for significant differences between different factor levels. P values <.05 were considered significant. Results from the statistical model are reported as mean (SE), all other results are reported as mean ± SD. Statistical analyses were performed in R version 2.9.1. | 3. Results
Seven (17.5%) of the recorded episodes of hypoglycemia were excluded because the subject had a blood glucose ≤3.5 mmol/L at t −30 ( n = 6) or because of instrumentation issues ( n = 1). Thus a total of 33 episodes were used in the data analysis with a minimum of two episodes available from each subject. Hypoglycemia was reached 45 ± 32 minutes after insulin was administered with blood glucose at nadir of 2.4 ± 0.3 mmol/L. The measured variables summarized for each time point are shown in Table 1 .
Using the MA method significant prolongation of QTcB from t baseline to t hypo was observed (ΔQTcB: 7(2); P < .05) but no prolongation of QTcF was seen (ΔQTcF: 1(2); P > .05). With the SI method both QTcB and QTcF prolonged significantly (ΔQTcB: 42(6); P < .001, ΔQTcF: 35(6); P < .001) ( Table 2 ). Heart rate and T/R ratio decreased significantly with both the MA and SI methods ( P < .001). The changes in variables from t baseline to t 15 were similar to the change from t baseline to t hypo ( Table 2 ). At t post , QTcB and QTcF had returned to t baseline levels while RR interval and T/R ratio remained decreased for both MA and SI methods ( Table 2 ).
There was a significant difference between ΔQTcF measured by the SI and MA methods (34 ms; P < .001) ( Table 3 , Figure 3 ). The difference between ΔQTcB and ΔQTcF was also significant (8 ms; P < .001) ( Table 3 , Figure 4 ). No significant difference between HI and Iasp was found on any of the measured ECG variables. | 4. Discussion
The MA method showed modest increase in QTcB and no increase in QTcF during hypoglycemia, while the SI method showed considerable prolongation of both QTcB and QTcF. Comparing the two methods directly showed that SI underestimated the QT interval at baseline and overestimated it at hypoglycemia compared to MA.
The use of the SI method for measuring the end of the T wave is known to be sensitive to changes in T wave amplitude, although the method was originally meant for cases with partial T-U fusion [ 11 , 16 ]. In particular a flattened T wave will cause an overestimation of the QT interval with the SI method when compared to the MA method ( Figure 2 ). Since there exists no gold standard of measuring the QT interval, neither of the measurement techniques can from this study be judged more correctly than the other. However, the discrepancy between the two methods illustrates that comparing studies of hypoglycemia using different QT measurement methods may be problematic. Also, it is apparent from the results that the SI method produces significantly longer QT intervals than the MA method which could indicate a higher probability of false positives with the SI method. An approach which could eliminate the bias associated with measuring the QT interval is to use alternative T wave morphology variables. Xue and Reddy [ 17 ] used principal component analysis of the T wave and showed that this approach had superior reproducibility than several QT measurement methods. Alternative T wave morphology parameters might therefore be better at characterizing changes in repolarization during hypoglycemia.
A pathologically prolonged QTc interval is usually defined as >450 ms for men and >470 ms for women [ 18 ]. In the present study, mean QTc did not exceed these thresholds with any of the methods. This could indicate that prolongation of the QTc cannot in itself explain the mechanism implicated in the dead-in-bed syndrome.
The differences between QTcB and QTcF in this study were larger at hypoglycemia than at baseline. It is known that Bazett's formula tends to overcorrect the QTc at higher heart rates [ 10 ]. In this study we observed a significant increase in heart rate during hypoglycemia which may have contributed to an overcorrection by Bazett's formula compared to Fridericia's formula. Similar findings of differing results using the two correction formulas have been reported [ 6 ] although they in other cases produce similar results [ 19 ].
One of the main limitations of the study is the absence of a control group. Without a control group it is less clear if the observed QT prolongation is caused by hypoglycemia per se. Indeed, insulin could act as a confounding variable as it has been shown to cause moderate QT interval prolongation [ 20 ]. To account for the effect of insulin, we measured the ECG variables of interest 15 minutes after insulin injection where the subjects were still normoglycemic. We anticipated that this measurement would quantify the effect of hyperinsulinemia alone. The results show that the change in ECG variables 15 minutes after insulin injection is comparable to the change at hypoglycemia. This could indicate that the observed changes during hypoglycemia may not be caused by hypoglycemia per se but rather by hyperinsulinaemia. We acknowledge that the small number of subjects in the study limits its generalisability to the general population, although measurements on each subject were repeated to reduce intrasubject variation. Additionally, the use of only one ECG lead for QT measurement may have introduced some variation in the measurements, which could have been mitigated by the use of several leads.
The results of this study are in agreement with previous studies of experimentally induced hypoglycemia using the MA method [ 4 , 6 ] although some studies also report a QTc prolongation [ 5 , 11 ]. Studies using the SI method consistently find significantly prolonged QTc during hypoglycemia [ 2 , 3 , 12 , 19 , 21 ]. We found no difference in the observed variables between HI and Iasp which is in line with previous findings [ 12 ]. Ireland and colleagues [ 11 ] compared the SI and MA methods and concluded that the SI method was preferred over the MA method because of a lower inter observer difference despite an overestimation of the QT interval at hypoglycemia. We cannot infer on inter-observer differences from our study but our results confirm that the SI method overestimates the QT interval during hypoglycemia. | 5. Conclusion
Conclusively, our results suggest that the methodology used for measuring and heart rate correcting the QT interval during hypoglycemia may have a significant impact on the measured prolongation of QTc. The SI method overestimates the QT interval compared to the MA method at hypoglycemia while Bazett's formula overcorrects the QTc compared to Fridericia's formula. The type of insulin for inducing hypoglycemia does not influence QT prolongation. Prolongation of QTc in this study did not reach pathologic values which suggest that additional factors play a role in the pathogenesis of the dead-in-bed syndrome. | Academic Editor: Chim Choy Lang
Introduction . Several studies show that hypoglycemia causes QT interval prolongation. The aim of this study was to investigate the effect of QT measurement methodology, heart rate correction, and insulin types during hypoglycemia. Methods . Ten adult subjects with type 1 diabetes had hypoglycemia induced by intravenous injection of two insulin types in a cross-over design. QT measurements were done using the slope-intersect (SI) and manual annotation (MA) methods. Heart rate correction was done using Bazett's (QTcB) and Fridericia's (QTcF) formulas. Results . The SI method showed significant prolongation at hypoglycemia for QTcB (42(6) ms; P < .001) and QTcF (35(6) ms; P < .001). The MA method showed prolongation at hypoglycemia for QTcB (7(2) ms, P < .05) but not QTcF. No difference in ECG variables between the types of insulin was observed. Discussion . The method for measuring the QT interval has a significant impact on the prolongation of QT during hypoglycemia. Heart rate correction may also influence the QT during hypoglycemia while the type of insulin is insignificant. Prolongation of QTc in this study did not reach pathologic values suggesting that QTc prolongation cannot fully explain the dead-in-bed syndrome. | Acknowledgment
This study has been funded by Novo Nordisk. | CC BY | no | 2022-01-13 01:45:13 | Cardiol Res Pract. 2010 Dec 22; 2010:961290 | oa_package/3c/82/PMC3014697.tar.gz |
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PMC3014700 | 21234407 | 1. Introduction
The enzyme β -galactosidase (EC.3.2.1.23), most commonly known as lactase, which hydrolyses lactose into its monomers that is glucose and galactose has potential applications in food processing industry. Because of low levels of the enzyme in intestine, large fraction of the population shows lactose intolerance and they have difficulty in consuming milk and dairy products. Lactose has a low relative sweetness and solubility, and excessive lactose in large intestine can lead to tissue dehydration due to osmotic effects, poor calcium absorption due to low acidity, and fermentation of the lactose by microflora resulting in fermentative diarrhea, bloating, flatulence, blanching and cramps, and watery diarrhea [ 1 ]. Furthermore, lactose is a hygroscopic sugar and has a strong tendency to absorb flavours and odours and causes many defects in refrigerated foods such as crystallization in dairy foods, development of sandy or gritty texture, and deposit formation [ 2 ].
Technologically, lactose gets easily crystallized, which sets the limits of its applications to certain processes in the dairy industry. Cheese manufactured from hydrolyzed milk ripens more quickly than that made from normal milk. Treatment of milk and milk products with lactase to reduce their lactose content seems to be an appropriate method to increase their potential uses and to deal with the problems of lactose insolubility and lack of sweetness. Furthermore, this treatment could make milk, a most suitable food, available to a large number of adults and children that are lactose intolerant. Moreover, the hydrolysis of whey converts lactose into a very useful product like sweet syrup, which can be used in various processes of dairy, confectionary, baking, and soft drink industries [ 3 , 4 ]. Therefore, lactose hydrolysis not only allows the milk consumption by lactose intolerant population but can also solve the environmental problems linked with whey disposal [ 5 – 7 ].
The enzyme β -galactosidase can also be used in transglycosylation of lactose to synthesize galacto-oligosaccharides (GOSs). These were widely recognized as the nondigestible oligosaccharides, not hydrolyzed or absorbed in the upper intestinal tract, and they pass onto the colon where they are fermented selectively by beneficial intestinal bacteria. Besides their prebiotic effects, these GOSs have low cariogenicity, low caloric values, and low sweetness [ 8 , 9 ]. GOSs occur naturally in trace amounts in breast milk, cow milk, honey, and a variety of fruits and vegetables [ 10 ]. As a result, increased production of GOS is useful. GOS can be readily manufactured by enzymatic transgalactosylation of β -galactosidase from whey lactose, which is available in abundance as a by-product of cheese industry.
Thus, the application of β -galactosidase in the hydrolysis of lactose in dairy industry has attracted the attention of researchers. Although most industries still hydrolyze lactose with free enzyme, the immobilization of β -galactosidase is an area of great interest because of its potential benefits [ 11 ]. The use of immobilization technology is of significant importance from economic point of view since it makes reutilization of the enzyme and continuous operation possible and also precludes the need to separate the cells from the whey following processing. It can also help to improve the enzyme stability. Nowadays, immobilized β -galactosidase is intensively being used in lactose hydrolysis of milk/whey and has been tested for the production of galacto-oligosaccharides. | 3.2. Entrapment Method
Entrapment method is the physical enclosure of enzymes in a small space. Matrix and membrane entrapment (including microcapsulation) are the major methods of entrapment. The major advantage of the entrapment technique is the simplicity by which spherical particles can be obtained by dripping a polymer-cell suspension into a medium containing positively charged ions or through thermal polymerization [ 86 ]. Further, beads formed particularly from alginate are transparent and generally mechanically stable. The major limitation of this technique for the immobilization of enzymes is the possible slow leakage during continuous use in view of the small molecular size compared to the cells. However, improvements can be made by using suitable linking procedures. The matrices used for the immobilization are usually made up of polymeric materials such as Ca-alginate, agar, k- carragenin, polyacrylamide, and collagen. However, some solid matrices such as activated carbon, porous ceramic, and diatomaceous earth can also be used for the immobilization. The membranes commonly used for the entrapment of enzymes are nylon, cellulose, polysulfone, and polyacrylmide.
Fungal β -galactosidase immobilized in polyvinyl alcohol gel was more thermostable than free enzyme and retained 70% of activity after 24 h at 50°C and 5% activity at 60°C [ 93 ]. The glutaraldehyde-treated K. bulgaricus cells having β -galactosidase were entrapped in alginate using BaCl 2 solution [ 57 ]. The alginate beads obtained after treatment with polyethyleneimine followed by glutaraldehyde solution were stable.
E. coliβ -galactosidase has been immobilized in polyacrylamide gels and through the preparation of cross-linked derivatives of E. coliβ -galactosidase by treating the enzyme with bisimidoesters. The combination of three protective agents, namely, bovine serum albumin, cysteine, and lactose, during immobilization gave an increased yield of 190% in the case of dimethyladipimidate (DMA) cross-linked preparation [ 58 ]. K. marxianus cells having lactase activity were entrapped in calcium pectate gel (CPG) and in calcium alginate gel (CAG) hardened by polyethyleneimine and glutaraldehyde. Permeabilized cells entrapped in CPG hydrolyzed lactose more than 80% in semicontinuous and continuous processes [ 94 ].
The comparison of the various methods of immobilization of β -galactosidase from Thermus aquaticus indicated that immobilization by cross-linking followed by entrapment in agarose beads can be beneficial for high enzyme loading with good activity yield [ 59 ]. The entrapment of A. oryzae β -galactosidase in a spongy polyvinyl alcohol cryogel increased the stability towards temperature, pH, and ionic strength more than the free enzyme [ 60 ]. The fibers composed of alginate and gelatin hardened with glutaraldehyde retained 56% relative activity of β -galactosidase for 35 days without any decrease. Moreover, the optimum conditions were also not affected by immobilization [ 95 ]. Another approach for immobilization of β -galactosidase is the use of liposomes and in this direction response surface methodology was applied to optimize the entrapment of the enzyme in liposomes by dehydration-rehydration vesicle method, which resulted in an entrapment efficiency of 28% [ 96 ].
It has been observed that entrapped cross-linked concanavalin A- β -galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations because it retained 95% activity after seventh repeated use and 93% of its original activity after 2-month storage at 4°C [ 97 ]. A. oryzaeβ -galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate-starch beads. The maximum activity of the immobilized β -galactosidase has been obtained at 60°C, approximately 10 degrees higher than that of the free enzyme. It has been also observed that the immobilized β -galactosidase exhibited significantly higher stability to heat, urea, MgCl 2 , and CaCl 2 than the free enzyme [ 98 ]. Calcium alginate-entrapped β -galactosidase preparations have been used for the hydrolysis of lactose from synthetic solution, milk, and whey in batch processes as well as in continuous packed bed columns. From the kinetic studies, it was observed that the Michaelis constant ( K m ) for the free and immobilized β -galactosidase was 2.51 mM and 5.18 mM, respectively. Moreover, the V max for the soluble and immobilized enzyme was 4.8 × 10 −4 mol/min and 4.2 × 10 −4 mol/min, respectively [ 99 ].
The main problems associated with this type of immobilization process are desorption of β -galactosidase from immobilization matrix and the leakage of the entrapped enzyme due to a small molecular weight compared to pores of gel in matrices, which can be overcome by cross-linking using bifunctional or multifunctional reagents [ 100 ]. The conditions for polyethyleimine- (PEI-) coating of agarose supports to achieve a β -galactosidase derivative have been optimized that allows a high lactose conversion from whey in a steady bed-reactor with no enzyme leakage, together with good elution properties [ 101 ]. Various cross-linking reagents used for improvement of β -galactosidase stability in immobilized state are described in Table 3 [ 102 – 110 ]. | 6. Conclusions
β -galactosidase is one of the most important enzymes used in food processing, which offers nutritional, technological, and environmental applications. Enzyme immobilization provides enzyme reutilization and may result in increased activity by providing a more suitable microenvironment for the enzyme. Moreover, immobilized systems can provide better enzyme thermostability and pH tolerance. However, major problems associated with the immobilized enzyme system are microbial contamination, protein adherence, and channeling. The periodic washing and pasteurization and flow direction of feed can solve these problems to great extent. The problem of microbial contamination can also be solved by exploiting the temperature property of the enzyme. The immobilized enzyme preparations showed up to 99% hydrolysis, and thus it can be applied successfully for the hydrolysis of lactose in milk or whey. The isolation of pyschrophilic bacteria with cold active β -galactosidase has opened up the possibility of processing of milk and whey even at low temperatures. On the other side, thermostable enzymes have the unique ability to retain their activity at higher temperatures for prolonged periods, and the process is less prone to microbial contamination due to higher operating temperature. Thus, cold active and thermostable enzymes will have the great potential in the lactose hydrolysis and of particular interest to the researchers. Thus, immobilization enzyme systems will certainly find greater role in future times for the hydrolysis of milk, whey, and synthesis of galacto-oligosaccharides. | Academic Editor: Cristina M. Rosell
The enzyme β -galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals. The use of β -galactosidase for the hydrolysis of lactose in milk and whey is one of the promising enzymatic applications in food and dairy processing industries. The enzyme can be used in either soluble or immobilized forms but the soluble enzyme can be used only for batch processes and the immobilized form has the advantage of being used in batch wise as well as in continuous operation. Immobilization has been found to be convenient method to make enzyme thermostable and to prevent the loss of enzyme activity. This review has been focused on the different types of techniques used for the immobilization of β -galactosidase and its potential applications in food industry. | 2. Microbial Sources of Enzyme
The enzyme β -galactosidase can be obtained from a wide variety of sources such as microorganisms, plants, and animals; however, according to their sources, their properties differ markedly [ 11 , 12 ]. Enzymes of plants and animal origin have little commercial value but several microbial sources of β -galactosidase are of great technological interest. Microorganisms offer various advantages over other available sources such as easy handling, higher multiplication rate, and high production yield. As a result of commercial interest in β -galactosidase, a large number of microorganisms [ 13 – 26 ] have been assessed as potential sources of this enzyme ( Table 1 ).
2.1. Production and Purification
Microorganisms are considered potential source of β -galactosidase for industrial applications. However, they differ in their optimum conditions for the enzyme application especially pH range. A recovery cost of the enzyme depends on the level of production and purification. Therefore, there has been increasing interest in finding microorganisms with adequate properties for industrial use, higher production capacity, and less expensive purification methods of this enzyme. A wide variety of bacterial, yeast, and fungal cultures have been reported for β -galactosidase production.
2.1.1. Bacterial Enzymes
The enzyme β -galactosidase can be produced by a large number of bacteria but Streptococcus thermophilus and Bacillus stearothermophilus are considered as potential bacterial sources. The enzyme from Escherichia coli serves as a model for understanding the catalytic mechanism of β -galactosidase action, but it is not considered suitable for use in foods due to toxicity problems associated with the host coliform [ 11 ]. Hence, the β -galactosidase from E. coli is generally not preferred for use in food industry [ 13 – 15 ].
β -galactosidase has been isolated from an extremely thermophilic Gram-negative anaerobe. Thermoanaerobacter has been purified by chromatography through DEAE-cellulose [ 27 ]. The optimization of the ultrasonication methods for the maximum cell disruption of Escherichia coli for the release of β -galactosidase has also been reported [ 28 ]. Lactobacillus delbrueckii subsp. bulgaricus cultures were subjected to treatments using sonication, a high-speed bead mill, and a high-pressure homogenizer for the release of β -galactosidase [ 29 ].
β -galactosidase has also been purified from psychotropic Pseudoalteromonas sp. isolated from Antarctica and a high yield of purification has been reported by a rapid purification scheme using extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafilteration techniques [ 30 ].
An intracellular β -galactosidase from a thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii has been purified using precipitation (with ammonium sulphate), gel permeation, ion-exchange, and affinity chromatography and preparative electrophoresis [ 26 ]. Further, a thermostable β -galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600 and recombinant enzyme has been purified by a combination of heat treatment, ammonium sulfate fractionation, ion exchange, and gel filtration chromatography techniques [ 31 ]. The intracellular β -galactosidase from thermophile B1.2 was purified by ion-exchange and affinity chromatography with a fold purification of 2.2 and 3.9, respectively. The molecular mass of the purified enzyme as determined by native PAGE was approximately 215 kDa, by SDS-PAGE was 75 kDa, and by gel filtration was 215 kDa [ 32 ]. The efficiency of different cell disruption methods on the extraction of intracellular β -galactosidase enzyme from Streptococcus thermophilus and Lactobacillus delbrueckii subsp . thermophilus has been tested [ 33 ]. Lysozyme enzyme treatment was determined as the most effective method, which resulted in approximately 1.5 and 10 times higher enzyme activity than glass bead and homogenization treatment, respectively.
The activity and the stability of the partially purified β -galactosidases from Thermus sp strain T2 and K. fragilis have been compared [ 34 ]. Both enzymes showed a remarkable hydrolytic activity and a weak transgalactosilation activity, even in the presence of high concentrations of lactose. The thermophilic enzyme showed a higher resistance to hydrophobic agents and a higher stability at different temperatures, pHs, and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, showing that some residues important for its stability were affected by oxidation. The enzyme from K. fragilis was strongly inhibited by o-nitrophenol in a acompetitive way but it was weakly and competitively inhibited by galactose. The thermophilic enzyme was competitively inhibited by galactose much strongly than its mesophilic counterpart but the inhibition did not change with the temperature. A novel thermostable chimeric β -galactosidase was constructed by fusing a poly-His tag to the N-terminal region of the β -galactosidase from Thermus sp. strain T2 to facilitate its overexpression in E. coli and its purification by immobilized metal-ion affinity chromatography [ 35 ]. To improve the enzyme purification a selective one-point adsorption was achieved by designing tailor-made low-activated Co-iminodiacetic acid (Co-IDA) or Ni-IDA supports. The new enzyme was not only useful for industrial purposes but also has become an excellent model to study the purification of large multimeric proteins via selective adsorption on tailor-made immobilized metal-ion affinity chromatography supports. Furthermore, β -galactosidase from Thermus sp. Strain T2 has been purified and immobilized in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification [ 36 ].
2.1.2. Fungal Enzymes
The optimum pH range for the fungal enzyme is 2.5–5.4, which makes them suitable for processing of acid whey and its ultrafiltration permeate. The optimum temperatures for these enzymes are high and can be typically used at temperatures up to 50°C. The purification of β -galactosidase from different fungal sources has been carried using a variety of purification techniques.
β -galactosidase from Aspergillus niger has been purified and resolved into three multiple forms, using molecular sieving, ion exchange, and hydrophobic chromatography [ 37 ]. The purification of β -galactosidase has been carried from a cellular extract of Fusarium oxysporum var. lini by heat shock and successive chromatography on DEAE-cellulose DE-52 and sephadex G-100 [ 18 ]. The purification of β -galactosidase by ammonium sulphate precipitation and CM-sephadex chromatography from cell-free extracts of fungus Beauveria bassiana has also been reported [ 38 ]. An extracellular β -galactosidase from a themophilic fungus Rhizomucor sp. has been purified by successive DEAE-cellulose chromatography, followed by gel filtration on sephacryl S-300 [ 39 ]. The precipitation with ammonium sulfate, ion-exchange chromatography on DEAE-sephadex, affinity chromatography and chromatofocusing has also been used for purification of β -galactosidase from Penicillium chrysogenum NCAIM 00237 [ 40 ].
β -galactosidase produced by submerged culture of Aspergillus japonicus showed 2.95 U mg −1 protein specific activities with an approximate molecular weight of 27 kDa [ 41 ]. The enzyme was purified 6.43-fold with 24.02% yield and a specific activity of 18.96 U mg −1 protein. An intracellular β -glycoside hydrolase with β -glucosidase and β -galactosidase activity designated β -glucosidase BGL1 has been purified from the thermophilic fungus Talaromyces thermophilus . The monomeric enzyme has a molecular mass of 50 kDa (SDS-PAGE), an isoelectric point of 4.5-4.6. β -galactosidase activity of β -glucosidase BGL1 is activated by various mono and divalent cations including Na + , K + , and Mg 2+ , and it is moderately inhibited by its reaction products that is glucose and galactose [ 42 ].
2.1.3. Yeast Enzymes
Yeast has been considered as an important source of β -galactosidase from industrial point of view. With neutral pH optima, these are well suited for hydrolysis of lactose in milk and are widely accepted as safe for use in foods. Much work has been carried on the production of β -galactosidase from different yeast strains for its potential use. The most commercially available yeast β -galactosidase under the trade name of Maxilact (DSM Food Specialties, Delft, The Netherlands) and Lactase (SNAM Progetti, Italy) is preparations extracted from K. lactis and Lactozym (Novo, Nordisk A/S, Bagsvaerd, Denmark) from K. fragilis [ 11 , 14 , 17 ].
Biermann and Glantz [ 43 ] first attempted the purification of β -galactosidase from Sacharomyces lactis by gel filtration on sephadex G-100, followed by ion exchange chromatography on DEAE-sephadex. The homogenizer (800 bar pressure, pH 7.5) was used for the disruption and extraction of β -galactosidase of K. marxianus [ 44 ]. Further, the partial purification of the enzyme from K. marxianus was carried using DEAE-sepharose column. The studies on the use of fed batch culture techniques to achieve high culture productivity in K. fragilis have also been carried out [ 45 ].
The optimization of β -galactosidase production by K. lactis using deprotienized whey as fermentation medium has been reported. The optimized condition for the enzyme production was reported as follows: temperature 30.3°C, pH 4.68, agitation speed 191 rpm, and fermentation time 18.5 hours [ 46 ]. The studies on two commercial β -galactosidase (Lactozym and Maxilact) preparations indicated that the enzyme activities of both enzyme preparations present similar behaviour with different pH and temperature and similar kinetic parameter values, which suggest that both enzymes are probably the same [ 47 ]. Response surface methodology has also been applied in the production of β -galactosidase using K. lactis NRRL Y-8279 [ 48 ] and maximum specific enzyme activity of 4218.4 U g −1 was obtained at the optimum levels of process variables (pH 7.35, agitation speed 179.2 rpm, initial sugar concentration 24.9 g L −1 , and incubation time 50.9 hours).
3. Immobilization of β -Galactosidase
Although the enzyme β -galactosidase has numerous applications in the food and dairy industries, but the moderate stability of enzyme is one of the limitations that hinder general implementation of biocatalysts at industrial scale. Thus, there is a need to explore their full potential as catalyst by adopting suitable strategies for enzyme stabilization. The multimeric enzyme can be stabilized by using proper experimental conditions and genetic tools to cross link or to strengthen the subunit-subunit interaction [ 49 ]. The stability of monomeric or multimeric enzymes can also be enhanced by multipoint and multi-subunit covalent immobilization and enzyme engineering via immobilization [ 50 ]. The enzyme has been immobilized by various methods such as physical absorption, entrapment, and covalent binding method [ 51 – 85 ] on different supports ( Table 2 ).
3.1. Physical Adsorption
Physical adsorption is considered as the simplest method of immobilization in which an enzyme is immobilized onto a water-insoluble carrier and the biocatalysts are held on the surface of the carriers by physical forces (van der waals forces). Frequently, however, additional forces are involved in the interaction between carrier and biocatalyst principally hydrophobic interactions, hydrogen bridges, and heteropolar (ionic) bonds [ 86 ]. This method has the advantage of being simple to carry out and has little influence on the conformation of the biocatalyst. However, the disadvantage of this technique is the relative weakness of the adsorptive binding forces. Different inorganic (alumina, silica, porous glass, ceramics, diatomaceous earth, clay, bentonite, etc.) and organic (cellulose, starch, activated carbon and ion-exchange resins, such as Amberlite, Sephadex, Dowex) support materials can be used for enzyme adsorption. Further adsorption of enzyme may be stabilized by glutaraldehyde treatment.
Immobilization of β -galactosidase on hydrophobic cotton cloth indicated that the enzyme adsorbed on the cloth was about 50% active as free enzymes [ 87 ]. The immobilization of β -galactosidase active yeast K. fragilis and K. lactis onto chitosan showed an enzyme activity of 0.9–2.2 U/mg dry cell wt [ 88 ]. Enzyme activity of immobilized enzyme from K. fragilis was higher but the operational stability of A. oryzae enzyme was 5–14 times higher depending upon the mode of immobilization [ 89 ]. When adsorption method was used, the highest activity was obtained with yeast enzyme and support Ostsorb-DEAE. The enzyme from A. oryzae immobilized on polyvinyl chloride (PVC) and silica gel membrane has been used for the hydrolysis of lactose in skim milk in an axial-annular flow reactor [ 51 ]. Further, maximum immobilization occurred at pH 5.5 and optimal results were obtained with citrate/phosphate buffer during immobilization of β -galactosidase from E. coli by physical adsorption on chromosorb-W [ 51 ]. A novel reactor consisting of β -galactosidase from B. circulans immobilized on a ribbed membrane composed of PVC and silica has also been used for skim milk lactose hydrolysis [ 53 ]. The immobilization of partially purified Bullera singularisβ -galactosidase in Chitopearl BCW 3510 bead (970 GU/g resin) by simple adsorption has also been carried out [ 90 ].
The studies on the kinetic behaviour of β -galactosidase from Kluyveromces marxianus ( Saccharomyces ) lactis , immobilized on to different oxide supports, such as alumina, silica, and silicated alumina indicated that the immobilized enzyme activity strongly depends on the chemical nature and physical structure of the support [ 53 ]. In particular, when the particle sizes of the support are increased, the enzymatic activity strongly decreases. Immobilization of β -galactosidase from Thermus sp. preceded very rapidly onto PEI-Sepabeads and conventional DEAE-Agarose. However, the adsorption strength was much higher in the case of PEI-Sepabeads [ 53 ].
A recombinant thermostable B. stearothermophilusβ -galactosidase was immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized β -galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized β -galactosidase showed greater relative activity in the presence of Ca 2+ than the free enzyme and was stable during the storage at 4°C for 6 weeks, whereas the free enzyme lost 31% of the initial activity under the same storage conditions [ 91 ]. Response surface methodology (RSM) and centre composite design (CCD) have been used to optimize immobilization of β -galactosidase (BGAL) from Pisum sativum onto two matrices: Sephadex G-75 and chitosan beads. The immobilization efficiency of 75.66% and 75.19% was achieved with Sephadex G-75 and chitosan, respectively [ 56 ].
A. oryzaeβ -galactosidase was immobilized on an inexpensive bioaffinity support, concanavalin A-cellulose. Concanavalin A-cellulose adsorbed and cross-linked β -galactosidase preparation retained 78% of the initial activity. The optimum temperature was increased from 50 to 60°C for the immobilized β -galactosidase. The cross-linked adsorbed enzyme retained 93% activity after 1-month storage while the native enzyme showed only 63% activity under similar incubation conditions [ 92 ].
3.3. Covalent Binding Method
Covalent binding is the retention of enzymes on support surface by covalent bond formation. Enzyme molecules bind to support material via certain functional groups such as amino, carboxyl, hydroxyl, and sulfydryl groups. These functional groups must not be in the active site. It is often advisable to carry out the immobilization in the presence of its substrate or a competitive inhibitor so as to protect the active site Functional groups on support material usually activated by using chemical reagents such as cyanogen bromide, carbodimide, and glutaraldehyde.
Eggshells ground into pieces can be good carrier for immobilization of β -galactosidase because of its low cost, good mechanical strength, and resistance to microbial attack [ 61 ]. Fungal enzyme from A. oryzae has been immobilized onto powdered nylon-6 and zeolite [ 62 ]. Zeolites were nonideal since its coupling yield was low whereas nylon resulted in a stable matrix. The derivatives obtained either by diazo or by carbodiimide coupling showed the highest activities during immobilization of the enzyme on glycophase-coated porous glass [ 103 ].
E. coliβ -galactosidase immobilized onto gelatin using chromium (III) acetate and glutaraldehyde retained the relative activities of 25% and 22% for glutaraldehyde and chromium (III) acetate immobilized enzyme, respectively [ 64 ]. The enzyme immobilized on silica-alumina was more stable than the free form at acidic pH [ 65 ]. The ratio of protein to polymer also plays an important role during enzyme immobilization and 100% binding of protein to polymer can be obtained using optimal conditions [ 66 ].
The performance of immobilization of the thermostable β -galactosidase from Thermus sp. strain T2 on a standard Sepabeads-epoxy support with other Sepabeads-epoxy supports partially modified with boronate, iminodiacetic, metal chelates, and ethylenediamine was compared [ 109 ]. Immobilization yields depended on the support, ranging from 95% using Sepabeads-epoxy-chelate, Sepabeads-epoxy-amino, or Sepabeads-epoxy-boronic to 5% using Sepabeads-epoxy-IDA. The immobilized β -galactosidase derivatives showed very improved but different stabilities after favoring multipoint covalent attachment by long-term alkaline incubation, the enzyme immobilized on Sepabeads-epoxy-boronic being the most stable. The optimal derivative was very active in lactose hydrolysis even at 70°C, maintaining its activity after long incubation times under these conditions. Recently, the cross-linking of β -galactosidase on magnetic beads prepared from different sources (Artemisia seed gum, chitosan, and magnetic fluid) was done in the presence of glutaraldehyde and the effects of various preparation conditions on the activity of the immobilized β -galactosidase were studied. The immobilized β -galactosidase resulted in an increase in enzyme stability [ 110 ].
The heat stability of lactase can be increased through immobilization [ 66 , 111 , 112 ]. The effect of temperature and pH on the catalytic activity of immobilized β -galactosidase from K. lactis indicated that the temperature-activity curves are similar for both the free and immobilized enzymes [ 113 ]. However, the maximum activity of the immobilized enzyme was shifted from 40°C to 50°C compared with the free enzyme.
The comparison of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) to conventional epoxy supports to immobilize β -galactosidase from A. oryzae showed that the enzyme stability can be significantly improved by the immobilization on this support, suggesting the promotion of some multipoint covalent attachment between the enzyme and the support [ 67 ]. The immobilization of thermophilic β -galactosidase on Sepabeads for lactose hydrolysis showed decrease in product inhibition, which can be helpful in improving the industrial performance of the enzyme [ 55 ].
Alginate-chitosan core-shell microcapsules have also been used for the immobilization of β -galactosidase [ 68 ]. The rate of 2-nitrophenyl β -galactopyranoside conversion to 2-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37°C relative to calcium alginate-chitosan microcapsules or free enzyme.
Among the three different models (without protection and molecular imprinting technique pretreatment) accomplished for the encapsulation of β -galactosidase, the highest enzymatic activity of enzyme was obtained with molecular imprinting technique [ 114 ]. The free lactase has been cross-linked into Fe 3 O 4 -chitosan magnetic microspheres for lactulose synthesis by dual-enzymatic method in organic-aqueous two-phase media using lactose and fructose as the raw materials [ 115 ]. The organic-aqueous media can significantly promote the transglycosidation activity of lactase and therefore improves the lactulose yield.
Immobilization technology has shown promising role in reducing the product inhibition of β -galactosidase. A. oryzae enzyme immobilized on chitosan beads was more effective as compared to nylon membranes to reduce the galactose inhibition [ 116 ]. Immobilization of the enzyme on heterofunctional epoxy Sepabeads (boronate-epoxy-Sepabeads and chelate-epoxy-Sepabeads) has shown considerable results in reducing the product inhibition [ 55 ]. The effect of internal mass transfer and product (galactose) inhibition on a simulated immobilized enzyme-catalyzed reactor for lactose hydrolysis has been studied [ 104 ]. A general mathematical model has been developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis-Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed-bed-immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion and internal and external mass transfer limitations. The effects of intraparticle diffusion resistances, external mass transfer, and axial dispersion have been studied and their effects were shown to reduce internal effectiveness factor. A. oryzae β -galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate-starch beads. The immobilized β -galactosidase had a much higher K iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose [ 98 ].
4. Applications of Immobilized β -Galactosidase
Immobilized β -galactosidases can be used in a number of ways to hydrolyze lactose in milk, whey/whey permeate, and oligosaccharides synthesis. The choice of process technology depends on the nature of the substrate, the characteristics of the enzyme, economics of production, and marketing of the product. The primary characteristic, which determines the choice and application of a given enzyme, is the operational pH range. Acid-pH enzymes from fungi are suitable for processing of acid whey and whey permeate whereas the neutral-pH enzymes from yeasts and bacteria are suitable for processing of milk and sweet whey.
4.1. Hydrolysis of Milk/Whey
Lactose-hydrolyzed milk has been used for the preparation of flavoured milk, cheese, and yoghurt. The hydrolysis of lactose in milk for food processing also prevents lactose crystallization in frozen and condensed milk products. Moreover, the use of hydrolyzed milk in yoghurt and cheese manufacture accelerates the acidification process, because lactose hydrolysis is normally the rate-limiting step of the process, which reduces the set time of yoghurt and accelerates the development of structure and flavour in cheese [ 1 ]. The quality of ice milk and ice-cream was significantly improved by addition of lactozyme. It prevents the crystallization of lactose by breaking into glucose and galactose and reduces sandiness [ 117 ].
High concentration of lactose in whey is a major environmental problem since its disposal in local water streams increases the biological oxygen demand manifolds. The hydrolysis of whey lactose is another important application of β -galactosidase in dairy industry. Concentrated hydrolyzed whey or whey permeates can be used as a sweetener in products such as canned fruit syrups and soft drinks [ 1 ]. Various immobilizing agents employed for the immobilization of β -galactosidase along with hydrolysis of lactose have been summarized in Table 4 [ 118 , 119 ].
Fungal β -galactosidase (Miles Chemie) immobilized in polyvinyl alcohol gel was found more thermostable than soluble enzyme, retaining 70% of the activity after 24 h at 50°C and 5% activity at 60°C. A lactose hydrolysis of 75% was obtained in 5-6 h and the degree of conversion decreased to 50% after 30 runs [ 93 ]. The studies on the hydrolysis of lactose using immobilized β -galactosidase ( Aspergillus niger ) on phenol-formaldehyde resin indicated that the optimal temperature was found to be dependent on the operating time but not on the lactose concentration or the conversion [ 69 ].
The immobilized β -galactosidase from A. niger displayed 70% hydrolysis in skim milk at 40°C, with a space time of 10 min [ 51 ]. The β -galactosidase enzyme of fungal sources immobilized on hydrogels was used for whey hydrolysis and 70%–75% hydrolysis was achieved within 7 h [ 118 ]. The immobilization of β -galactosidase from A. oryzae on activated silica gel resulted in the most active immobilized preparation from TiCl 3 and FeCl 3 -activated silica gel and resulted in 81 and 84% hydrolysis, respectively, in 4% lactose solution [ 77 ].
The β -galactosidase from Bacillus circulans immobilized onto Duolite ES-762 displayed lactose conversion of >70% in a continuous stirred tank reactor [ 120 ]. The immobilized β -galactosidase from A. oryzae in a packed bed reactor displayed 80% of lactose hydrolysis in whey [ 121 ] whereas the immobilized β -galactosidase from Saccharomyces fragilis resulted in a hydrolytic rate of 50% within 3 h in a recycling packed bed reactor [ 78 ]. Further, the operational stability was tested, with the system being used up to 5 times before any significant drop in the activity. The addition of Mg 2+ and Mn 2+ enhances the hydrolysis of ONPG and lactose by β -galactosidase from K. lactis , but the rates of activation by each metal on both substrate were not the same [ 122 ]. The immobilized K. lactisβ -galactosidase from onto CPC-silica (silanizated and activated with glutaraldehyde) and agarose (activated with cyanylating agent) displayed 90% lactose conversion in packed bed minireactors [ 72 ]. β -galactosidase entrapped in a copolymer gel of N -isopropylacrylamide and acrylamide was effective in hydrolysis of lactose at 5°C for production of low lactose milk. It has been observed that lactose conversion decreased the stability of milk casein particles and increased its dispersity [ 123 ].
The kinetic model for the lactose using immobilized β -galactosidase from K. fragilis has also been developed. The immobilized enzyme was active at a low temperature of 5°C and it could also be applied for the production of freeze dairy products to avoid lactose crystallization and to enhance the digestibility and flavour of such products [ 64 ].
K. fragilisβ -galactosidase immobilized on silanized porous glass modified by glutraldehyde binding retained more than 90% of its activity [ 124 ]. A lactose saccharification of 86%–90% in whey permeate was achieved both in a batch process and in a recycling packed-bed bioreactor. K. lactisβ -galactosidase immobilized onto graphite surface and glutaraldehyde has been used as the cross-linking reagent with the specific activity yield of 17% and 25% while the enzyme loading was 1.8 and 1.1 U/cm 2 of the graphite external surface area, respectively. It was observed that specific activity yield decreased with the increase of the enzyme loading [ 113 ]. Lactose hydrolysis by a β -galactosidase from Thermus sp. both in solution and immobilized on a commercial silica-alumina was studied [ 34 ]. Both the free and the immobilized enzymes are competitively inhibited by galactose, while glucose inhibited only the action of free enzyme, in an uncompetitive way. The immobilization step helped to eliminate the inhibition by glucose. Moreover, the immobilization reduced to a half the inhibitory action of galactose. In general, the immobilization reduced the activity of the enzyme but increased its thermal stability.
The Lactozym (a commercially available enzyme preparation of β -galactosidase obtained from K. fragilis ) immobilized on cellulose beads has been used for the hydrolysis of whey lactose (>90% conversion) and milk lactose (60% conversion) in 5 h and the immobilized enzyme could be reused three times without any change in the performance of the fluidized bed reactor [ 14 ]. The immobilized preparations of β -galactosidase from Thermus sp. resulted in hydrolysis yield higher than 99%. These immobilized forms of β -galactosidase could be used in the total hydrolysis of lactose in milk or dairy whey even at 70°C [ 55 ]. The hydrolysis of lactose by immobilized β -galactosidase has also been studied in a continuous flow capillary bed reactor by various temperatures. Based on the observed thermal deactivation rate constants, at an operating temperature of 40°C, only 10% of the enzyme activity loss could be there in one year [ 125 ].
The β -galactosidase entrapment in liposomes showed superior thermal stability at various ranges of temperature. Moreover, the proteolytic stability of the β -galactosidase was enhanced by encapsulation in liposomes [ 126 ]. The entrapment of β -galactosidase in liposomes by dehydration-rehydration vesicle method has also been used to prevent an immediate hydrolysis of lactose in milk [ 96 ]. A. oryzaeβ -galactosidase was immobilized by three different techniques: adsorption on celite, covalent coupling to chitosan, and aggregation by cross-linking and comparing the yield of immobilized preparation, enzymatic characteristics, stability, and efficiency in oligosaccharide synthesis. Cross-linked enzyme aggregates of β -galactosidase were found effective in lactose hydrolysis yielding 78% monosaccharide in 12 h [ 75 ]. K. lactisβ -galactosidase immobilized on cotton fabric using glutaraldehyde as the cross-linking reagent was used for hydrolysis of lactose in whole milk and 95% of lactose conversion has been observed after 2 h of batch operation [ 23 ].
K. lactisβ -galactosidase was covalently immobilized onto a polysiloxane-polyvinyl alcohol magnet, using glutaraldehyde as activating agent that presented a higher operational and thermal stability than the soluble enzyme; so this immobilized β -galactosidase was also effectively used for the hydrolysis of lactose from milk [ 83 ]. A. oryzaeβ -galactosidase was immobilized on silica, the enzyme activity as well as stability has been evaluated, and the best immobilization results were obtained by using glutaraldehyde as support's activator and enzyme stabilizer. Among the different treatments (microfiltration, thermal treatment, and ultrafiltration) of whey, ultrafiltration was the best treatment towards a proper substrate solution for feeding the reactor [ 84 ].
A recombinant thermostable β -galactosidase from Bacillus stearothermophilus immobilized onto chitosan using Tris (hydroxymethyl) phosphine (THP) and glutaraldehyde resulted in >80% lactose hydrolysis in milk after 2 h of operation in a packed bed reactor. Thus, THP-immobilized recombinant thermostable β -galactosidase from Bacillus stearothermophilus has the potential application for the production of lactose-hydrolyzed milk [ 54 ]. Calcium alginate entrapped β -galactosidase used for the hydrolysis of lactose from solution, milk, and whey in batch processes as well as in continuous packed bed column. It was also observed that entrapped cross-linked concanavalin A- β -galactosidase was more efficient in the hydrolysis of lactose present in milk (77%) and whey (86%) in batch processes as compared to the entrapped soluble β -galactosidase [ 93 ]. Among the two matrices (Sephadex G-75 and chitosan beads) tested for immobilization β -galactosidase (BGAL) from Pisum sativum for lactose hydrolysis, chitosan-PsBGAL displayed higher rate of lactose hydrolysis in milk and whey at room temperature and 4°C than Sephadex-PsBGA and is better suited for industrial application based on its broad pH and temperature optima, high temperature stability, reusability, and so forth [ 56 ]. β -galactosidases (from K. lactis and A. oryzae ) were also immobilized in poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats used for the production of D-galactose from lactose (200 g L −1 ) in the batch mode of a simultaneous saccharification and fermentation process [ 7 ].
A. oryzaeβ -galactosidase was immobilized on an inexpensive bioaffinity support, and concanavalin A-cellulose was used for the continuous hydrolysis of lactose from milk and whey. It was observed that the optimum pH for soluble and immobilized β -galactosidase is 4.8 but the optimum temperature increased from 50 to 60°C for the immobilized β -galactosidase. The immobilized enzyme had higher thermal stability at 60°C [ 92 ]. Recently, a packed bed reactor together with alginate entrapped permeabilized cells ( K. marxianus ) has been used for hydrolysis of milk lactose in a continuous system, which resulted in 87.2% hydrolysis of milk lactose [ 127 ].
4.2. Synthesis of Galacto-Oligosaccharides (GOSs)
Besides hydrolytic action, β -galactosidase has also transferase activity by which the enzyme produces and hydrolyses a series of oligosaccharides, which have a beneficial effect on the growth of desirable intestinal microflora [ 12 ]. Moreover, the transferase reaction can be used to attach galactose to other chemicals, resulting in formation of galacto-oligosaccharides (GOSs), and consequently have potential application in the production of food ingredients, pharmaceuticals, and other biological active compounds. Nowadays, oligosaccharide production becomes the interesting subject for the researchers, because the oligosaccharides have beneficial effect on human intestinal as “bifidus factor”—promoting growth of desirable intestinal microflora. Oligosaccharides are recognized as useful dietary tools for the modulation of the colonic microflora toward a healthy balance. This usually involves selectively increasing the levels of gut Bifidobacteria and Lactobacilli at the expense of less-desirable organisms such as Escherichia coli , Clostridia , and proteolytic bacteroides [ 128 ]. The amount and nature of oligosaccharides formed depend upon the several factors including the enzyme source, the concentration and nature of the substrate, and reaction conditions [ 12 , 129 , 130 ]. The yield of oligosaccharides can be increased by using higher substrate concentrations or decreasing the water content [ 31 ].
Although β -galactosidase catalyzes both hydrolysis and transgalactosylation reactions, however, the process conditions for lactose hydrolysis and GOS synthesis are different. The reaction conditions for transgalactosylation should be high lactose concentration, elevated temperature, and low water activity in the reaction medium [ 130 ]. The temperature, concentration of substrate, and enzyme origin play an important role in the enzymatic synthesis of oligosaccharides [ 131 ]. However, the influence of the initial lactose concentration can be much larger [ 132 , 133 ]. In general, more and larger galacto-oligosaccharides (GOSs) can be produced with higher initial lactose concentrations. The higher temperatures can be beneficial in higher oligosaccharide yields. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Hence, immobilized β -galactosidase should be stable at high temperature, low water content, and giving high transgalactosylation activity [ 134 ].
Partially purified β -galactosidase from Bullera singularis ATCC 24193 immobilized in Chitopearl BCW 3510 bead has been used for the production of galacto-oligosaccharides (GOSs) from lactose in a packed bed reactor, which resulted in 55% (w/w) oligosaccharides with a productivity of 4.4 g/(L-h) during a 15-day operation [ 132 ]. The enzyme immobilized on tosylate cotton cloth was used in plug-flow reactor for continuous production of galacto-oligosaccharide from lactose. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOSs produced in the reactions. The chitosan-immobilized A. oryzae β -galactosidase gave maximum trisaccharides yield (17.3% of the total sugar) using 20% (w/v) of lactose, within 2 h as compared to 10% with free enzyme and 4.6% with cross-linked aggregates [ 73 ].
An immobilized-enzyme system using polyethyleneimine, glutaraldehyde, and cotton cloth was studied and compared the galacto-oligosaccharide production in free-enzyme ultrafiltration and in immobilized-enzyme systems [ 135 ]. In the immobilization process, approximately 50% to approximately 90% enzyme inactivation was found with the combination of PEI and GA. Equivalent free- and immobilized-enzyme systems showed very similar maximum GOS production of approximately 22% and approximately 20% (w/v) at approximately 15 to 17 min, 50% conversion for free- and immobilized-systems, respectively.
The synthesis of galacto-oligosaccharides was optimized with respect to lactose concentration and enzyme to substrate ratio using immobilized A. oryzaeβ -galactosidase [ 136 ]. In the sequential batch production of galacto-oligosaccharides, biocatalyst efficiency was increased by 190% with respect to the free enzyme in solution, and 8500 g of galacto-oligosaccharides per gram of enzyme preparation were produced after 10 batches. The immobilized A. oryzaeβ -galactosidase enzyme on magnetic Fe 3 O 4 –chitosan (Fe 3 O 4 –CS) nanoparticles as support resulted in 15.5% (w/v) maximum yield of galacto-oligosaccharides [ 137 ]. The synthesis of galacto-oligosaccharides (GOSs) using A. oryzae β -galactosidase (free and immobilized) on magnetic polysiloxane-polyvinyl alcohol (mPOS-PVA) has also been carried out [ 138 ]. A maximum of 26% (w/v) of total sugars was achieved at near 55% lactose conversion from 50% (w/v) lactose solution at pH 4.5 and 40°C. Trisaccharides accounted for more than 81% of the total GOS produced. GOS formation was not considerably affected by pH and temperature. The concentrations of glucose and galactose encountered near maximum GOS concentration greatly inhibited the reactions and reduced GOS yield.
The packed bed reactor and a plug-flow reactor have been successfully used for continuous production of GOS from lactose using immobilized β -galactosidase [ 63 , 90 ]. The selectivity for GOS synthesis can be increased several-fold under microwave irradiation, using immobilized β -glucosidase and with added cosolvents such as hexanol [ 139 ]. Recently, a new type of ceramic membrane reactor system using immobilized β -galactosidase ( Kluyveromyces lactis ) has been proposed for continuous enzymatic production of galactosyl-oligosaccharides (GOSs) from lactose, which resulted in maximum oligosaccharide (38%, w/w) when the average residence time was 24 min, with an initial 30% (w/w) lactose concentration [ 140 ].
5. Scale-Up Issues
For the production of lactose-free milk, the enzyme β -galactosidase can be added directly to whole milk, but after complete lactose hydrolysis at a desired level, the enzyme can be deactivated by heat treatment. Since the free enzyme cannot be reused, thus the resulting operation is not cost effective. To overcome this problem, immobilized β -galactosidase is used for the hydrolysis of skim milk. After the desired lactose hydrolysis is achieved, cream is added to the hydrolysed milk to adjust its fat content. Although numerous hydrolysis systems have been investigated, only few of them have been scaled up and even fewer have been applied at an industrial or semi-industrial level.
The first company for the commercial hydrolysis of lactose in milk by using immobilized lactase was Centrale del Latte of Milan, Italy, utilizing the SNAM Progetti technology. The process used an immobilized Saccharomyces ( Kluyveromyces ) lactis lactase entrapped in cellulose triacetate fibres. Sumitomo Chemical, Japan, has also developed an immobilization process to immobilize β -galactosidase of fungal origin on the rugged surface of an amphoteric ion-exchange resin of phenol formaldehyde polymer and this technology was used by Drouin Cooperative Butter Factory for producing market milk and hydrolyzed whey [ 141 ].
Snow Brand's factory has developed a rotary column reactor that could be used both as a stirred tank reactor and as a packed bed reactor [ 142 ]. The reaction rate was greatly affected by the packing density of immobilized β -galactosidase in the rotary column. This reactor can also overcome the problem of channeling or severe pressure drop. If the hydrolysis of lactose was carried out in horizontal rotary column, 70%–80% lactose hydrolysis was observed and washing of the immobilized enzyme was carried out for 36 cycles, which indicated that horizontal rotary column reactor was well suited for hydrolyzing lactose in milk with fibrous immobilized enzyme. From the pilot plant experimentations, a commercial plant was set up at Snow Brand's factory [ 142 ]. Although the immobilized β -galactosidase was washed with phosphate buffer solution and pasteurized with Tego-51, the standard plate count of lactose hydrolyzed milk increased sharply.
Thus, immobilized β -galactosidase technology is an effective process for successful hydrolysis of lactose and it can overcome the problems associated with costs of soluble enzyme. However, major problems associated with the immobilized enzyme system are microbial contamination, protein adherence, and channeling. Therefore, for long-term operations using immobilized system, periodic washing, and pasteurization are indispensable processes [ 143 – 145 ]. In immobilized enzyme system, protein adhered to the enzyme can be easily dissolved by using high and low pH solutions, because the immobilized enzyme has high durability over a wide range of pH. The immobilized enzyme can be pasteurized with benzalkonium chloride (quaternary ammonium salt) after removing the proteins. The use of acetic acid solution as a cleaning and pasteurizing agent instead of lactic acid can also be effective. The problem of channeling observed in the packed column system can be overcome by changing the flow direction of feed during the operation [ 142 , 144 , 145 ].
Galacto-oligosaccharides are produced simultaneously during lactose hydrolysis due to transgalactosylation activity of β -galactosidase. Oligosaccharides/Bifidobacteria provide a wide variety of health benefits, including anticarcinogenic effects, reduction in serum cholesterol, improved liver function, reduction of the colon cancer risk, and improved intestinal health [ 146 , 147 ]. Therefore, the public demand for their production is significantly increased together with the development of an effective and inexpensive GOS production. Major companies dealing with oligosaccharides production (including GOS) are in Japan [ 148 ]. Recently, there is also an increasing trend of GOS production in Europe. Besides lactulose and soybean oligosaccharides, all oligosaccharides are prepared by transglycosylation from mono and disaccharides or by controlled hydrolysis of polysaccharides [ 147 ]. | Acknowledgment
The authors acknowledge the financial support given by the Council for Scientific and Industrial Research (CSIR), New Delhi, India. | CC BY | no | 2022-01-13 02:52:24 | Enzyme Res. 2010 Dec 27; 2010:473137 | oa_package/34/77/PMC3014700.tar.gz |
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PMC3014701 | 21234408 | 1. Introduction
Hereditary gingival fibromatosis (HGF) is a rare benign, nonhemorrhagic fibrous enlargement of gingival tissue [ 1 ]. Males and females are equally affected at a phenotype frequency of 1 : 750,000 with varying intensity and expressivity even in individuals within the same family [ 1 , 2 ]. The disease may be found as an autosomal dominant or autosomal recessive mode of inheritance [ 2 – 4 ].
The hyperplastic gingiva usually presents a normal color and has a firm consistency with abundant stippling. The gingival enlargement usually coincides with the eruption of the permanent dentition although it may occur during the eruption of primary dentition or rarely at birth. It may be localized (nodular) or generalized (symmetric), thus potentially interfering with speech, closure of the lips, and mastication resulting in both aesthetic and functional problems [ 2 , 5 ].
HGF is usually seen as an isolated disorder, but it may also develop as one feature of several rare multisystem syndromes such as Zimmerman-Laband (ZLS), Jones, Ramon and Rutherford syndrome, Juvenile hyaline fibromatosis, systemic infantile hyalinosis, and mannosidosis. HGF has been recorded in association with hypertrichosis, mental retardation, epilepsy, progressive sensorineural hearing loss, and abnormalities of extremities, particularly of fingers and toes [ 6 ].
Conditions associated with Zimmerman-Laband syndrome are gingival fibromatosis, abnormal fingers, fingernails, nose, and ears. Other findings associated with ZLS are Splenomegaly, hepatomegaly, and hyperextensible metacarpophalangela joint [ 7 ]. In Jones syndrome, gingival fibromatosis associated with progressive sensoneural hearing loss was found [ 8 ]. In Ramon syndrome, the findings were gingival fibromatosis, cherubism, seizures, mental deficiency, hypertrichosis, stunted growth, and juvenile rheumatoid arthritis [ 9 ]. In Rutherford syndrome conditions associated with gingival hypertrophy include corneal opacity, mental retardation, failure of tooth eruption, and aggressive behavior [ 10 ]. In juvenile hyaline fibromatosis, gingival fibromatosis was associated with multiple subcutaneous tumors, dysseborrhea, sclerodermiform atrophy, whitish nodules, and osteolytic and osteoclastic skeletal lesion [ 11 ]. In systemic infantile hyalinosis, gingival fibromatosis was associated with thickened skin, focal skin nodularity, restricted movement, joint contractures, and osteoporosis and most of the individuals fail to thrive [ 12 ]. In Mannosidosis, gingival hypertrophy was associated with deafness, muscle hypotonia, craniofacial dysmorphism, mental retardation, and Immunoglobulin deficiency [ 13 ]. | 5. Discussion
HGF may be found as an autosomal dominant or autosomal recessive mode of inheritance with variable penetrance and expressivity [ 2 – 4 ]. The mode of genetic transmission in this patient points to an autosomal dominant gene because family members of both sexes were affected, and the condition was present in successive generations (grandfather, mother, and children). The enlargement began with the emergence of deciduous dentition and gradually increased to cover the teeth completely, delaying the exfoliation of primary molars. Patient exhibited a more common generalized (symmetric) gingival enlargement. Severe growth resulted in crowding of underlying teeth, speech impediment, difficulty with mastication, and prevented normal closure of lips. Syndromic abnormalities commonly seen in association with HGF were not observed in this patient. To date, three different loci are associated with isolated form of HGF: two map to chromosome 2 (GINGF on 2p21-22 and GINGF3 on 2p22.3-p23.3) and one maps to chromosome 5 (GINGF2 on 5q13-HGF1 locus 2p13-p21) [ 2 , 14 ]. Of these loci, only the SOS1 ( son-of-sevenless-1 ) gene that codes for guanine nucleotide exchange factor for ras proteins has been described [ 15 , 16 ].
The histopathologic features observed in the present case had the typical appearance of the gingival lesions in HGF, and the provisional diagnosis was confirmed. Although the mechanism that leads to the accumulation of abnormal amounts of gingival tissue in HGF is still unknown, there is some evidence that certain defects may lie in the anabolism of gingival tissue products.
HGF cannot be cured but may be controlled with varying degree of success. The best time to initiate treatment to HGF is when all of the permanent dentition has erupted because the risk of recurrence is higher before it. Treatments vary according to the degree of severity of gingival enlargement. When the enlargement is minimal, thorough scaling of teeth and home care may be sufficient. However, excessive gingival tissue and esthetic and functional impairment dictate the need for surgical intervention [ 2 , 5 ]. Because of the severity of the involvement with no attachment loss and pocketing in this case, an external bevel gingivectomy was the favored treatment. In areas with inadequate attached gingiva (right lower posterior region), flap surgery was carried out. Psychological benefits because of cosmetic improvement outweigh the probability of recurrences in such severe cases. In the present case, because of periodic appointments with good plaque control measures with appropriate and timely orthodontic treatment, recovery is expected to be uneventful. A multispeciality approach involving a periodontist, orthodontist, oral surgeon, and oral pathologist helped to provide a successful treatment in this case. Recurrence of gingival over growth in HGF is not uncommon. Therefore, more frequent followup might be required. | Academic Editor: Dimitris N. Tatakis
Background . Hereditary gingival fibromatosis is a fibrotic enlargement of the gingiva. It may exist as an isolated abnormality or as part of multisystem syndrome. This paper reports a case of 16-year-old male with generalized severe gingival overgrowth, involving the maxillary and mandibular arches and covering almost all teeth. Methods . Periodontal management of gingival enlargement included gingivectomy in both arches except in the lower right molar region where flap surgery was done under general anesthesia. After a 2-month followup period, orthodontic treatment was started with fixed appliances. Monthly periodontal checkups and maintainance (scaling and polishing) were scheduled to control the gingival inflammation. Results . Reevaluation of the patient of surgical treatment after two months did not show any recurrence of condition; however, minimal overgrowth was noted 1 month after the beginning of orthodontic treatment which was treated nonsurgically. Conclusions . Although the risk of recurrence is high with this condition, surgical treatment with correction of malocclusion and regular followup can provide excellent outcome as seen in this case. | 2. Case Report
A 16-year-old male presented at the Department of Periodontology and Implantology, Meenakshi Ammal Dental College and Hospital, Chennai, with the chief complaint of gingival swelling covering both mandibular and maxillary teeth. The swelling caused difficulties in speaking and eating, and he also had obvious implications for his aesthetic appearance. The patient's medical history appeared to be noncontributory to the development of the gingival enlargement. The patient in this report had no history of using drugs such as phenytoin, nifedipine, or cyclosporine. The patient revealed a family history which was apparently significant to the present finding. His maternal grandfather and maternal uncle had similar gingival enlargements but were deceased. The patient's mother (58 years old) ( Figure 5 ), maternal aunt (59 years old), and sister (26 years old) ( Figure 6 ) had similar gingival enlargement involving, to various extents, the maxilla as well as the mandible, which was treated at various points of time.
The intraoral examination revealed generalized, severe gingival overgrowth involving both the mandibular and maxillary arches (Figures 1(a) , 1(c) , 2(a) , and 2(b) ). The gingival overgrowth was seen as firm, dense, fibrous, and painless enlargement with normal gingival color. Panoramic radiographic examination revealed complete permanent dentition with retained deciduous molars (54, 64, 75, and 85) ( Figure 1(b) ). Teeth were malaligned with minimal alveolar bone loss. In the light of patient, family history, and these clinical observations, a provisional diagnosis of HGF was given based on the family history and clinical examination.
3. Treatment
Functional and esthetic disability indicated a need for surgical intervention which was carried out under general anesthesia after informed consent was obtained from the patient's parent. The treatment consisted of an external bevel gingivectomy at all quadrants using scalpel and electrocautery (Colorado tip) with total excised tissue weighing approximately 160 g. Postoperative bleeding did not occur. The deciduous molars were removed at the time of surgery, as they were held in place only by the bulk of gingiva. Postoperatively, the patient was advised to continue the antibiotic (amoxillin-500 mg tds) for 5 days, analgesic (ibuprofen 400 mg) as and when needed and to use 0.2% chlorhexidine digluconate mouth rinse for 2 weeks postoperatively ( Figure 3 ). The patient was recalled for checkup at 1, 3, and 6 weeks intervals postoperatively. The postsurgical healing was excellent as the patient maintained good oral hygiene. Orthodontic treatment was started after two months and she is still in progress ( Figure 4 ).
4. Histopathological Examination
The fixed tissue specimens (10% buffered formalin) showed highly fibrous connective tissue with dense collagen bundle arranged in haphazard manner with moderate number of spindle-shaped fibroblasts. Focal areas of chronic inflammatory cell infiltrate with lymphocytes, plasma cells, neutrophils, and few mast cells were seen. Blood capillaries showed compressed lumen and some were engorged with RBCs. The overlying epithelium appeared hyperkeratotic stratified squamous of variable thickness with irregular rete ridges showing hyperplasia in some areas and atrophy in others. Superficial layers of epithelium showed features of edema. | Acknowledgments
The authors would like to thank Dr. Biju mameen, Dr. Manikandan, Dr. Vivek, Dr. Deepika (Meenakshi Ammal Dental College and Hospital) for their excellent assistance in surgical and orthodontic treatment. | CC BY | no | 2022-01-13 02:32:44 | Int J Dent. 2010 Dec 23; 2010:575979 | oa_package/be/e0/PMC3014701.tar.gz |
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PMC3014702 | 21234409 | 1. Introduction
Cytoplasmic crystalline inclusions and pathological calcification of liver have been described occasionally in humans. There are only a few reports of crystalline inclusions in hepatocytes of dogs [ 1 ], cattle [ 2 , 3 ], chimpanzees [ 4 ], rats [ 5 ], rabbits [ 6 ], and Ohrid trout [ 7 ]. They have never been described in antelopes. The current report describes the histological and electron microscopic features of hepatocellular crystalline inclusions in a gazelle. | 3. Discussion
Hepatocellular cytoplasmic crystalline inclusions have been described in cattle with mucosal disease [ 2 ], 8-month-old steer with shipping fever [ 3 ], dogs and rats with Escherichia coli endotoxin [ 5 ], and liver of normal dogs [ 8 ] and rabbits [ 6 ]. The presented case was an 11-year-old gazelle; therefore, it appears that there is unknown relationship between hepatocellular inclusions and age of the animal. Crystalline inclusions have been observed in the hepatocytes of normal human liver, but more frequently in Gilbert's syndrome [ 9 ]. Gilbert's syndrome is a congenital hyperbilirubinemia seen in humans, inherited as an autosomal dominant trait [ 10 ]. It was suggested that disruption of mitochondria liberates the inclusions into the cytoplasm or that crystals represent some form of hepatic degeneration in humans [ 9 ] or crystallized form of viruses [ 11 ]. Nonmembrane bound hepatocellular intracytoplasmic crystalline inclusions have never been reported in an antelope. Crystalline inclusions in the mitochondria of dog's liver have been documented in animals kept on low-protein diet [ 12 ]. However, the crystalline inclusions in the present study were extramitochondrial, and their pattern was different from the structure of intramitochondrial inclusions described in dogs. SEM/EDXA analyses on the liver of gazelle provided evidence of accumulations of calcium, oxygen, phosphorus, and smaller amounts of magnesium and sodium. Infrared spectroscopy showed the hepatocellular material consistent with protein and carbohydrate. There was no evidence of bacterial or viral infection microscopically and ultrastructurally. Bacteriological culture on liver revealed no growth. Initially, storage disease was considered as a possible differential after examining the tissue sections of liver stained with hematoxylin and eosin. Storage diseases can be either inherent/spontaneous or acquired/toxin induced (such as due to ingestion of toxic plants). There was no clinical history of toxin exposure in the zoo, but it cannot be completely excluded.
The possible cause of severe emaciation of this gazelle was considered due to severe parasitism and concurrent thyroid hyperplasia. At the time of necropsy, there was a moderate parasitic burden in this animal based on fecal examination. However, with the history of recent deworming, the number of worms and ova remaining could be indicative of a severe infestation earlier. Hepatocellular calcification has been reported following a wide range of injuries such as inflammatory state, benign, and malignant neoplasm [ 13 ]. In the majority of the cases, calcification was consequence of massive hepatocellular necrosis or apoptosis [ 14 ]. However, there was no evidence of necrosis and/or apoptosis in the present case. The hepatic lesions in this gazelle were most likely due to a combination of thyroid hyperplasia and severe/chronic parasitism leading to hypoproteinemia. This finding is in partial agreement with a previous study in which dogs fed on low-protein diet developed intramitochondrial crystalline inclusions [ 12 ]. The cytoplasmic hepatocellular crystalline inclusions in the current report could be due to altered lipoprotein synthesis through disturbed oxidative metabolism in addition to disorganization of the membranous compartments in the cytoplasm. The other possible mechanism is that crystalline inclusions may have been derived due to failure of normal physiological hepatocellular clearance associated with a severe chronic disease which was confirmed histologically in the present case. It is also intriguing to speculate that increased protein production and increased permeability of nuclear membrane caused cytoplasmic crystallization of the protein. Moreover, the coexistence of calcium and phosphorus on SEM/EDXA analyses in the liver is suggestive of precipitation of calcium and phosphorus within the cytoplasm of hepatocytes. Whether the crystalline inclusions are de novo or degenerative products of the preexisting material in the cells remains to be elucidated. This case report is the first to describe cytoplasmic hepatocellular crystalline inclusions in an antelope. | Academic Editor: Tomoyuki Shibahara
This case report describes intracytoplasmic crystalline inclusions in the hepatocytes of a 13-year-old female Thomson's gazelle. Histologically, multifocal to coalescing areas of many hepatocytes contained large cytoplasmic vacuoles filled with pale eosinophilic homogeneous material and rare fine basophilic granules. Von Kossa staining showed the presence of calcium within cytoplasm, mainly in the inclusions, of hepatocytes. Transmission electron microscopy, scanning electron microscopy, energy dispersive X-rays analyses, and infrared spectroscopy on the liver showed the hepatocellular material consistent with protein and carbohydrate with secondary accumulation of calcium and phosphorus. It was concluded that crystalline inclusions may have been derived due to failure of normal physiological hepatocellular clearance associated with a severe chronic disease. To the authors' knowledge this is the first reported case of hepatocellular crystalline inclusions in an antelope. | 2. Case Description
A 13-year-old female Thomson's gazelle ( Eudorcas thomsoni ) from a zoo was presented to Louisiana Animal Disease Diagnostic Laboratory, Louisiana State University (LSU) (Baton Rouge, LA, USA) for necropsy examination. The animal was found dead and had a history of severe parasitism. Postmortem examination revealed sparse body fat store in the gazelle. There was bilateral enlargement of thyroid glands (right: 6 × 3 × 2 cm; left: 2 × 2 × 1 cm), and the cut surfaces were pale tan with multifocal red areas. The liver was firm, rounded, and the cut surface was mottled tan and red ( Figure 1 ). There was marked serous atrophy of fat in the bone marrow and epicardial fat. Histologically, multifocal to coalescing areas of many hepatocytes contained large cytoplasmic vacuoles filled with pale eosinophilic homogeneous material and rare fine basophilic granules with hematoxylin and eosin stain ( Figure 2(a) ). Periportal areas had moderately increased fibrous connective tissue with marked biliary hyperplasia, mild oval cell hyperplasia, and moderate infiltrates of predominant lymphocytes and plasma cells with occasional neutrophils. Multifocal small aggregates of neutrophils occasionally admixed with plasma cells, lymphocytes, and rare degenerative hepatocytes were present in the hepatic parenchyma. There were multifocal areas of hepatocellular nodular regeneration. The histological findings in liver were diagnosed as chronic multifocal to coalescing severe hepatocellular vacuolation with unknown material and mineral, and multifocal moderate lymphoplasmacytic cholangiohepatitis, bridging portal fibrosis and biliary hyperplasia. There was no evidence of hepatocellular necrosis. The histopathological findings in liver were interpreted as severe chronic hepatic disease. The majority of intracytoplasmic inclusions failed to stain for fat with oil red O ( Figure 2(b) ), glycogen with periodic acid-Schiff stain (with and without diastase digestion) ( Figure 2(d) ), and acid mucopolysaccharides. The hepatocytes stained strongly with von Kossa staining consistent with calcium (mineral), mainly in the intracytoplasmic inclusions ( Figure 2(c) ). Additional stains including Hall's for bile pigment, fibrinogen, phosphotungstic acid hematoxylin for fibrin, Prussian blue for iron, auramine-rhodamine for acid fast organisms, Mayer's mucicarmine for polysaccharide, alcian blue (pH 2.5) for mucin, and congo red for amyloid failed to stain the pale eosinophilic cytoplasmic inclusions. Additionally, immunohistochemical staining for fibrinogen, kappa, and lambda light chains yielded negative results. An undetermined storage disease was considered as a differential in the tissue sections of liver. The enlarged thyroid glands were diagnosed as thyroid hyperplasia. No other significant microscopic findings were seen in this animal. Fecal floatation revealed moderate mixed parasitic burden including Trichostrongylus spp., Haemonchus spp., and Nematodirus spp. parasites eggs.
For transmission electron microscopic examination, 1-mm 3 fragments of liver were fixed in 3% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated in ethanol, and embedded in liquid epoxy resin. The ultrathin sections (70–90 nm) were stained with lead citrate and uranyl acetate and examined with an electron microscope (JEOL JEM-1011). Ultrastructurally, nonmembrane bound, irregular shaped crystalline inclusions were seen in the cytoplasmic vacuoles of hepatocytes ( Figure 3(a) ). Lipid with/without mineral deposits was noted within the vacuoles ( Figure 3(b) ). Scanning electron microscopy and energy dispersive X-rays analyses (SEM/EDXA) on the liver provided evidence of accumulations of calcium, oxygen, phosphorus, and smaller amounts of magnesium and sodium. Infrared spectroscopy showed the hepatocellular material consistent with protein and carbohydrate. | CC BY | no | 2022-01-13 03:21:04 | Vet Med Int. 2010 Dec 27; 2010:373698 | oa_package/72/cc/PMC3014702.tar.gz |
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PMC3014703 | 21234410 | 1. Introduction
Propylthiouracil (PTU) has been implicated in drug-induced liver injury (DILI) in patients with hyperthyroidism treated with this medication. Reported injury has ranged from mild asymptomatic elevation of aminotransferases to acute liver failure (ALF). Although asymptomatic elevations in hepatic enzymes have been described in patients with untreated hyperthyroidism, recognition of hepatic dysfunction in a patient taking PTU requires immediate discontinuation of the drug and close followup. | 3. Discussion
PTU is a thioamide derivative widely used for the treatment of hyperthyroidism which exerts its pharmacologic effects by two different mechanisms. It inhibits reactions catalyzed by the enzyme thyroid peroxidase expressed in the thyroid follicles and blocks iodine organification, and it also inhibits the enzyme 5′-deiodinase accountable for the peripheral conversion of T4 into the active T3 moiety.
PTU-induced hepatitis was first reported by H. J. Livingston and S. F. Livingston in 1947 [ 1 ], shortly after the FDA had approved this medication for the treatment of hyperthyroidism. This patient was successfully managed with supportive care after discontinuation of the drug. Six years later, Eisen [ 2 ] reported the first case of fulminant hepatic failure attributed to PTU, an ominous adverse reaction that since then has been observed in an extremely small number of patients receiving this medication. Based on published data about the annual incidence of hypothyroidism, the reported frequency of PTU therapy (15,000 adults per year), and the incidence of PTU-induced severe liver injury (approximately 0.1% in the adult population), approximately 15 adults will develop this complication annually in USA and 10% of them (1 : 10,000 adults) will progress to ALF. Asymptomatic elevations of alanine aminotransferase (ALT) are observed in 14–28% of patients started on PTU during the first 2 months of therapy and usually resolve with no intervention [ 3 ]. Acute hepatitis associated with this medication has been reported to occur in 0.1–1.2% of patients [ 4 ]. The mortality associated with PTU-induced acute hepatitis can be as high as 25% [ 5 ]. Data suggest that the risk of severe hepatotoxicity is greater in children treated with PTU (1 : 1,000 children), but the overall incidence is significantly lower because fewer children are treated with this drug (1,500–4,000 children per year) [ 6 ]. For example, a significant reduction in PTU use by pediatric endocrinologists has been observed over the past several years, and some authors advocate that PTU should never be used as a first-line therapy in children due to the potential risk severe hepatotoxicity [ 7 ].
Liver biopsy remains the gold standard for diagnosis of PTU-induced hepatic injury, but the diagnosis is often inferred from the time course after initiation of PTU therapy [ 5 , 8 ]. PTU hepatotoxicity has been reported to cause a variety of histological changes including portal and periportal inflammation with eosinophilic, lymphocytic, and plasmacytic infiltration in varying combinations, chronic active hepatitis, and submassive or massive hepatic necrosis [ 9 ]. These histological features resemble autoimmune hepatitis type 1 (AIH-1) and are referred to as drug-induced AIH-1. A multifactorial hypothesis has been proposed as an explanation for PTU-induced hepatopathy in which inhibition of glucuronyl transferase, reduced bile acid synthesis, and increased oxygen consumption by the hepatocytes could be implicated in more or less extent [ 10 , 11 ]. Severe PTU-induced hepatotoxicity is postulated to be a dose independent, idiosyncratic hypersensitivity reaction. This theory is supported by positive lymphocyte sensitization studies [ 3 , 4 , 12 ]. Some authors have noted an association between this pathologic condition and positive antineutrophil cytoplasmic antibodies (ANCA); however, ANCA positivity has been reported in up to 50% of asymptomatic patients receiving PTU, and this finding has been considered to be incidental in the majority of cases and not directly related to DILI or ANCA-associated vasculitis [ 13 , 14 ].
Treatment options for PTU-induced liver failure are limited. Immediate discontinuation of the drug is imperative, but progression of liver injury may occur, particularly in cases with severe forms of DILI, and aggressive supportive care is usually required. The milder form of PTU-induced hepatotoxicity, characterized by symptomatic hepatitis, has been associated with complete recovery after discontinuation of the drug [ 15 ]. This outcome has also been reported in cases with cholestatic liver injury, although the time required for normalization of biochemical markers was longer in the latter group [ 4 ]. Some authors have suggested that the coincidental clinical improvement in patients receiving systemic corticosteroids for the treatment of thyrotoxicosis might reflect a therapeutic response [ 16 ]. Plasmapheresis was apparently successfully used, in one case report of fulminant hepatic failure; nonetheless, concomitant use of prednisone confounded evaluation [ 8 ]. Liver transplantation has been reported in severe, life-threatening cases of PTU-induced hepatotoxicity with good outcomes [ 5 , 15 ]. Between 1990 and 2007, the United Network for Organ Sharing (UNOS) reported 23 liver transplants (16 in adults and 7 in children) for PTU-induced ALF. Population-based estimates of liver transplantations for ALF using the UNOS database indicate that drug-induced ALF accounts for approximately 12–15% of liver transplants for ALF in USA [ 17 , 18 ]. PTU is the third most common single drug responsible for DILI requiring liver transplantation in the general population, preceded by acetaminophen and isoniazid in the adult population and by acetaminophen and valproic acid in pediatric patients [ 17 ]. The mean age of patients with PTU-induced ALF is 28 years; over two thirds of these patients are females and almost half of them are African Americans. Therefore, African American ethnicity may be a risk factor for severe form of PTU-induced hepatotoxicity; nonetheless, there are no data supporting ethnicity-specific or genetic variations responsible for these observations, and specific recommendations about the use of PTU in African Americans cannot be made. ALF secondary to PTU and herbal substances is associated with higher bilirubin levels (23.2 mg/dL and 23.6 mg/dL on average, resp.) compared to other drugs, including acetaminophen (9.8 mg/dL on average). The one-year graft survival rate after liver transplantation for PTU-induced ALF is 84%; however, no significant difference has been found in graft survival rates after ALF caused by PTU, acetaminophen, isoniazid, or phenytoin [ 17 ].
We add to the body of literature two more cases of severe PTU-induced liver failure successfully treated with OLT. The first patient had an uneventful posttransplant recovery, but the second case had a complicated clinical course requiring retransplantation and multiple interventions to prevent loss of the second allograft. Extended criteria for donor organs, including advanced-age donors, may be used due to the emergency need for OLT in patients with fulminant hepatic failure; nonetheless, higher rates of severe complications requiring specialized care underscore the importance of treating these patients in experienced, high-volume transplant centers. There are no official recommendations regarding monitoring for serologic markers of liver injury during PTU therapy; however, some authors have recommended monthly monitoring of serum transaminases for the first six months of therapy, which is the period where most cases of PTU-induced DILI occur [ 19 , 20 ]. Although there are no evidence-based data to support this recommendation, we agree with the suggested time intervals and duration of monitoring and emphasize the importance of early discontinuation of therapy when early signs of liver injury are detected. | 4. Conclusions
Clinicians taking care of patients being treated with PTU should have a low threshold for suspecting medication-related adverse reactions, particularly for PTU-induced DILI. Routine monitoring of serum transaminases and close followup is recommended; nonetheless, the appropriate intervals and duration of followup have not been established. Early identification of clinical signs and abnormalities in biochemical markers of hepatic injury followed by immediate discontinuation of PTU, aggressive supportive care, and transfer of patients to centers capable of performing emergent liver transplantation is strongly advised due to the high mortality associated with severe forms of this complication. | Academic Editor: Furio M. Pacini
Propylthiouracil- (PTU-) induced hepatotoxicity is rare but potentially lethal with a spectrum of liver injury ranging from asymptomatic elevation of transaminases to fulminant hepatic failure and death. We describe two cases of acute hepatic failure due to PTU that required liver transplantation. Differences in the clinical presentation, histological characteristics, and posttransplant management are described as well as alternative therapeutic options. Frequent monitoring for PTU-induced hepatic dysfunction is strongly advised because timely discontinuation of this drug and implementation of noninvasive therapeutic interventions may prevent progression to liver failure or even death. | 2. Case Presentations
Case 1 A 29-year-old African American woman with Graves' disease unsuccessfully treated with methimazole was prescribed propylthiouracil (PTU) 50 mg PO every 8 hours. Aminotransferase levels were normal before therapy was started; however, mild elevations of these enzymes were initially noticed by the fourth week of therapy (AST: 64 U/L, ALT: 94 U/L, alkaline phosphatase: 170 U/L) and continued to progressively increase by week eight. By week ten of treatment, she reported jaundice, fatigue, epigastric abdominal pain, nausea, and vomiting. She denied the use of over-the-counter or herbal medications. Her past medical history and family history were not contributory. She denied excessive alcohol consumption and recreational drug use. Upon subsequent transfer to our institution, initial laboratory workup revealed a prothrombin time of 39.1 seconds, INR 3.9, total bilirubin 19.3 mg/dL, direct bilirubin 12.1 mg/dL, AST 503 U/L, ALT 443 U/L, alkaline phosphatase 509 U/L, TSH 0.013 μ IU/ml, and free-T4 0.7 ng/dL. Serum markers for viral hepatitis A, B, and C were negative. Antinuclear antibodies, antismooth muscle antibodies, and antimitochondrial antibodies were negative. PTU was discontinued, but the coagulopathy worsened, and she subsequently developed hepatic encephalopathy. She underwent orthotopic liver transplantation (OLT) eight days following admission. Histologic examination of the explanted liver revealed submassive, confluent necrosis with parenchymal hemorrhage, bile duct proliferation, intracellular and canalicular cholestasis, bile plugging, and severe lymphoplasmacytic and eosinophilic infiltrates. Immunostain for IgG4 was positive ( Figure 1 , microphotographs (a) and (b)). The patient was discharged home in stable condition on postoperative day twelve. Her liver function become normal 6 months following OLT.
Case 2 A 34-year-old African American woman with Graves' disease and without an antecedent history of liver disease was prescribed PTU 150 mg PO twice daily. Baseline levels of aminotransferases were normal at the time PTU was started. Six weeks later she started to complain of malaise and generalized weakness followed by progressive jaundice, but she did not seek medical care until two weeks later when she had developed confusion, nausea, and vomiting. Blood tests at that time were INR 4.3, total bilirubin 22.8 mg/dL, direct bilirubin 10.8 mg/dL, AST 1081 U/L, ALT 1227 U/L, alkaline phosphatase 272 U/L, TSH 61.2 μ IU/ml, and free-T4 0.6 ng/dL. Serum markers for autoimmune and viral hepatitis A, B, and C were negative. PTU was discontinued; however, her mental status continued to deteriorate with progression to severe hepatic encephalopathy. A transjugular liver biopsy revealed extensive parenchymal necrosis, collapse of the lobular architecture, bile duct proliferation, and periportal inflammation. Two days after admission, she was listed for OLT and received a liver transplant three days later. Histological examination of the native liver showed submassive confluent necrosis with prominent eosinophilic, neutrophilic, and lymphoplasmacytic infiltrate with canalicular and intracellular cholestasis and numerous lobular acidophilic bodies ( Figure 1 , microphotographs (c) and (d)). The allograft was retrieved from a 77-year-old deceased donor, due to the recipient's critical status. The posttransplant course was complicated by the development of graft dysfunction due to severe rejection, which did not improve with aggressive immunosuppressive therapy. The patient was relisted for OLT two weeks after the initial transplant and received a second allograft 6 days later. The postoperative course after the second transplant was complicated by a biliary leak that required reconstruction of the biliary anastomosis, as well as multiple episodes of rejection which were treated with antilymphocyte antibodies, plasmapheresis, and administration of rituximab. The patient eventually recovered. She was discharged on postoperative day 155 from the first liver transplant and is currently at home doing well 6 months after the second liver transplant. | Conflict of Interest Disclosure
None; Dr. Martin is a consultant and speaker for Roche, Gilead, and Bristol-Myers Squibb Pharmaceuticals.
Declaration of Funding Source
There are no sources of pharmaceutical or industry support related to this report. | CC BY | no | 2022-01-13 02:35:08 | Int J Endocrinol. 2010 Dec 23; 2010:910636 | oa_package/56/2b/PMC3014703.tar.gz |
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PMC3014705 | 21234412 | 1. Introduction
Papillary lesions of breast have varied morphological, radiological, and pathological features. Such lesions are characterized by formation of epithelial fronds that have both the luminal epithelial and the outer myoepithelial cell layers, supported by a fibrovascular stroma [ 1 ]. Papillomas of the breast can be divided into solitary papillomas, juvenile papillomatosis, and multiple papillomatosis [ 2 ]. Their malignant potentials vary and may have an impact on patients' decision making process. We report a case of multiple papillomatosis of breast where patient's choice of treatment was affected by the anxiety of risk of malignancy and decided to opt for prophylactic mastectomy. | 3. Discussion
Pathologically, a papilloma is a mass-like projection that consists of papillary fronds attached to the inner mammary duct wall by a fibrovascular core that is covered with ductal epithelial and myoepithelial cells [ 2 ]. The epithelial component can be subject to a spectrum of morphologic changes ranging from metaplasia to hyperplasia, atypical intraductal hyperplasia, and in situ carcinoma [ 2 , 3 ].
Intraductal papilloma is a discrete benign tumour of the epithelium of mammary ducts. It shows a predilection for the extreme ends of the ductal system the lactiferous sinuses and the terminal ductules [ 4 ]. Clinically, solitary papillomas commonly occur in perimenopausal women, who usually present with spontaneous nipple discharge. Solitary papillomas are associated with a slightly increased risk (1.5–2.0 times) of developing breast carcinoma [ 5 ]. A solitary papilloma occasionally appears on mammography as a circumscribed subareolar mass or as a solitary dilated retroareolar duct [ 2 ]. On sonography, a papilloma is seen as an intraductal mass in a dilated duct, an intracystic mass, or a solid mass with a well-defined border [ 5 ]. Ductography may show an intraluminal filling defect or ductal dilatation due to partial or complete ductal obstruction. Recently, MRI has been reported to be an additional useful technique for detecting intraductal papillomas of breast [ 6 ].
On the other hand, papillomatosis is defined as a minimum of five clearly separate papillomas within a localized segment of breast tissue [ 1 ]. Juvenile papillomatosis (JP) of the breast is defined as severe ductal papillomatosis occurring in women less than thirty years old. Pathologic findings consist of papillomatosis and extensive cyst formation [ 7 ]. Patients typically present with a painless, circumscribed, mobile mass, which is easily confused with a fibroadenoma [ 7 ]. As the lesions usually occur in young women, patients are usually first evaluated with sonography. Juvenile papillomatosis is seen on sonography as an ill-defined heterogeneous mass with multiple peripheral small cystic spaces. Mammography usually shows dense breast tissue with no detectable lesion or asymmetric density [ 8 ]. Follow-up studies have suggested that JP is associated with an increased risk of breast cancer. Patient's female relatives and the patient herself may be at increased risk for developing carcinoma, particularly if the lesion is bilateral and the patient has a family history of breast cancer [ 9 – 11 ]. Therefore, long-term followup is recommended both for the patient and the family [ 10 , 12 ].
Multiple papillomatosis occurs in approximately 10% of cases of intraductal papillomas, tends to occur in younger patients than solitary papillomas, and is usually peripheral in location. Multiple papillomatosis arises in the terminal ductal lobular units and are more frequently associated with hyperplasia, atypia, DCIS, sclerosing adenosis, and radial scar [ 3 ]. Cardenosa et al. noted that the incidence of atypical ductal hyperplasia was 43 percent [ 13 ]. Clinically, patients commonly present with palpable masses [ 2 ]. Nipple discharge is less commonly seen, occurring as the presenting complaint only in approximately 20% of patients [ 13 ]. The patient in this case presented with a lump in the breast, and there was no history of nipple discharge. Mammographic findings of multiple papillomatosis are variable and include round, oval, or slightly lobulated well-circumscribed or spiculated masses with or without calcification, foci of microcalcification, clusters of nodule, and asymmetric density. On sonography, multiple papillomatosis lesions are seen as round, oval, or lobulated circumscribed solid masses or complex masses [ 14 ]. Indeed, mammogram in the case presented showed round soft tissue opacity and the ultrasound showed a solid lesion. However, early or small lesions may remain radiologically occult. Micropapillary DCIS without calcification is difficult to recognize on mammography, or findings may be nonspecific. This would explain the lack of radiological signs of “incidental” lesions noted in the left breast in the case reported [ 2 , 13 ].
Bilateral disease and recurrences after surgical treatment are more common in multiple papillomatosis [ 14 , 15 ]. Association of multiple papillomatosis with in-situ carcinomas ranged from 10% to 37.5% [ 15 – 17 ]. Actual size of the lesion may be larger than that appreciated by clinical and imaging findings. This may give rise to the potential of incomplete excision and recurrence. In one series, bilateral disease was reported in as many as 14% of patients, and 24% had recurrences after surgical treatment [ 15 ]. Invasive carcinoma has also been noted, but is rare. Therefore, if treated conservatively (i.e., wide local excision), patients with multiple papillomatosis should be kept under annual review. In addition to digital mammography, magnetic resonance can be also used in surveillance in view of its high sensitivity in detecting papillomas and demonstrating multicentric nature of the disease [ 18 , 19 ].
Prognosis of papillary lesions, even though associated with DCIS, remains excellent. Even those with papillary carcinoma have a better prognosis, with less axillary nodal involvement, than those with other forms of ductal carcinomas [ 3 , 20 , 21 ]. Multifocality and sizes of lesions, associated risk factors and patients' wishes may help decide the type of surgery, such as breast conservative surgery or mastectomy. It should be emphasised that patients who undergo breast conservative surgery do not necessarily have a worse prognosis than patients treated by a mastectomy. It is recommended that if breast conservative treatment is undertaken, a clear margin of at least 10 mm should be adhered to [ 22 , 23 ].
Patient's choice in decision making is paramount in such cases, as demonstrated in the report. As part of the informed consent process, patients must receive sufficient information, in a way that they can understand, to enable them to exercise their right to make informed decisions about their care [ 24 ].
The patient under consideration could not cope with the thought of the risk of papillomatosis (and hence, cancer) to the contralateral breast and opted for bilateral mastectomy. Her decision was vindicated by the finding of incidental DCIS on the asymptomatic side (left breast). We have not come across any previous reported case of multiple papillomatosis with DCIS of breast, along with incidental synchronous papillomatosis of contralateral breast with DCIS, which makes this case unique. | 4. Conclusions
Clinicians should be aware of various papillary lesions of breast. Multiple papillomatosis of breast remains a distinct entity, has a high propensity of being bilateral and recurrent, and is associated with in-situ carcinomas. It is important to emphasise such characteristics to the patient while discussing treatment options so that patient can make an informed choice. | Academic Editor: I-Tien Yeh
Papillary lesions of breast represent a range of lesions. Intraductal papilloma and its association with nipple discharge are well known. However, multiple papillomatosis has quite distinct characteristics and decision making can be somewhat challenging. We report a case of multiple papillomatosis in association with ductal carcinoma in situ (DCIS). Patient opted for ipsilateral mastectomy and prophylactic mastectomy of contralateral breast. Her decision of having prophylactic mastectomy was vindicated by presence of incidental DCIS in the contralateral breast. To our knowledge, this is the first reported case of multiple papillomatosis with DCIS of breast, along with incidental synchronous papillomatosis of contralateral breast with DCIS. The case illustrates few distinct features of multiple papillomatosis of breast and exemplifies how a patient's choice is so paramount in decision making process. Patients should be fully informed of the treatment options of the condition, and their wishes should be fully taken into account while making the final decision. | 2. Case Report
A 41-year-old woman presented with chief complaint of a lump in right breast of eight weeks duration. Her maternal aunt had breast cancer at age fifty-five. On examination, a firm 2.5 × 2 cm 2 mass was felt in upper outer quadrant (UOQ) of right breast.
Mammogram showed round soft tissue opacity in OUQ of right breast (M2). Ultrasound scan showed a solid nodule suggestive of fibroadenoma measuring 21 × 17 × 17 mm 3 in the lateral aspect (U3) of right breast ( Figure 1 ).
Fine-needle aspiration cytology of UOQ lesion of the right breast was noted to be C4, but core biopsy showed benign papillary proliferation (B3). Excision biopsy of the lesion demonstrated multiple papillomatosis containing areas of atypical ductal hyperplasia and DCIS, forming a cribriform pattern (20 mm in size). The excision was stated to be incomplete.
The results were discussed with patient. She was explained about the risk of developing cancer in future, and various options were given. She requested for bilateral mastectomy (prophylactic on left side). Following appropriate psychological assessment, she underwent bilateral mastectomy along with immediate breast reconstruction using expander prosthesis. Her postoperative recovery was uneventful.
Histology of right mastectomy specimen showed residual multiple papillomatosis with low-grade DCIS ( Figure 2 ). Taking previous excision into consideration, the sizes of papillary lesion and DCIS amounted to 24 mm and 32 mm, respectively. Histology of left mastectomy specimen showed an area of papillomatous lesion (9 mm) in the upper outer quadrant of breast with transition to low-grade DCIS (15 mm) ( Figure 3 ). Excision was complete on both sides. No invasive malignancy was noted on either side. | Acknowledgment
We would like to thank the Radiology Department, Victoria Hospital, Romford, RM1 4HL, the United Kingdom, for providing the ultrasound image. | CC BY | no | 2022-01-13 03:14:42 | Patholog Res Int. 2010 Dec 26; 2010:540590 | oa_package/ca/eb/PMC3014705.tar.gz |
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PMC3014707 | 21234414 | 1. Introduction
Diabetes Mellitus is one of the leading causes of death, illness, and economic loss in the United States [ 1 ]. Not surprisingly, an extensive amount of research is ongoing which is directed at the cause, diagnosis, and treatment of this crippling disorder [ 2 ]. Animal experimentation has a long history in the field of diabetes research. To induce diabetes in animals, toxic chemicals such as streptozotocin [ 3 ] and alloxan [ 4 ] are popularly used. Transgenic rodent models of diabetes are gaining popularity. A major criticism of using rodents is that, due to their short life span, they may not develop the clinical complications seen in humans that have been diabetic for a number of years. Cats, dogs, swine, and nonhuman primates are occasionally used [ 5 ]. Rabbits are a potentially overlooked, manageable species for chronic diabetes experiments. They are considered lower on the phylogenetic scale than cats, dogs, swine, and nonhuman primates, and they have an average life-span of 5–8 years which is significantly longer than rodents.
Studies have demonstrated that rabbits possess characteristics desirable in a laboratory animal model including convenient size, longer life span, strain specific, have good temperaments, are easily handled and are relatively inexpensive [ 6 ]. We searched PubMed and in the past decade more than 1000 studies have been conducted using rabbits as a model of diabetes [ 7 ]. However, most of these experiments used rabbits that were diabetic for only several weeks to 2 months [ 8 ]. Studies of these durations have severely limited diabetic research capabilities as many complications of diabetes require years to develop [ 5 ]. In this study, we created a long-term rabbit model of type 1 diabetes mellitus with reasonably good health for experimental usage for one year or longer. This paper reports our management strategies and physical, biochemical, and histological findings of these rabbits. | 2. Methods
The study was conducted in accordance with the National Institutes of Health guidelines for the care and use of animals in research, and the protocol was approved by the Institutional Animal Care and Use Committee of the University of Louisville, an AAALAC accredited program. A total of 32 young (8–10 weeks, 2.13 ± 0.24 kg), male New Zealand white rabbits, Oryctolagus cuniculus (Myrtle's Rabbitry, Thompson Station, TN) were divided into two groups. The first group consisted of 20 animals with chemically induced type 1 diabetes mellitus; the second group consisted of 12 age-matched control (orthoglycemic) animals. Animals were singly housed in stainless steel caging with slotted floors (40 × 60 × 80 cm) in a temperature (16–22°C) and humidity (30–70%) controlled room on a 12 : 12 hour light : dark cycle. Cage pans were cleaned three times per week and cage racks were cleaned once weekly. Laboratory Rabbit Diet HF 5326 (LabDiet, PMI Nutrition International) supplemented with certified timothy hay cubes (Bio-Serv, Frenchtown, NJ) and filtered tap water was provided ad libitum through an automated watering system from arrival throughout the end of the study. Environmental enrichment consisted of a variety of fresh vegetables at least once weekly, dumbbells and stainless steel rattles (Bio-Serv, Frenchtown, NJ). Vendor microbiological monitoring health reports indicated that the rabbits were serologically negative for the following pathogens: CAR Bacillus, Encephalitozoon cuniculi, Pasteurella multocida, Treponema cuniculi, and Oral Papilloma Virus. The rabbits were also negative for Bordetella bronchiseptica, Salmonella, Klebsiella, Citrobacter, and Clostridium piliformis. Animals were free of internal and external parasites. Before any experimental manipulations were initiated the rabbits were allowed to acclimate for at least 7 days.
2.1. Induction and Management of Diabetes Mellitus
Rabbits were weighed weekly throughout the study and the weights were recorded. For the alloxan injection, rabbits were lightly anesthetized with ketamine hydrochloride 30 mg/kg and xylazine 3 mg/kg (IM). Artificial tears ointment (Butler Animal Health Supply, Dublin, OH) was applied to the surface of both eyes to prevent drying. Body temperature was maintained with a warm water circulating blanket (Gaymar T Pump, Gaymar Industries Inc., Orchard Park, NY). Heart rate, respiratory rate, body temperature, and SpO 2 were monitored while the animals were under anesthesia and through recovery. Alloxan monohydrate (Sigma Aldrich Chemical, Saint Louis, MO, USA) was dissolved in sterile normal saline to achieve a concentration of 5% (W/V), and 100 mg/kg was immediately administered intravenously via the marginal ear vein over a period of 2 minutes with a 25 gauge butterfly catheter. The rabbits were allowed to recover from anesthesia. To avoid mortalities during the hypoglycemic phase, alloxan was administrated to nonfasted animals, and food and water were offered to animals immediately upon recovery after anesthesia. At 4, 8, and 12 hours following alloxan injection, 10 ml of glucose (5% W/V) was administered subcutaneously, and an oral solution of 20% glucose in tap water was provided via water bottle ad libitum for 1-2 days after confirmation of hypoglycemia (less than 70 mg/dl) to prevent hypoglycemic shock. During this period access to the automated watering system was restricted to encourage intake of the oral glucose solution. Rabbits whose blood glucose levels remained <300 mg/dl for more than one week following the initial injection of alloxan received a second dose of alloxan (100 mg/kg IV) to maintain a blood glucose level >300 mg/dl for the duration of the study. Blood glucose concentration was measured 1-2 times per day using a blood glucose meter (LifeSpan, Inc. Milpitas CA) for the first 4 weeks and once weekly thereafter in the morning. If morning blood glucose levels (BGL) were higher than 350 mg/dl, regular insulin (Novolin-R, Novo Nordisk Pharmaceuticals Inc. Princeton, NJ) was administered subcutaneously (SC) once daily with a 30 gauge needle. The insulin dose was adjusted according to the blood glucose level for the individual rabbit based on the following: (1) BGL < 400 mg/dl received 1 U/kg, (2) BGL = 400–500 mg/dl received 2 U/kg, (3) BGL = 500–600 mg/dl received 3 U/kg, and (4) BGL > 600 mg/dl received 4 U/kg.) In animals with BG levels > 500 mg/dl, a second insulin injection (half of the morning dose) was administered in the late afternoon. Blood and urine samples were collected for biochemical analysis weekly for two months then monthly until the end of experiment. Blood was collected from the central auricular artery with a 25 gauge needle. For urine collection, clean cage pans were placed under the cages without liners, and urine was collected for 24 hours.
2.2. Determination of Insulin Dose
Due to individual variation and sensitivity to insulin, the insulin dose for each rabbit was determined shortly after the induction of diabetes. A blood glucose curve was performed by administering a test dose of insulin (based on the aforementioned dosing rate) after measuring the blood glucose concentration in the morning. Blood glucose concentration was then measured every hour for 10 hours when it was clear that blood glucose levels were continuing to rise to obtain a glucose curve. If the lowest blood glucose was less than 50 mg/dl or higher than 200 mg/dl, a new glucose curve was obtained the next day by decreasing or increasing the insulin dosage until the best dose was obtained. The final insulin dose was determined when a glucose curve was obtained where the peak BGL was >350 mg/dl and the trough BGL was ≥50 mg/dl. If rabbits developed symptoms of illness (such as a loss of appetite, ptyalism, or severe weight loss) during the experiment, a new BG curve was obtained and a new insulin dose was implemented.
2.3. Pinna Wound Healing Model
To evaluate the effect of long-term diabetes on wound healing, we created full thickness skin wounds on the ventral surface of the pinnae in both diabetic and nondiabetic age-matched rabbit groups at 2 weeks and one year using a minimally invasive surgical technique [ 9 , 10 ]. Briefly, one hour before surgery, rabbits were injected subcutaneously with buprenorphine (0.03 mg/kg), and were anesthetized and monitored as described above. Under aseptic conditions, four circular full-thickness wounds were created on each ear with a 6-mm stainless steel punch. Prior to recovery a 25 μ g/hr Fentanyl transdermal patch (Duragesic, Ortho-McNeil, Raritan, NJ), was applied to the skin over the shoulders for analgesia. Sterile normal saline was used to dress all wounds. The wounds were then covered with Tegaderm (3M, St. Paul, MN, USA). Wound dressings were changed and digital photos were taken daily until the wounds were closed. Wounds were considered closed when they were totally reepithelialized [ 9 , 10 ] and further confirmed by histology and morphometric analysis [ 11 , 12 ] using Nikon Eclipse Ti microscope and Nikon Element software.
2.4. Biochemistry Analysis
Plasma was obtained by centrifugation of blood samples at 2500 g for 20 min at 4°C, and was stored at −20°C until analysis. The activity of plasma lactate dehydrogenase (LDH), creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, cholesterol, triglycerides (TG), blood urea nitrogen (BUN), and creatinine was assayed using a Roche Cobas Mira Plus chemistry analyzer (Roche Diagnostic System, Inc, Branchburg, NJ). Beta-hydroxybutyrate (BHBA) was also analyzed in some samples. All regents used in the analysis were purchased from CATACHEM (Bridgeport, CT, USA).
Urine samples were collected for 24 hours as previously described. The volume of urine collected was recorded, and its protein content was analyzed with a Roche Cobas Mira Plus chemistry analyzer. Ketones were analyzed by IDEXX VetLab (One IDEXX Drive, Westbrook, ME, USA).
2.5. Preparation of Tissue Samples
At the end of 12 months, rabbits were euthanatized with 100 mg/kg pentobarbital (IV). Tissue samples were taken from the pancreas, liver, kidney, aorta, and pinna for histological studies. The samples were fixed in 4% buffered formaldehyde and embedded in paraffin. Six μ m slices were made from the paraffin blocks which were stained with hematoxylin and eosin and evaluated for histological changes.
2.6. Statistical Analysis
Results are reported as mean and standard deviation (SD). Student's t -test was used to compare the two groups and ANOVA was used for repeated longitudinal measurements with commercially available statistical software (GraphPad Software, Inc. San Diego, CA). A P value of <.05 was considered significant. | 3. Results
3.1. Animals
Of the 20 alloxan-treated rabbits, two died at 6 and 9 months secondary to renal failure. Four animals were used for a 2-week wound healing study, and 6 of the animals were used for a one-year wound healing study. The remaining 8 rabbits are still alive after 12 months.
3.2. Alloxan Dose and Hyperglycemia
Baseline nonfasting blood glucose levels were normal for all animals (124 ± 17 mg/dl). After the administration of alloxan there was a characteristic response in blood glucose level [ 4 ]. In the first 2 hours, blood glucose increased. The second phase, which was observed approximately 6 hours following alloxan injection, was a hypoglycemic phase which persisted for 24–48 hours. By that time the animals were alert, active, eating and drinking glucose water unassisted. It was determined that 24 hour continuous observation was not necessary. During our study, the blood glucose of some rabbits decreased to life threatening levels (less than 50 mg/dl). However, no animals died in the hypoglycemic phase of this study. The third phase was persistent hyperglycemia. Of the 20 alloxan-treated rabbits, blood glucose levels generally increased up to 300 mg/dl within 48 hours of receiving alloxan. After two weeks, the diabetic animals were divided into three groups according to their morning blood glucose levels: 300–400 mg/dl ( n = 7), mild diabetic group; 401–500 mg/dl ( n = 7), moderately severe diabetic group; greater than 501 mg/dl ( n = 6), severe diabetic group. We found that animals in the mild group exhibited body weight increases rather quickly however their blood glucose levels decreased below 300 mg/dl 3–8 weeks after the first injection of alloxan, and a second injection of alloxan was required. Twelve animals required a second injection of alloxan to maintain persistent hyperglycemia (>300 mg/dl). An interesting finding is that the second injection of alloxan did not induce the characteristic hypoglycemic that was observed following the initial dose of alloxan. Therefore animals that received a second dose of alloxan did not require glucose supplementation. All animals that received a second dose of alloxan survived until the end of the experiment.
3.3. Insulin Dose and Glucose Concentration
In most of the rabbits, blood glucose concentrations followed a relatively predictable pattern. After insulin administration, blood glucose reached a trough (70–150 mg/dl) 2 hours after injection. By four hours after insulin BG levels began to rise and were >300 mg/dl within 6 hours and peaked the following morning before the next insulin injection. The mean time that rabbits were maintained in a hyperglycemic state was 16 hours. The mean time that rabbits were normoglycemic was 8 hours per day.
3.4. Body Weight
Baseline mean body weight of the rabbits was 2.13 ± 0.24 kg. Food intake was not measured in this study, however fecal output was assessed by husbandry staff daily and any decrease from normal production was brought to the attention of the study director and a clinical veterinarian. Although the blood glucose levels were very high, there was a tendency toward weight gain in all of the diabetic rabbits. There appeared to be a negative relationship between the highest blood glucose levels observed and body weight gain. At the end of 12 months, their body weights ranged from 3.12 to 4.18 kg, while the body weights of the age-matched nondiabetic rabbits ranged from 4.86–5.96 kg. The body weight increase of the diabetic rabbits was significantly lower than that of age-matched nondiabetic rabbits ( P < .05).
3.5. Polyuria and Proteinuria
Both Polyuria and Proteinuria are characteristics of diabetes. Following diabetes induction in the rabbits, there were significant increases in urine volume ( P = .0019, Figure 1(a) ) and urine protein ( P = .0004, Figure 1(b) ) compared with the baseline.
3.6. Renal Dysfunction
Plasma levels of blood urea nitrogen (BUN) and creatinine increased following diabetes induction (Figures 1(c) and 1(d) ). There were significant differences between the diabetic and age-matched nondiabetic rabbits at 9 months ( P < .0001 for BUN and P = .0008 for creatinine) and 12 months ( P = .0002 for BUN and P < .0001 for creatinine).
3.7. Hyperlipidemia
Plasma cholesterol and triglyceride levels increased in all diabetic rabbits. There were significant differences between the diabetic and age-matched nondiabetic rabbits at 9 months ( P < .0001 for cholesterol and P < .0001 for triglyceride) and 12 months ( P < .0001 for cholesterol and P < .0001 for triglyceride, Figures 2(a) and 2(b) ).
3.8. Plasma Enzymes Levels
The majority of the plasma levels of ALT, AST, CK, and LDH were within normal range; however levels were elevated at times in individual rabbits. In addition, plasma total protein was within normal range although proteinuria was observed ( Table 1 ).
3.9. Ketoacidosis and Urine Ketosis
Diabetic ketoacidosis was demonstrated by increased blood beta-hydroxybutyrate (BHBA) [ 13 ]. When peak blood glucose levels were above 500 mg/dl, BHBA levels at times increased to more than 10 mg/dl or even greater than 40 mg/dl (ketoacidosis) ( Figure 3 ). Ketones were also present in the urine of some severely diabetic rabbits whose peak blood glucose levels were greater than 500 mg/dl.
3.10. Wound Healing
In rabbits wounded at two weeks following the induction of diabetes, wound closure time tended to be longer (16.5 ± 3.5 days) than the age-matched nondiabetic rabbits (15.6 ± 1.1 days), but the difference was not statistically significant ( P = .3865). However, in rabbits that had been diabetic for one year, wound closure time (19.7 ± 3.7 days) was significantly longer than in age-matched nondiabetic rabbits (15.1 ± 1.0 days) ( P < .01, Figure 4 ).
3.11. Other Complications
Throughout the study some animals exhibited anorexia, severe weight loss, ptyalism, weakness, and lethargy. These were caused by excessive hyper- or hypoglycemia, and all of them were treated by adjusting insulin dosages. Four rabbits suffered from slight anemia, with hemoglobin between 7–9 g/dl. The animals were not treated for anemia since it was not severe.
3.12. Pathologic Changes
Histopathologic changes in animals that had diabetes for one year are shown in Figure 5 . The pancreas showed marked beta-cell damage and thickened arterial walls. The kidney showed hyaline arteriolosclerosis. The amorphous, homogeneous eosinophilic material is seen in the thickened vascular wall of afferent glomerular arteriole and the lumen is narrowed markedly (arrow). Glomerular atrophy is also present. In the liver, there was lipid accumulation in the cytoplasm of the hepatocytes which appeared as vacuoles (hepatic fatty degeneration). The aortic media demonstrated mild spot calcifications in small areas. | 4. Discussion
To our knowledge, this is by far the largest group of diabetic rabbits kept for one year or longer for scientific research. A long-term diabetic animal model has many potential benefits. Our experience has shown that, with careful management, alloxan-induced diabetic rabbits can be kept for one year or longer in reasonably good health. The biochemical and histological changes indicated well-developed diabetes.
Many studies have been conducted in diabetic rabbits over short periods of time in the past several decades. However, short-term diabetic rabbits have severely limited the diabetic research capabilities because human diabetes lasts for decades and many diabetic complications take years to develop. A PubMed search revealed only a few reports in which diabetic rabbits were kept alive for 3–6 months for pharmacology or growth factor studies [ 14 – 18 ]. Only one article was found in which 3 rabbits were kept for 12 months to study the aortic intima-media of alloxan-diabetic rabbits [ 19 ]. Due to many medical complications, the management of long-term diabetic rabbits is labor intensive, expensive, and technically demanding which reduces the incentive to perform these studies over long periods of time.
The toxin-induced diabetic rabbits were first administrated alloxan by Dunn and Mcletchie [ 20 ] who by studying the crush syndrome in rabbits investigated the effects of a series of uric acid derivatives including alloxan with regard to kidney damage. The animals which received alloxan became comatose after 12 hours, and were hypothermic with high blood urea and low blood glucose values. Shortly after the injection there was a marked hyperglycemia. Histological examination revealed early signs of expected kidney damage. An unexpected finding was a partial or total necrosis of the pancreatic islets. Other tissues showed no damage, and alloxan was gradually adopted to generate diabetes in animals. Over the course of our study, alloxan-induced diabetic animals exhibited classic symptoms of human diabetes, such as hyperglycemia, glucosuria, polydipsia, and polyuria, loss of body weight despite polyphagia, hyperlipemia, ketonuria, and acidosis.
Following the administration of alloxan there is a characteristic response in blood glucose level [ 4 ]. In the first 2 hours, blood glucose rises. This transient hyperglycemia is thought to be due to sudden glycogen breakdown in the liver [ 21 ]. The reason for the breakdown of liver glycogen during this phase is unknown, but may be a secondary effect of epinephrine release. The second phase is a hypoglycemic phase which may be severe enough to lead to death if it is not prevented or treated with supplemental glucose [ 4 , 22 ]. The hypoglycemia is due to a sudden outpouring of insulin from dying beta cells [ 21 ]. The hypoglycemia generally appears after 6 hours [ 23 ]. Hypoglycemia is more pronounced in fasted animals [ 24 ].
The susceptibility to both toxic and diabetogenic doses of alloxan varies widely not only in different species but also among animals of the same species [ 25 , 26 ]. The range of the safe diabetogenic dose of alloxan in a particular animal is quite narrow and even light overdosing may be toxic and result in death. This loss is likely due to kidney tubular cell necrotic toxicity, in particular when high doses of alloxan are administered [ 27 ]. The dose of alloxan (100 mg/kg) utilized in this study is diabetogenic and less toxic. However, this dose does not necessarily produce stable diabetes in rabbits [ 28 ]. Sixty percent of the rabbits in this study required a second dose of alloxan to maintain persistent hyperglycemia, and all of these animals survived until the end of the experiment. The recovery from diabetes has been proposed to be the consequence of either a multiplication of beta cells that survive the initial alloxan injection or the formation of new beta-cells from the duct epithelium of the exocrine portion of pancreas [ 29 , 30 ]. Another possible explanation is that there may have been a stress-induced hyperglycemia at the time of the initial alloxan injection initiated by handling of the animals. It has been shown that hyperglycemia can provide protection against alloxan-induced injury [ 31 ]. It has also been shown that male rabbits are more likely to experience stress-induced hyperglycemia than female rabbits [ 32 ] and this may have contributed to the high percentage of rabbits requiring a second dose of alloxan. In future studies we plan to measure the blood glucose level of the rabbits immediately prior to the administration of alloxan to be able to better predict those animals that may require a second dose of alloxan as well as increase the amount of handling that they receive during the acclimation period in an attempt to decrease the percentage of rabbits that require a second dose. Since untreated diabetics have higher complication rates than regulated diabetics, we chose to treat the rabbits in this study with insulin to increase the survival time of the rabbits, but we did not fully regulate the diabetes. Normal blood glucose levels were maintained a mean of 8 hours per day, and a hyperglycemic state was maintained for a mean of 16 hours per day. Therefore this model may more closely represent a model of poorly regulated diabetes because glucose fluctuation has been shown to cause more tissue damage than stable hyperglycemia [ 33 ].
Based on the severity of hyperglycemia, we divided the rabbits into three groups, those that had mild, moderately severe, and severe diabetes. Each group exhibited certain characteristic physiologic changes throughout the course of the study. In the mild diabetic group, body weights increased rather quickly, the biochemical values were usually in the normal range, the animals BG returned to levels <300 mg/dl and they required a second injection of alloxan. In the severe diabetic group, body weights increased very slowly and biochemical values were mostly abnormal including elevated BUN, anemia due to renal dysfunction [ 34 ], dyslipidemia [ 35 ], and at times ketoacidosis [ 36 ]. In the moderately severe diabetic group, the changes observed were a combination of the above two groups and their blood glucose levels were more manageable.
In addition to hyperglycemia in the long-term diabetic rabbits, we observed other characteristics of diabetes. While all of the rabbits in this study gained body weight, the gain in the diabetic rabbits was less than the gain observed in the age-matched animals. Prior to alloxan administration, their body weights ranged from 2.0 to 2.5 kg. After one year, they only increased to 3.5–4.0 kg, a weight comparable to a normal 4-5-month old rabbit. However, the body weight of age-matched nondiabetic rabbits in this study increased to more than 5.0 kg after one year which is consistent with the normal growth rate provided by the vendor. The increase in body weight in this study, albeit small, appeared to be different from many of the previous studies in which diabetic rabbits lose weight. A large number of previous studies utilized rabbits that were 3-4 months of age and were considered to be young adults. Our finding of an increase in body weight is similar to that reported by Goseki et al. [ 16 ] who utilized young rabbits that were 10 weeks of age similar to those used in our study. Because body weight change is a reflection of whole body function, maintenance of appropriate body weight might be an important factor for the rabbits to survive long-term in the diabetic state. Plasma cholesterol and triglyceride levels increased in all diabetic rabbits in this study. These findings are in agreement with previously published studies [ 35 ]. We also found a few diabetic animals that suffered from anemia. These rabbits all had poor renal function, which is similar to clinical human patients due to a decrease in erythropoietin production by the damaged kidneys [ 37 ].
The histological changes observed in animals kept for one year were typical of diabetic damage. In addition to pancreatic islet damage, microscopic cross-sections of the pancreatic arteries showed thickening of vessel walls which indicates atherosclerosis in this organ. In the aorta, microscopic sections of the aortic media demonstrated mild spot calcifications in small areas which are also indicative of atherosclerosis. Similar changes were also seen in the kidney, in which the afferent glomerular arteriole wall was markedly thickened and the lumen was narrowed. Glomerular atrophy was also present in the kidney. In the liver, lipid accumulated in the cytoplasm of the hepatocytes as vacuoles. All of these histological changes are consistent with those seen in long-term diabetics which may not be present in animals that are diabetic for shorter periods of time.
One of the most common complications of human diabetes is ulceration of the extremities, which has been subjected to numerous experimental studies. However, all of these studies suffer from several limitations that make it difficult to extrapolate animal results to clinical settings. One is the time during which the animal has been diabetic, and another is the etiology of diabetic ulcers. It is well known that human diabetic ulcers are the result of years, even decades of diabetes mellitus. Neuropathy, vasculopathy, immune dysfunction, and biochemical abnormalities all contribute to the development of chronic wounds in diabetic patients [ 38 , 39 ]. To date, none of the animal models used for diabetic wound research have all of the same features of human diabetic wounds with the exception of hyperglycemia. The length of time that rabbits have been maintained in a hyperglycemic state in the majority of rabbit wound healing studies has been very limited, with the shortest being several days and the longest being two months. These short periods of persistent hyperglycemia simply cannot reproduce the changes observed in human diabetes. In order to test whether the time of hyperglycemia had any effect on wound healing, we compared wound healing times in diabetic rabbits and age-matched nondiabetic animals at two weeks and one year. To date little is known about the difference between wound healing in short-term hyperglycemia and long-term diabetes [ 8 ]. In the present study, short-term hyperglycemia (2 weeks) appeared to delay the healing process to a degree as compared to nondiabetic rabbits, but this difference was not statistically significant. However, in the one-year diabetic rabbits, the closure time of wounds was significantly longer than age-matched nondiabetic wounds. Our results appear to indicate that wound healing time is proportionate to the length of diabetic times. A state of systemic hyperglycemia, as seen in diabetes, may influence wound closure in numerous ways. Several hypotheses have been described in the literature such as the formation of glycation end products [ 40 ], hyperosmolarity [ 41 ], and altered insulin signaling in various ways [ 42 ]. However, hyperglycemia alone plays a limited role in the status of many nonhealing chronic wounds, while other pathophysiological changes take much longer to develop. If a rabbit's life span is 5–8 years, one year may be comparable to 15–20 years of human life, although such an approximation is an oversimplification. The biochemical and histological changes found in this study provide some evidence to mimic human diabetic changes; an animal model that is diabetic for a longer period of time than that which is more commonly published provides more features closer to human diabetic changes.
In summary, with careful management which includes adaptive and repeated injections of alloxan, accurate adjustment of insulin dose, monitoring body weight and biochemical values, performing blood glucose curves, and adjusting insulin dosage as soon as animals exhibit abnormal symptoms, alloxan-induced diabetic rabbits can be kept for one year or longer in reasonably good health. Such a model may prove useful in scientific research on diabetes. | Academic Editor: Bernard Portha
This study was to create a long-term rabbit model of diabetes mellitus for medical studies of up to one year or longer and to evaluate the effects of chronic hyperglycemia on damage of major organs. A single dose of alloxan monohydrate (100 mg/kg) was given intravenously to 20 young New Zealand White rabbits. Another 12 age-matched normal rabbits were used as controls. Hyperglycemia developed within 48 hours after treatment with alloxan. Insulin was given daily after diabetes developed. All animals gained some body weight, but the gain was much less than the age-matched nondiabetic rabbits. Hyperlipidemia, higher blood urea nitrogen and creatinine were found in the diabetic animals. Histologically, the pancreas showed marked beta cell damage. The kidneys showed significantly thickened afferent glomerular arterioles with narrowed lumens along with glomerular atrophy. Lipid accumulation in the cytoplasm of hepatocytes appeared as vacuoles. Full-thickness skin wound healing was delayed. In summary, with careful management, alloxan-induced diabetic rabbits can be maintained for one year or longer in reasonably good health for diabetic studies. | Acknowledgments
This study was partly supported by NIH Grants DK74566 and AR52984. The authors would like to acknowledge the outstanding support and veterinary care provided for the animals in our studies by the Research Resources Facilities veterinary technical staff. | CC BY | no | 2022-01-13 02:21:51 | Exp Diabetes Res. 2010 Dec 27; 2010:289614 | oa_package/ff/6e/PMC3014707.tar.gz |
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PMC3014712 | 21234419 | 1. Introduction
Fertilization in mammals requires a successful series of events involving a profound remodeling of the nucleus and cytoplasm of both spermatozoa and oocytes. Microtubules and actin microfilaments have been demonstrated to dynamically play an important role during fertilization and cleavage in a number of species. The microtubules actively involve in the process of fertilization by the formation of microtubule networks that facilitate the migration and apposition of male and female pronuclei. These microtubules are paternally inherited in most mammalian species, including human [ 1 , 2 ], sheep [ 3 ], rabbit [ 4 ], porcine [ 5 ], bovine [ 6 – 8 ], and rhesus monkey [ 9 ]. On the other hand, the paternal centrosome in the ooplasm is functionally absent in mice, and thus the syngamy of the two pronuclei requires the maternal centrosome [ 10 , 11 ]. In addition, the evidence that a reversible microfilament depolymerizer (cytochalasin B) fails to inhibit the movement of male and female pronuclei but it adversely affects the syngamy and cell division [ 12 ] suggests an important role of actin microfilaments during cellular cleavage [ 3 , 4 ]. However, these events on gamete interaction and early embryo development especially during fertilization have been poorly reported in the swamp buffalo. It has only been morphologically studied in vivo [ 13 ]. Understanding the redistribution patterns and role of microtubules and actin microfilaments during fertilization in vitro will provide fundamental knowledge of early embryo development and may improve in vitro embryo production techniques principally by the characterization of factors associated with fertilization failure in this species. The present research was designed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. | 2. Materials and Methods
2.1. Chemicals
All chemicals used in this study were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise stated.
2.2. In Vitro Maturation (IVM)
Swamp buffalo ovaries were obtained from animals of unknown reproductive status at a local slaughterhouse, then they were transported to the laboratory within 4 h in 0.9% (w/v) normal saline supplemented with 100 IU/mL penicillin G and 100 μ g/mL streptomycin at 28–35°C. The ovaries were washed once in 70% (v/v) alcohol and 0.9% (w/v) normal saline [ 14 ]. The oocytes were later aspirated from 2–8 mm antral follicles with an 18-gauge needle attached to a 10 ml syringe. The cumulus oocyte complexes were morphologically selected under a stereomicroscope at 400x magnifications. Cumulus-oocyte complexes (COCs) with homogenous ooplasm and surrounded by compact multiple layers of cumulus cells were submitted to in vitro maturation. Groups of 10 COCs were cultured in 50 μ L droplets of NaHCO 3 -buffered tissue culture medium 199 covered with mineral oil supplemented with 10% (v/v) buffalo follicular fluid, 50 IU/mL human chorionic gonadotropin (Intervet, Boxmeer, The Netherlands), 0.02 IU/mL follicle stimulating hormone, 1 μ g/mL estradiol-17 β , 100 μ M cysteamine, 20 ng/mL epidermal growth factor, 100 IU/mL penicillin G, and 100 μ g/mL streptomycin. Three pools of follicular fluid were obtained from 2–8 mm follicles, then sterilized by filtering through the 0.22 μ m syringe driven filter, and then stored in sterile microcentrifuge tubes at −80°C. IVM was performed at 38.5°C for 22 h in a humidified atmosphere of 5% CO 2 in air.
2.3. Partial Digestion of Zona Pellucida (ZP)
After in vitro maturation, oocytes were denuded and were transferred into 30 μ L droplet of an acid Tyrode's solution (pH 3.1) for 45 sec at room temperature (28–30°C). They were washed immediately two times with 2 ml of a modified Tyrode's (TALP) medium. ZP-digested oocytes were submitted to fertilization and culture procedures as mentioned above. Percentage of pronuclear formation was recorded at 18 h after IVF. Non-Tyrode treated oocytes served as control.
2.4. In Vitro Fertilization and In Vitro Culture (IVF and IVC)
Frozen semen from a fertile bull was used in this study. The semen was thawed at 37°C for 30 sec and then submitted to swim-up procedure for 45 min in TALP medium supplemented with 10 μ g/mL heparin as described by Parrish et al. [ 15 ]. Groups of 10–15 COCs in TALP medium, supplemented with 20 μ M penicillamine, 10 μ M hypotaurine, and 1 μ M epinephrine, were fertilized with sperm at a final concentration of 2 × 10 6 sperm/mL [ 16 ]. IVF was performed at 38.5°C in a humidified atmosphere of 5% CO 2 , 5% O 2 for 12 h. Excessive cumulus cells and sperms were then removed by repeated pipetting in culture medium containing 1 mg/mL hyaluronidase. Ten to fifteen presumptive zygotes were then cultured in 50 droplets of synthetic oviductal fluid containing 1% (v/v) fetal calf serum, 100 IU/mL penicillin G, and 100 μ g/mL streptomycin at 38.5°C in an atmosphere of 5% CO 2 , 5% O 2 .
The presumptive zygotes were randomly fixed and examined at 6, 12, 18, 24, and 30 h after IVF. Prior to fixation, they were incubated for 45 min at 37°C in a glycerol-based microtubule-stabilizing solution that contained 25% (v/v) glycerol, 50 mM MgCl 2 , 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 50 mM imidazole, 4% Triton-X-100, and 25 μ M phenylmethylsulfonyl fluoride at pH 6.7 [ 17 ]. Subsequently, they were fixed and stored in 4% (w/v) paraformaldehyde in PBS until analysis.
2.5. Fluorescent Labeling of Oocytes and Presumptive Zygotes
To label the microtubules, the presumptive zygotes and embryos were first incubated at 25°C for 1 h in a 1 : 100 solution of monoclonal anti- α -tubulin (clone B1-5-1-2) in 0.1% (v/v) Triton-X-100 in PBS-BSA. They were subsequently washed in PBS-BSA and incubated for 1 h in a 1 : 100 solution of a goat antimouse second antibody conjugated with tetramethylrhodamine isothiocyanate (TRITC) in PBS-BSA. After washing twice in PBS-BSA, the actin microfilaments were stained by incubation for 30 min in a solution of 0.165 μ M Alexa Fluor 488 phalloidin (Molecular Probes, Invitrogen, OR, USA) in PBS-BSA. In addition, they were subsequently stained for 10 min with Alexa Fluor 594 wheat germ agglutinin (WGA; Molecular Probes) to locate lectin-rich ZP. Finally, the presumptive zygotes and embryos were incubated for 15 min with 20 μ M DAPI to label the chromatin. Labeled samples were mounted on a glass microscope slide in a 2 μ L droplet of antifade medium (Vectashield, Vector Lab, CA, USA) to retard photobleaching.
2.6. Confocal Laser Scanning Microscopy (CLSM)
Confocal laser scanning microscopy (C1, Nikon, Japan) was used to demonstrate the presence or absence of sperm within ooplasm (sperm penetration rate) at 6 h after IVF. Three laser sources from Diote 408 nm, Argon 488 nm, and HeNe 543 nm were used to simultaneously excite the fluorescent signals from DAPI, AlexaFluor 488 phalloidin (microfilaments), and Alexa Fluor 594 (ZP), respectively. The digital micrographs produced using the sequential scanning mode for the 3 separate colors were merged into single panel using EZ-C1 software (Nikon, Japan). The resulting multicolor micrographs were subsequently examined using Adobe Photoshop CS (Adobe System Inc., Mountain View, CA, USA).
2.7. Experimental Design
A total of 63 presumptive zygotes were examined at 6 h after IVF for sperm penetration using confocal laser scanning microscopy. Phalloidin and WGA were used to localize the boundary of the ooplasm and lectin-rich ZP in order to facilitate the visualization of sperm within the ooplasm, and DAPI was used to label the chromatin. The presence of a spermatozoon within ooplasm indicated the sperm penetration, and the oocytes having sperm head(s) bound onto or within the ZP were considered as nonfertilized oocytes.
To test whether partial digestion of ZP prior to IVF would improve the sperm penetration rate, thereby improving IVF efficiency, a total of 64 oocytes were treated with acid Tyrode's solution and then fertilized in vitro . Fertilization rate was assessed by percentage of male and female pronuclear formation at 18 h after IVF.
For the distribution pattern of the cell cytoskeleton and chromatin configurations during fertilization and early embryo development, 378 presumptive zygotes and/or embryos were fixed at 12, 18, 24, and 30 h after IVF. Following immunolabeling with monoclonal anti- α -tubulin-TRITC, Phalloidin, and DAPI to identify microtubules, microfilaments, and chromatin, respectively, they were examined using immunofluorescent microscopy (BX51, Olympus, Tokyo, Japan). The characteristics of the chromatin, polar body, cytoskeleton, sperm heads, and pronuclear formation were recorded. Oocyte activation was defined by the progression of chromosomal development after IVF from metaphase II (MII) through telophase II and also the formation of female pronucleus. Activated oocytes having decondensing sperm head and/or formation of male pronucleus and cell cleavage were identified as fertilized oocytes/zygotes. Metaphase I (MI), anaphase I, or telophase I oocytes were classified as nonmatured oocytes. The oocytes that had a dispersed pattern of chromatin and cytoskeleton were classified as degenerate oocytes.
2.8. Statistical Analysis
Descriptive data was used to describe the chronology of early embryo development in terms of the redistribution of cytoskeleton (microtubules and actin microfilaments) and chromatin configurations. Differences in percentage data of maturation and fertilization among stages were presented as mean and analyzed by Fisher's exact test (SAS 9.1, The SAS Institute Inc., Cary, NC, USA). P -values less than.05 were interpreted as significant. | 3. Results
3.1. Sperm Penetration
Spermatozoa penetrated into ZP and were found in the cytoplasm of MII oocytes by 6 h after IVF (28/63, 44.4%, Figure 1(a) ; Table 1 ). Although a large proportion of these penetrated oocytes were still arrested at MII stage, 2 of the 28 oocytes (7.1%) had already resumed the second meiosis and were in telophase II. Eighteen MII oocytes (18/63, 28.6%) were classified as nonfertilized oocytes as determined by the absence of a spermatozoon within the ooplasm, or spermatozoa were only attached at the ZP of the oocytes. Two of 12 MI oocytes were also penetrated by spermatozoa.
3.2. In Vitro Fertilization of Partial Zona-Digested Oocytes
Tyrode's solution dissolved the ZP of buffalo oocytes. After exposure for 45 sec, approximately one-third of ZP was digested. Fertilization rate, in terms of pronuclear formation, of ZP-digested oocytes (60.4%), was not significantly different compared to 54.8% of control ( P > .05, Table 2 ).
3.3. Redistribution of Cytoskeleton and Chromatin Configurations
Percentage of maturation and fertilization of swamp buffalo oocytes at 12, 18, 24, and 30 h after IVF are shown in Table 3 ; the maturation rate did not significantly differ among groups ( P > .05). However, the rate of fertilization at 12 h after IVF was significantly lower than the other time points ( P < .05). The results demonstrated that the presumptive zygotes showed decondensation of both male and female chromatin by 12 h after IVF. A small proportion of MII oocytes (18/63, 28.6%) underwent activation. Of these activated oocytes, 5 and 13 oocytes were in telophase II ( Figure 1(b) ) and pronuclear stage, respectively. During decondensation of sperm chromatin, densely stained microtubules were observed and continually elongated to form the sperm aster that radiated from the nucleation site of the sperm centrosome at the base of the decondensing sperm head ( Figure 1(c) ). It revealed that the developmental rate of the male and female pronuclei was asynchronous at 12 h after IVF (a lack of synchrony between the male and female pronuclei). By 18 h after IVF, 33 of 65 MII oocytes (50.8%) were recorded as being fertilized, and 30 of them had already reached the pronuclear stage. At this time, microtubule networks of sperm aster simultaneously increased in size and extended throughout the ooplasm of the fertilized oocytes.
The percentage of fertilized oocytes at 24 h after IVF was 49.2% (29/59). Most of fertilized oocytes (25/29) were also in pronuclear stage. Apposition of the pronuclei (syngamy) was observed in 14 of these 25 fertilized oocytes. At this stage, microtubules were intensely stained between the two pronuclei ( Figure 1(d) ). Cleavage was firstly observed by 30 h after IVF (14 of 27 fertilized oocytes). A dense array of microfilaments formed between two presumptive blastomeres ( Figure 1(e) ) and later formed an intensely labeled layer beneath the plasma membrane of each blastomere after cell cleavage ( Figure 1(f) ). In addition, the percentage of “nonfertilized” MII oocytes was clearly demonstrated in the present study. Most MII oocytes that failed to progress through the second meiotic division had only a number of spermatozoa bound onto or within the zona pellucida, suggesting the important role of spermatozoa on the failure of fertilization. | 4. Discussion
The chronology of early embryonic development in terms of cytoskeleton redistribution and chromatin configurations in swamp buffalo embryos during the first cell cycle was examined and firstly described in this study. Until recently, overall success of in vitro embryo production in swamp buffalo has remained relatively poor when compared to the results obtained from riverine buffalos and bovines, for example, due to the limitation of fundamental knowledge associated with gamete interaction at fertilization. Oocyte maturation rates in this study ( Table 3 ) were similar to the range of 47 to 85% in other observations [ 18 – 20 ] and fertilization rates of these MII oocytes (ranging from 28.6–50.8%) in terms of oocyte activation and cell cleavage were variable, which is also in accordance with other previous studies [ 16 , 21 – 24 ]. While a number of factors have been demonstrated to be involved in the fertilization rate of swamp buffalo oocytes including sperm quality [ 25 ], concentration [ 16 ], and culture media used [ 26 , 27 ]; intrinsic factors within the cytoplasm of MII oocytes (as usually referred to as cytoplasmic maturation) also play a critical role during the activation of both male and female gametes during fertilization and early embryo development [ 28 – 30 ]. During IVF, spermatozoa rapidly undergo several modifications including a remodeling of the sperm plasma membrane, and spermatozoa then become hyperactivated [ 31 ] and capable of binding to mature oocyte via specific sperm-binding ZP3 receptors [ 32 ]. As a consequence, spermatozoa undergo the acrosome reaction and finally penetrate the ZP and fuse with the oolemma [ 33 ]. In this study, spermatozoa penetrated through the ZP of mature oocytes by 6 h after IVF which was similar to previous reports in bovines [ 34 , 35 ]. However, it has been reported that a spermatozoon was already present in the cytoplasm of bovine oocytes as early as 2-3 h after IVF [ 36 ]. Many factors were likely involved in the difference in speed of sperm penetration among studies such as type of spermatozoa (fresh or frozen), coincubation time, and capability of spermatozoa to respond to the capacitating medium during IVF [ 6 , 35 , 36 ]. In addition, our study demonstrated that sperm penetration in MI swamp buffalo oocytes was also possible which was similar to reports in bovine [ 37 , 38 ] and canine [ 39 ]. However, it appears that intrinsic factors within the ooplasm of oocyte also play a crucial role in determining the fate of sperm decondensation such as immature oocytes that cannot support postfertilization events of sperm head decondensation [ 40 ].
This study revealed that a dense network of a thread-like structure of tubulin (referred to as the sperm aster) was formed at the base of decondensing sperm head during gamete activation. The evidence that elongated radial sperm aster was involved in the movement and apposition of male and female pronuclei of buffalo's zygote suggested that the centrosomal material is primarily paternally inherited and is similar to previous reports in other mammalian species such as human [ 1 , 2 ], sheep [ 3 ], rabbit [ 4 ], porcine [ 5 ], bovine [ 6 – 8 ], and rhesus monkey [ 9 ]. However, this is different in mouse because cytoplasmic microtubules in the cytoplasm originate from maternal centrosomes and sperm astral microtubules were not detected in the decondensing paternal chromatin [ 10 , 41 ].
We found the significant differences in the percentage of oocytes being fertilized between 12 h and the others ( P < .05). The low numbers of fertilized oocytes presented in Table 3 indicate that a large proportion of penetrated sperms underwent pronuclear formation during 12–18 h after IVF, while formation of female pronucleus in oocytes occurred before the decondensation of sperm head. After decondensation of sperm chromatin, the proportion of pronuclear stage embryos was asynchronously observed by 12–18 h after IVF. Although not exhaustively examined, this asynchronous development of zygotes has been postulated to be caused by several factors such as delayed decondensation of sperm chromatin [ 35 ], variation of individual animals [ 6 , 42 ], and variation of cell cycle transition (M phase to G2 stage) following sperm entry [ 43 , 44 ]. In this study, the development of buffalo zygotes was more synchronized around 18 h which was similar to a report in bovine [ 6 ].
In buffalo, pronuclei movement was clearly influenced by the sperm aster. We found that the male and female pronuclei were positioned at the center of the oocyte, in which the sperm aster mediated by microtubules was concentrated between the two pronuclei until the zygote entered the first mitotic phase and cleaved to the two-cell stage. This result is in agreement with previous report in sheep [ 3 ], but in contrast to rabbit and mouse. The sperm aster of the latter species was a transitory structure that dispersed rapidly around the male pronucleus [ 4 , 11 ]. In addition to the role of microtubules during fertilization and early embryo development, this study also indicated that microfilaments also played an important part in the fertilization and cleavage in swamp buffalo embryos. These filaments were concentrated as a cell furrow predominantly in the middle line of the dividing cell and subsequently located just beneath the plasma membrane as previously reported in Xenopus [ 45 ], mouse [ 46 ], porcine [ 12 ], and bovine [ 47 ] embryos. Actin microfilaments were regulated by actin-related protein such as profilins [ 48 ] and have also been documented to be actively involved in the redistribution of mitochondria [ 49 , 50 ], polarization of embryos, and also pronuclear apposition [ 51 ].
In our study, we firstly found that fertilization failure was associated with the absence of spermatozoa in the MII ooplasm, even though a number of spermatozoa were tightly bound to the zona pellucida. Although partial digestion of ZP using Tyrode's acid did not affect sperm penetration, this approach also failed to improve fertilization rate when compared with non-Tyrode treated control. It is postulated that other factors such as quality of frozen-thawed sperm [ 52 ] and culture environment (low versus high oxygen tension) should be taken into account rather than only assessing the sperm motility prior IVF. Other advance sperm parameters, such as functional membrane integrity, mitochondrial membrane potential, and acrosome integrity, may be additionally required [ 25 ].
In summary, this study is the first paper to examine and describe the chronology of swamp buffalo embryo development in terms of redistribution of cytoskeleton and chromatin configurations during the first cell cycle. The study demonstrates that a microtubule organizing center is formed at the area of sperm centrosome and plays an important role in the migration and apposition of pronuclei, whereas actin microfilaments actively involves in cellular cleavage. Fertilization failure of buffalo oocytes, at least in our current culture system, is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate in this species. Other factors associated with fertilization failure in buffalo oocytes are needed to be characterized. | Academic Editor: Pedro J. Ginel
This paper aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments) and chromatin configurations during the first cell cycle in swamp buffalo embryos. Oocytes were matured and fertilized in vitro , and they were fixed at various time points after IVF. At 6 h after IVF, 44.4% matured oocytes were penetrated by spermatozoa. Partial ZP digestion, however, did not improve fertilization rate compared to control ( P > .05). At 12 h after IVF, the fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the paternal chromatin continued to decondense. A sperm aster was observed radiating from the base of the decondensing sperm head. At 18 h after IVF, most presumptive zygotes had reached the pronuclear stage. The sperm aster was concurrently enlarged to assist the migration and apposition of pronuclei. Cell cleavage was facilitated by microfilaments and firstly observed by 30 h after IVF. In conclusion, the cytoskeleton actively involves with the process of fertilization and cleavage in swamp buffalo oocytes. The centrosomal material is paternally inherited. Fertilization failure is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate. | Acknowledgments
This study was supported by the TRF-Master Research Grants, the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), the CHE-TRF Senior Research Scholars RTA-5080010, and Chulalongkorn University Centenary Academic Development Project. V. Chankitisakul is Ph.D. candidate of RGJ-Ph.D. program. The confocal laser scanning microscopic examination was performed at Chula Medical Center, Faculty of Medicine, Chulalongkorn University. | CC BY | no | 2022-01-13 03:35:57 | Vet Med Int. 2010 Dec 22; 2010:382989 | oa_package/2b/05/PMC3014712.tar.gz |
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PMC3014713 | 21234420 | 1. Introduction
Although proliferation of various nonepithelial cells can occur in the urinary bladder, this phenomenon is rather rare. However, a fundamental understanding of the proliferation of these cells is important to determine the appropriate treatment approach for patients presenting with this phenomenon.
Interstitial cells of the urinary bladder belong to a group of nonepithelial mesenchymal cells that exhibit elongated or satellite-shaped cell bodies and express KIT [ 1 ]. Suburothelial ICCs are characterized by the expression of both KIT and a gap-junction protein, connexin 43 (Cx43), to form an interconnection with neighboring interstitial cells [ 2 ]. Piotrowska et al. reported that the ICCs were absent in the urinary bladder of patients with megacystis-microcolon-intestinal peristalsis syndrome, which is characterized by a distended unobstructed urinary bladder [ 3 ]. Roosen et al. also showed that the cell number of the Cx43-expressing ICCs was significantly increased in the urinary bladder of patients with detrusor overactivity [ 4 ]. These findings may support the idea that ICCs act as a pacemaker or a neurotransmitter in the urinary bladder.
KIT expression is clinical important because of the existence of a compound, imatinib mesylate that specifically inhibits tyrosine kinase receptors [ 5 ]. Therefore, it is important to know whether KIT-expressing ICCs could be a source for tumorous or pseudosarcomatous proliferation in the urinary bladder.
Here, we report a case of proliferation of suburothelial KIT- and Cx43-expressing mesenchymal spindle cells in an adult urinary bladder. | 4. Discussion
Here, we report a case of proliferation of mesenchymal spindle cells with a unique immunophenotype, that is, KIT and Cx43 expression.
KIT is well characterized as a receptor tyrosine kinase for stem cell factors [ 7 ]. The clinical importance of KIT expression is based on the successful treatment with a compound—imatinib mesylate—that specifically inhibits tyrosine kinase receptors [ 5 ]. Thus, it is important to recognize the presence of a KIT-expressing tumor to provide the optimum therapy for patients.
In the gastrointestinal tract, KIT-expressing ICCs are believed to act as pacemaker cells that generate spontaneous electrical slow waves and mediate inputs from motor neurons [ 8 ]. The findings of another study also indicate that KIT-expressing myofibroblast cells, generally referred to as ICCs, exist in the human urinary bladder [ 1 , 9 ]. The human urinary bladder contains 2 major types of ICCs, namely, suburothelial ICCs and detrusor ICCs. Suburothelial ICCs and lamina propria-ICCs form a network with neighboring ICCs via the gap-junction protein Cx43. The expression of Cx43 is recognized as a distinct feature of suburothelial ICCs [ 2 ]. As demonstrated in Figures 3(f) and 3(g) , the spindle cells also showed a cytoplasmic projection that connected them with other spindle cells. Therefore, we think that the proliferating spindle cells in the present case may have the immunophenotype of suburothelial ICCs.
Histopathologically, the present case may have features overlapping those of 4 well-established tumors or tumorous conditions. First, the general morphology somewhat resembled that of an IMT [ 10 ]. IMT of the urinary bladder is very rare; however, myxoid stroma with inflammatory change is often found in IMT of the urinary bladder [ 10 ]. Recently, a case of IMT, which represented the exophytic tumors arising from the dome of the bladder, in a patient with Von Recklinghausen's syndrome was reported [ 11 ].
Loosely arranged proliferating myofibroblastic cells are also observed in IMT. However, the myofibroblastic cells in the present case exhibited nuclear atypia, which may not be accepted as a characteristic of classical IMT by many pathologists. Inflammatory cells of the IMT are usually plasma cells or lymphocytes [ 10 ]. In contrast, eosinophils were predominant in the present case. Mitosis was reported to be minimal in IMT; however, we could see mitotic structures in the present case (the number of mitotic structures was approximately 2–4/10 high-power fields). IMT is also characterized by delicate capillaries in the inflamed myxoid stroma. However, the present mass lacked delicate capillary networks. The result of the immunohistochemical study was also not compatible with that of typical IMT. ALK expression has been noted in half of the cases of IMT of the urinary bladder [ 10 ]; however, we could not detect any significant ALK expression in the present case. KIT expression is also unusual for IMT [ 10 ].
Leiomyosarcoma could be considered in the differential diagnosis of the present case. The fascicular arrangement in the present case may be found in leiomyosarcoma. Myxoid change is also frequently found in leiomyosarcoma of the urinary bladder [ 12 , 13 ]. However, the cellularity of leiomyosarcoma is more uniform than that observed in the present case. Further, the immunophenotype of the present case, that is, KIT-positive, desmin- or h-caldesmon-negative, was not consistent with that of leiomyosarcoma [ 13 ].
The concept of extragastrointestinal stromal tumors has been suggested for various tissues. A case of an extragastrointestinal KIT-positive stromal tumor in the urinary bladder has been reported [ 14 ]. However, this tumor was found in the serosa of the urinary bladder and projected to the peritoneal cavity. By contrast, the primary location of the mass in the present case was in the bladder wall. Myxoid stroma, or fascicular arrangement of the proliferating cells, is unusual in extragastrointestinal KIT-positive tumors, which closely resemble gastrointestinal stromal tumors. Compact proliferating cells, which are often found in extragastrointestinal KIT-positive tumors, were not noted in the present case.
Finally, so-called pseudosarcomatous fibroepithelial stromal polyp could be the differential diagnosis of the present lesion. Atypical stromal cells of the pseudosarcomatous fibroepithelial stromal polyp formed short, intersecting fascicles mimicking a smooth muscle tumor like the present lesion. A series of pseudosarcomatous fibroepithelial stromal polyp of the lower female genital tract was reported [ 15 ]. Although fibroepithelial stromal polyp occur most commonly in the vagina, similar lesions have also been described in the urinary bladder [ 16 ]. However, the present lesion had no proliferating epithelial component, which characterized the fibroepithelial stromal polyp. It may be interesting to examine KIT and Cx43 expression in the proliferating atypical stromal cells of pseudosarcomatous fibroepithelial stromal polyp.
To the best of our knowledge, this is the first report which describes the proliferation of KIT- and Cx43-expressing cells in the urinary bladder. Although, we could not conclude whether the mass in our case represents a tumor with indolent proliferation and favorable outcome or pseudosarcomatous mesenchymal cell proliferation, further case studies might draw a definite conclusion. | Academic Editor: Kenneth R. Shroyer
The authors report a case showing proliferation of KIT- and connexin 43-expressing mesenchymal cells of the urinary bladder. A 75-year-old woman had an ulcerated endophytic mass (size, approximately 2 × 2 cm) in the left posterolateral wall. She underwent transurethral resection and subsequent partial cystectomy. The suburothelial mass extended to the muscularis propria. The histopathological analysis revealed spindle-shaped mesenchymal cells that were loosely arranged with myxoid stroma and showed a focal compact fascicular arrangement. In the immunohistochemical analysis, these spindle cells were stained with specific antibodies to KIT and connexin 43. The patient is currently free of disease at 5 years after operation. The proliferating spindle cells in the present case might represent a phenotype of interstitial cells of the lamina propria. | 2. Case Report
The patient was a 75-year-old woman with diabetes mellitus and hypertension, but without any history of instrumental treatment. She presented with lower abdominal pain that had persisted for 2 months. Macroscopic hematuria or lower urinary symptoms were not associated. The clinical examinations, including ultrasonography, computed tomography (CT) imaging, and cystoscopy, indicated that the patient had a mass in the left lateral wall of the urinary bladder ( Figure 1 ). CT imaging indicated that the size of mass was approximately 1.5 × 1.5 cm at the initial diagnosis. She underwent transurethral resection; however, the residual mass remained after transurethral resection. Notably, CT imaging, just prior to transurethral resection, showed that the size of the mass increased to 2.0 × 2.0 cm within 1 month. Pathological examination of the resected tumor could not exclude the possibility of leiomyosarcoma. The increase of the mass in a month's time also indicated an aggressive tumor. Ten days after the first transurethral resection, the patient underwent partial cystectomy. The surgical margin was less than 2 mm; however, the patient did not wish to undergo any additional treatments. The patient has been undergoing careful followup examinations including cystoscopy and biopsies. No local recurrence or metastasis has been found 5 years after the patient underwent partial cystectomy. Informed consent was obtained from patient.
3. Pathology Findings
Histopathological examination of the resected tissue specimens showed that loosely arranged spindle-shaped cells proliferated in the myxoid stroma without any significant epithelial cell proliferation. At least partially, these spindle cells showed nuclear atypia accompanied by a few mitotic figures. The spindle cells also showed focal fascicular arrangement. The representative findings are shown in Figure 2 .
We observed loosely arranged spindle-shaped cells proliferated in the lamina propria; these cells extended to the deep muscle propria in a dome-shaped formation and accompanied the inflamed myxoid stroma ( Figure 2 ). Various inflammatory cells, most of which were eosinophils and lymphocytes, were found in the myxoid stroma. Some spindle cells exhibited “cigar-shaped” nuclei, which are often found in leiomyosarcoma; further, fascicular arrangement of the spindle cells, which is also often found in leiomyosarcoma, was observed. The number of mitotic structures was approximately 2–4/10 high-power fields. Nuclear atypia was found in many proliferating spindle cells.
Immunohistochemical staining was performed as previously reported [ 6 ]. In brief, staining was performed using an automated immunostainer (Ventana; Tuscon, AZ). The representative immunohistochemical stains are shown in Figure 3 . In the immunohistochemical analysis, most of the proliferating spindle cells (Figures 3(a) and 3(b) ) as well as intact interstitial cells outside the mass ( Figure 3(c) ) stained positive for KIT (CD117) (Ventana). In addition, the spindle cells showed positive staining for α -smooth muscle actin (DAKO, Carpenteria, CA) ( Figure 3(d) ) and vimentin (DAKO) ( Figure 3(e) ). Notably, almost all proliferating spindle cells showed strong immunoreactivities to the anti-Cx43 antibodies (Abcam, Cambridge, UK) at the projections of the cytoplasm (Figures 3(f) and 3(g) ). In contrast, no significant immunoreactivity was detected with antianaplastic lymphoma kinase (ALK) antibody, anticytokeratin antibodies AE1/AE3 (Boehringer-Mannheim, Indianapolis, IN), CAM5.2 (Becton Dickinson, San Jose, CA), cytokeratin 5/6 (DAKO), desmin (DAKO), h-caldesmon (DAKO), HMB-45 (DAKO), or anti-S-100 (DAKO).
The final pathological diagnosis varied among pathologists, including 4 consultants. Some pathologists diagnosed the present case as an inflammatory myofibroblastic tumor (IMT). Others believed that the present case should be diagnosed as a low-grade myxoid leiomyosarcoma. | Acknowledgment
The authors deeply thank the late Mr. Takuya Yamaguchi for his skillful assistance. | CC BY | no | 2022-01-13 03:14:42 | Patholog Res Int. 2010 Dec 22; 2010:961325 | oa_package/6a/40/PMC3014713.tar.gz |
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PMC3014714 | 21234421 | 1. Introduction
Diabetes Mellitus (DM) is a common, chronic group of metabolic diseases which is associated with various vascular, neuronal, endocrine, and immune alterations at cellular, tissues and organ levels [ 1 ]. Macro- and microvascular disease, are currently accepted to be the most common determinants of morbidity and mortality in the course of DM [ 2 ]. However, the exact mechanisms of the development of vascular disease have not been completely elucidated until now.
Endothelial dysfunction and oxidative stress play a key role in the pathogenesis of diabetic vascular disease [ 3 ]. Under physiological conditions, vascular endothelium plays an important role in the formation of vascular smooth muscle tone by releasing relaxant mediators, such as nitric oxide (NO), endothelium derived hyperpolarizing factor, endothelium derived contracting factor, and prostacycline [ 4 ]. Impairment of endothelium-dependent relaxation has been demonstrated both in vessels of patients with insulin-dependent diabetes mellitus [ 5 ] and noninsulin-dependent diabetes mellitus [ 6 ]. Although the reason for this impairment is not entirely clear, some theories have been postulated. As is known, DM is characterized by hyperglycemia and hyperlipidemia, two cardinal biochemical features associated with inhibition of endothelial nitric oxide synthase (eNOS), leading to diminished NO production and increased formation of reactive oxygen species (ROS) in endothelial and vascular smooth muscle cells. Besides, impaired expression or activity of some antioxidant enzymes such as superoxide dismutase (SOD) and catalase contributes to the development of endothelial dysfunction in DM by increasing oxidative stress [ 7 ].
Endothelial dysfunction accompanied by upregulated proinflammatory and inflammatory mediators is thought to be another contributing factor to the pathogenesis of diabetic vascular complications. Multiple effects of inflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor- α (TNF- α ), which lead to prothrombotic and proinflammatory changes on the vascular endothelium, have been outlined in some reports [ 8 ]. Recently it has been suggested that myeloperoxidase (MPO), a “heme” protein derived from leukocytes, plays an important role in leukocyte-mediated vascular injury responses in inflammatory vascular diseases such as diabetic vasculopathy and atherosclerosis [ 9 , 10 ].
In diabetic patients, hyperhomocysteinemia is an independent risk factor for macroangiopathy and mortality [ 11 ]. Many ROS are generated during the auto-oxidation of homocysteine (Hcy) in diabetic patients. These oxygen-derived molecules initiate the lipid peroxidation in cell membranes that are responsible for endothelial injury and reduction of vascular NO production [ 12 ].
Peroxisome proliferator-activated receptor- α (PPAR- α ) is a hormonal activated nuclear receptor which plays an important role in the course of many vascular diseases such as DM, hypertension, and coronary heart disease [ 13 , 14 ]. In recent publications, it has been clearly demonstrated that activation of PPAR- α leads to an antiinflammatory effect by reducing plasma concentrations of TNF- α . On the other hand, it produces an antioxidant effect by reducing plasma concentrations of malonyldialdehyde, major indicator of oxidative stress, and by stimulating the expression of SOD, one of the major molecules of antioxidant defense [ 15 , 16 ]. In this context fenofibrate (FF), a third generation fibric acid derivative and a PPAR- α agonist, can be a beneficial choice for the treatment of diabetic vascular complications because of its antiinflammatory and antioxidant effects. Moreover, FF is a useful drug for the treatment of atherogenic dyslipidemias, producing a substantial decrease in the levels of triglyceride-rich lipoproteins and an increase in high density lipoprotein cholesterol levels. In contrast, several studies show that FF can significantly increase plasma Hcy levels [ 17 , 18 ]. However, the underlined mechanisms by which FF increase total Hcy levels and whether they have any adverse effects on endothelial function are unknown.
In light of the foregoing data, the primary aim of this study is to investigate the role of FF on diabetic endothelial dysfunction, to elucidate its antioxidant and anti-inflammatory effects, and to evaluate the contribution of FF-induced hyperhomocysteinemia in diabetic vascular complications in a rat model of streptozotocin- (STZ-) induced DM. As far as we know this is the first study in literature which discusses all these parameters in one single study protocol. | 2. Materials and Methods
2.1. Animals
Male Wistar rats (body weight 200–250 g, 10–12 weeks old) were used in this study. The animals were housed in individual cages at a constant temperature (22°C) with a fixed 12 : 12-h light-dark cycle. This study was approved by the Ethics Committee of the School of Medicine, University of Ege (Approval no: 2004-17).
The animals were randomised to four experimental groups: untreated control, untreated diabetic, FF-treated diabetic (150 mg kg −1 day −1 via oral gavage), and FF-treated control. Diabetes was induced by a single intraperitoneal injection of STZ (55 mg kg −1 ). STZ was dissolved in 1 ml cold fresh saline immediately before use. Seven days after STZ injection, blood glucose levels were determined using an Accu-Chek Go glucometer (Roche, Turkey). Rats with blood glucose levels of 250 mg dl −1 or above were considered to be diabetic. Control rats were injected intraperitoneally with 1 ml cold fresh saline. FF treatment was started 6 weeks after STZ or saline injection, and the treatment continued for 4 weeks.
2.2. Vascular Reactivity Studies
10 weeks after STZ or saline injection, rats were killed by the withdrawal of blood via cardiac puncture under anesthesia. Thoracic aortas were quickly removed into 4°C Krebs-Henseleit solution and cut into four 3-4 mm wide rings. In some rings, the endothelium was quickly removed mechanically by inserting a small forceps into the lumen and gently rolling. In each experiment, endothelium-intact and endothelium-denuded rings (two of each) were suspended horizontally under a resting tension of 2 g in 20 ml organ chambers containing Krebs-Henseleit solution of the following composition (mM): NaCl, 118.30; KCl, 4.70; MgSO 4 , 1.20; KH 2 PO 4 , 1.22; CaCl 2 , 2.50; NaHCO 3 , 25.00; glucose, 11.10; ph, 7.4; gassed with a 95% O 2 , 5% CO 2 mixture and maintained at 37°C. Each ring was connected to a force displacement transducer for the measurement of isometric force which was continuously displayed and recorded online on a personal computer via an 8-channel transducer data acquisition system (BIOPAC COMMAT İletişim Ltd., Ankara, Turkey) using a software (BIOPAC MP35 COMMAT İletişim Ltd., Ankara, Turkey) which also analysed the data.
After 15 min of equilibration, each ring was systematically stretched to the optimum of its length-active tension relation by exposure to incremental concentrations of KCl. Rings were then left to equilibrate in the bath for a total of 30 min and washed every 15 min. After the initial equilibration period of 60–90 min, endothelium-denuded rings were used to assess the contractile responses elicited by either incremental concentrations of phenylephrine (PE) (0.001–30 μ M) or a single concentration of KCl (120 mM).
Relaxant responses were determined using cumulative concentrations of acetylcholine (0.001–30 μ M, ACh), calcium ionophore A23187 (0.001–3 μ M, A23187), L-arginine (0.1–300 μ M), or sodium nitroprusside (0.0001–0.3 μ M, SNP) on endothelium-intact rings precontracted with submaximal concentration of phenylephrine. In order to maintain appropriate precontractile tension in all preparations, submaximal concentrations were determined using the prior cumulative concentration-response curves of PE. Contractile responses generated by cumulative concentrations of PE (0.001–30 μ M) were also assessed in these rings.
In endothelium-intact rings, the production of basal NO was also evaluated by calculating the ratio of additional contractions induced by a single concentration of NG-Nitro-L-arginine methyl ester (100 μ M, L-NAME) to the precontraction elicited by a single concentration of PE given at median effective concentration (EC 50 ).
2.3. Biochemical Measurements
After obtaining heparinized blood samples by intracardiac puncture, serum was immediately separated, and erythrocyte hemolyzates were prepared. For this purpose, the packed erythrocytes were washed two times with 9 g/L NaCI solution and haemolysed with ice-cold water (1/5, v/v). Erythrocyte SOD and catalase activities were determined immediately in hemolyzates. The haemoglobin values were measured by Drabkin's method. The erythrocyte SOD activities were measured based on the inhibition of autoxidation of epinephrine by SOD at 480 nm, with an LKB Ultraspec 2 spectrophotometer (LKB Biocrom Ltd, Cambridge, England). The assay was calibrated by using purified SOD, and done unit of enzyme was defined as the amount of enzyme, which inhibits 50% of autoxidation of epinephrine. The erythrocyte catalase activities were determined as described by Sözmen et al. [ 19 ]. According to this method, the degradation of hydrogen peroxide is recorded spectrophotometrically at 240 nm absorbance. One unit of catalase is defined as the amount of enzyme, which decomposes 1 μ mol hydrogen peroxide/min under specific conditions.
In addition, aortic and liver tissue samples were homogenized in phosphate buffer (0.5 M; pH = 7.0), (1/10 w/v). The homogenate was centrifuged for 5 min at 700 × g at 4°C to sediment unbroken cells and cellular debris. Determination of SOD and catalase activities in the supernatants, as defined above, and the determination of lipid peroxidation were carried out immediately. Lipid peroxidation was briefly measured by the determination of thiobarbituric acid reactive substances (TBARSs) which was performed by the incubation of tissue homogenates in TBARS solution (0,12 M thiobarbituric acid in 15% trichloroacetic acid and 1% hydrochloric acid mixture) for 30 min at 95°C. TBARS levels were calculated using 1, 1, 3, 3 tetramethoxypropane standard curve. All biochemical parameters were normalized to total protein content of the heamolyzate as measured by the Lowry method using bovine-serum albumin as standard [ 20 ].
2.4. Measurement of MPO and TNF- α Levels
Tissue MPO activities were measured according to the modified method of Grisham et al. [ 21 ]. Briefly, following homogenization of aortic tissue, homogenates were centrifuged at 10000 rpm for 15 min. Pellets were rehomogenized in 0.5 mM HETAB (hexadecyltrimethyl ammonium bromide) in phosphate buffer (50 mM, pH = 6.0). Following three freeze and thaw cycles, samples were centrifuged at 10000 rpm for 10 min. Supernatants were added to reactive solution containing 0.5 M o-dianisidin (in phosphate buffer). After addition of hydrogen peroxide solution (20 mM), absorbance of samples was recorded at 492 nm with a microplate reader for 3 minutes with 15-second intervals. MPO activities were calculated using a standard curve.
The serum content of TNF- α was determined spectrophotometrically according to the instructions of a commercially available ELISA kit (Rat TNF ELISA Kit BD Biosciences Inc.).
2.5. Measurement of Serum Total Cholesterol and Triglyceride Levels
Lipid levels were measured in the fasting serum samples by using ready-made cholesterol kits (Cormary Lot: 304-1910 A Lublin Poland) and triglyceride kits (Cormary Lot 301-141409 Lublin Poland) on the Hitachi 912 Automatic Analyzer machine.
2.6. Measurement of Serum Hcy Levels
Blood samples obtained by cardiac puncture were centrifuged (1000 × g for 10 min), and serum was separated. Serum total Hcy was measured by Fluorescence Polarization Immunoassay using an IMx automatic analyser (Abbott Laboratories, Diagnostic Division, Abbott Park, IL, USA).
2.7. Drugs
Streptozotocin, acetylcholine, phenylephrine, L-arginine, calcium ionophore A23187, sodium nitroprusside, and L -NAME were obtained from Sigma Chemical Co. (St. Louis, MO, USA). FF was supplied from Nobel Drug Co. (Interlab Ltd, Turkey).
2.8. Statistical Analysis
Data were given as mean ± SEM. Concentration response curves were configured with nonlinear regression analysis prior to the evaluation of EC 50 and p D 2 (−log EC 50 ) via Graphpad Prism 4.0 program. The contractile response of each aortic ring to phenylephrine was recorded in milligrams by using BIOPAC Mp35 software program. Relaxation responses were evaluated as percentage of phenylephrine precontraction. Repeated measures of one-way analysis of variance (ANOVA) were performed to analyze the repetitious concentration response curves, and when significancy was determined, post hoc analysis was carried out using Bonferroni test. Nonrepetitious data (E max , EC 50 , Hyc, TC, TG, Blood glucose) were evaluated by using nonparametric Kruskall-Wallis test. For all results a P value of <.05 was accepted to be significant. | 3. Results
3.1. Metabolic Parameters
Body weight, blood glucose concentrations, serum total cholesterol, and triglyceride levels were presented in Table 1 . Induction of diabetes with STZ resulted in a significant decrease in body weight and a significant increase in blood glucose levels compared to control rats. FF treatment affected neither high glucose levels nor weight loss in diabetic animals. Although diabetes was associated with significant increases in total cholesterol and triglyceride levels, FF treatment did not alter serum lipid levels significantly.
3.2. Vascular Functional Studies
3.2.1. Contractile Responses
PE (1 nM–10 μ M) induced concentration-dependent contractile responses in the endothelium-intact aortic rings of all rats ( Figure 1(a) ). Contractility of PE was significantly increased in diabetic animals when compared to control group. However, FF provided a significant decrease in PE contractility in the diabetic group ( Figure 1(a) ). In the endothelium-denuded aortic rings, no significant changes in the PE contractility were observed between the experimental groups ( Figure 1(b) ). In addition, contractile responses to KCl were similar in all experimental groups ( Figure 1(c) ).
3.2.2. Vasorelaxant Responses
Endothelium-dependent relaxant response to ACh was significantly impaired in nontreated diabetic aorta ( Figure 2(a) ) without a significant difference in the p D 2 value ( Table 2 ). FF treatment restored the impaired relaxations to ACh and increased E max in the diabetic rats ( Table 2 ). Concentration-dependent relaxant responses to A23187 ( Figure 2(b) ), L-arginine ( Figure 2(c) ), and SNP ( Figure 2(d) ), as well as p D 2 and E max values ( Table 2 ), did not show any significant differences between groups.
3.2.3. Effect of L-NAME on Aortic Rings
L-NAME was added to endothelium-intact aortic rings after precontracting the rings with the EC 50 concentration of PE. L-NAME responses were significantly impaired in the nontreated diabetic group; however, FF brought in a significant improvement in these responses ( Figure 3 ).
3.3. Antioxidant and Anti-Inflammatory Parameters
Erythrocyte-SOD and erythrocyte-catalase levels were markedly lower in the diabetic group than in the control group, and FF treatment caused a significant increase in these parameters ( Table 3 ). Although liver-SOD levels were decreased, liver-catalase levels remained unchanged in the diabetic group. FF treatment significantly increased liver-SOD levels ( Table 3 ).
In contrast, TBARS values in both erythrocytes and livers of the diabetic group were significantly higher than in the control group ( Table 1 ). Although FF significantly lowered the erythrocyte-TBARS levels, it had no effect on the liver-TBARS levels. Aorta TBARS level was significantly increased in the diabetic group, and FF treatment markedly prevented the increased aortic TBARS level in diabetic group ( Table 3 ).
Serum TNF- α and aortic MPO levels were significantly higher in the diabetic group than in the control group ( Table 3 ). FF treatment decreased the high MPO levels in the diabetic rats without a significant effect on the TNF- α values.
3.4. Homocysteine Levels
Serum total Hcy levels were markedly decreased in diabetic rats when compared to control group ( Table 3 ). FF treatment did not affect Hcy levels in diabetic rats; however, chronic administration of FF significantly increased Hcy levels in control rats. | 4. Discussion
Data obtained in the present study reveal that in STZ-induced diabetic rats, chronic administration of FF improves endothelium-dependent relaxation and increases basal NO production in the aorta, enhances the levels of antioxidant enzymes such as SOD and catalase, ameliorates the abnormal TBARS levels, and finally, prevents the increased tissue MPO levels without a significant change in serum levels of total cholesterol and triglyceride. FF is one of the major drugs used in the treatment of dyslipidemia, and it has recently been reported that FF decreases serum levels of cholesterol and triglyceride in STZ-induced diabetic rats [ 22 ] and it produces a considerable decrease in serum triglyceride levels, a moderate reduction in LDL cholesterol levels, and a significant enhancement in HDL cholesterol concentrations in a model of diabetic dyslipidemia [ 23 ]. All these effects of FF have been attributed to the activation of PPAR- α by FF. Contrarily, we failed to show any beneficial effect of FF on the increased total cholesterol and triglyceride levels in diabetic rats; however FF prevented the diabetes-induced impairment in the endothelium-dependent relaxation. Although FF treatment is expected to reduce lipid levels, controversial studies also exist presenting that FF treatment does not affect plasma triglyceride and total cholesterol concentrations at doses up to 300 mg kg −1 day −1 in normoglycemic rats [ 24 ]. Moreover, another fibric acid derivative, bezafibrate, has been reported to affect none of the increased lipid levels (namely, total cholesterol, LDL, and triglyceride) in hyperglycemic rats at 30 mg kg −1 day −1 dose [ 25 ]. Our findings indicate that mechanisms other than the inhibition of high circulating lipids such as triglycerides and total cholesterol and the correction of dyslipidemia may involve in FF-induced restoration of endothelial dysfunction.
It has been reported in various studies that increased oxidative stress, production of proinflammatory cytokines such as TNF- α and IL-6, leukocyte-mediated vascular injury with MPO, and hyperhomocysteinemia can be accepted as the crucial mechanisms responsible for the pathogenesis and progression of diabetic tissue damage [ 26 – 28 ]. Reduction in the acetylcholine-induced endothelium-dependent relaxation of diabetic rats in the present study is compatible with the results of several other studies [ 29 – 31 ]. In our study, induction of diabetes resulted in a marked decrease in acetylcholine response without a significant change in the p D 2 value, indicating that the sensitivity of the diabetic aortic tissue to acetylcholine was preserved. Furthermore, endothelium-dependent relaxant responses to neither calcium ionophore A23187 nor L-arginine were found to be impaired. Even sodium-nitroprusside-induced endothelium-independent relaxation was preserved in diabetic aorta. These findings suggest that the vascular damage in our animal model is limited to the endothelium per se ; however, the stimulated activation of eNOS seems not to be impaired nor does the utilization of the precursor L-arginine. In this case, it seems likely that endothelial dysfunction may be limited to the impairment in the homeostatic balance maintained by basal NO release. Indeed, PE contractility in endothelium-intact aorta was enhanced in diabetic vessels, and rings from diabetic animals responded to L-NAME with significantly greater tension, indicating a significant decrease of basal NO release in these vessels.
Enhanced contractility and decreased ACh responses may be associated with the deficient basal endothelial activity, besides, increased oxidative stress due to excessive production of oxygen-free radicals and decreased antioxidant defense systems may also involve in the process [ 32 ]. Oxidative degradation of lipids is a well-defined mechanism of cellular damage caused by excessive production of ROS, and TBARS is the most widely employed assay used to determine lipid peroxidation. Today, it is well known that TBARS level is increased in both plasma and aortic tissue of experimental diabetic models [ 33 , 34 ]. In the present study, we have demonstrated that enhanced aortic, erythrocyte, and liver levels of TBARS in diabetic rats accompany the defective vascular endothelial function. In addition, SOD and catalase are known to be the most important enzymes in the antioxidant defence system of the body. The major function of SOD is to catalyze the conversion of superoxide anion radicals to hydrogen peroxide in order to reduce their toxic effects [ 34 ]. On the other hand catalase is responsible for the removal of intracellular hydrogen peroxide produced by SOD. Some in vitro studies have shown that ertyhrocyte antioxidant defences protect endothelial cells against oxidant-induced damage [ 35 , 36 ]. In our study, we have observed that diabetic rats have decreased SOD levels both in the liver tissue and in the erythrocytes. Similarly, erythrocyte catalase levels were diminished without a significant change in the liver catalase levels.
The mechanisms of the beneficial effects of FF on vascular function have not been fully understood yet. However, direct activation of PPAR- α in the arterial wall, correction of lipid abnormalities, and increment in the formation, availability, and action of NO have all been postulated. There is only one study published so far claiming that FF increases acetylcholine response in STZ-induced diabetic animals and reduces oxidative stress [ 22 ]. Data obtained in our study also confirm these vascular effects of FF.
We have also questioned the potential inflammatory mechanisms of diabetic vasculopathy and among them are the aortic MPO and TNF- α levels. Myeloperoxidase is a leukocyte-derived heme protein. Recent studies have shown that MPO plays an important role in endothelial dysfunction [ 27 ]. Plasma MPO levels are shown to be increased both in type 1 and type 2 diabetic patients [ 37 ]. An important consequence of MPO activity is the consumption of NO and thereby induction of endothelial dysfunction. The enzyme MPO can convert NO into nitrating oxidants, which are potential inflammatory mediators in cardiovascular diseases [ 37 ]. Compatible with up-to-date data, we have showed that aortic MPO levels are significantly increased in diabetic rats when compared to the control group. Evidence from recent studies suggest that TNF- α impairs endothelium-dependent and NO-mediated vasodilatation in various vascular beds such as rat thoracic aorta, coronary arteries, and carotid arteries [ 38 ]. The increased TNF- α expression induces the production of ROS, leading to endothelial dysfunction in diabetes. Besides TNF- α appears to decrease the bioavailability of NO by diminishing its production and enhancing its removal. Indeed, serum TNF- α levels in diabetic rats have been noted to increase in our study. FF treatment successfully prevented the diabetes-induced increase in aortic MPO levels; however it failed to affect the increased serum TNF- α levels. Controversial reports exist regarding the effect of FF on TNF- α levels. For example, Tian-Lun Yang has reported that FF reduces serum TNF- α levels of rats with LDL-induced endothelial dysfunction [ 39 ]. On the contrary, Choj has demonstrated that FF does not affect serum TNF- α levels in OLETF rats [ 40 ]. So far to our knowledge, no scientific reports exist using STZ-induced diabetic rat model to investigate the potential effects of FF on TNF- α levels, and we claim that FF has no effect on serum TNF- α levels.
Despite its favourable effects on diabetic vascular dysfunction, FF treatment may result in elevation of serum total Hcy, which is known to induce oxidative stress and endothelial dysfunction [ 12 ]. Folate status has been associated with endothelial dysfunction in adolescents with type 1 diabetes, and elevated total Hcy is a risk factor for vascular disease in the nondiabetic population [ 41 ]. It is well known that FF increases total Hcy levels by PPAR- α mediated mechanism although it dose not affect unbound Hyc levels in rat serum [ 13 ]. In comparison to human beings, metabolism of Hcy is completely different in rats, and there is an extensive intrarenal Hcy metabolism in rats [ 42 ]. We have found decreased total Hcy levels in the serum of diabetic rats. It is probable that excessive protein loss due to diabetic nephropathy may cause a decrease in the protein-bound fraction of Hcy and the free form undergoes excessive metabolism resulting in a decrease in the total Hcy levels in diabetic rats. Indeed, our finding confirms to a previous report suggesting that serum total Hcy levels are found to be decreased in STZ-induced diabetic rat model [ 42 ]. In our study, FF did not affect serum total Hcy levels in the diabetic group, whereas it increased total Hcy levels in the control group probably through PPAR- α mediated mechanism; however the increase in total Hcy levels in FF-treated control rats did not impair the endothelial function. Therefore, we assume that fenofibrate-induced hyperhomocysteinemia does not involve in the pathogenesis of diabetes-induced endothelial dysfunction.
FF dose in the current study was selected from previous studies on rats [ 43 ]. It should be noted that this dose was much higher than those clinically used in the treatment of dyslipidemia (100–250 mg day −1 ). Additionally, it should be reminded that this is an ex vivo , but not an in vivo , study which is performed on the large, but not small, arteries of STZ-diabetic rats. Therefore, any potential antioxidant activity of FF requires to be investigated in well-conducted clinical trials performed on humans. Indeed, in humans, fibrate therapy has been reported to be associated with decreased levels of biomarkers of endothelial dysfunction in a few, but not all, studies presenting some clue on the preventory effect of fenofibrate in the development of diabetic microangiopathy [ 44 – 48 ]. Moreover, supplementation of FF treatment with antioxidants like coenzyme Q10 has been reported to have favourable vascular effects in the forearm microcirculation of dyslipidaemic type 2 diabetic patients, due to increase in the bioactivity of and/or responses to endothelium-derived relaxing factors, including NO, and this may entail synergistic stimulation of peroxisome proliferator-activated receptor [ 49 ].
In conclusion, treatment with FF produces a significant improvement in the endothelial dysfunction in STZ-induced diabetic rats without a significant effect on the serum total cholesterol and triglyceride levels. This effect of FF seems to be related to its potential antioxidant activity. | Academic Editor: Norman Cameron
Diabetic endothelial dysfunction is accompanied by increased oxidative stress and upregulated proinflammatory and inflammatory mediators in the vasculature. Activation of peroxisome proliferator-activated receptor-alpha (PPAR- α ) results in antioxidant and anti-inflammatory effects. This study was designed to investigate the effect of fenofibrate, a PPAR- α activator, on the endothelial dysfunction, oxidative stress, and inflammation in streptozotocin diabetic rats. Diabetic rats received fenofibrate (150 mg kg −1 day −1 ) for 4 weeks. Fenofibrate treatment restored the impaired endothelium-dependent relaxation and increased basal nitric oxide availability in diabetic aorta, enhanced erythrocyte/liver superoxide dismutase and catalase levels, ameliorated the abnormal serum/aortic thiobarbituric acid reactive substances, and prevented the increased aortic myeloperoxidase without a significant change in serum total cholesterol and triglyceride levels. It did not affect the decreased total homocysteine level and the increased tumor necrosis factor- α level in the serum of diabetic rats. Fenofibrate-induced prevention of the endothelial function seems to be related to its potential antioxidant and antiinflammatory activity. | Conflict of Interests
The authors declare that there is no conflict of interests associated with this paper. | Acknowledgments
The study was supported by the Turkish Diabetes Foundation and the Research Fund of Ege University (Project: 04 TIP-016). | CC BY | no | 2022-01-13 02:21:51 | Exp Diabetes Res. 2010 Dec 27; 2010:828531 | oa_package/e1/02/PMC3014714.tar.gz |
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PMC3014719 | 21234366 | 1. Introduction
Ochratoxin A (OA), a nephrotoxic mycotoxin mainly produced by Aspergillus ochraceus and Penicillium viridicatum, has been shown to contaminate a wide variety of cereals and feed stuffs and is extremely toxic to domestic fowls [ 1 ] and swine [ 2 ]. Spontaneous occurrence of OA in feed and feed stuffs have been reported and OA has been implicated in field outbreaks of mycotoxicosis resulting in poor growth rate and poor feed efficiency [ 3 ]. The effects of OA in poultry were found to be quite pronounced in young broiler chicks [ 4 ].
An attempt is, therefore, made to study the nature of effect of experimental OA toxicity in young broiler chicks. This investigation seeks to study the pattern of change elicited by OA in diet on feed intake, feed conversion, and body weight in broiler chicken. | 2. Material and Methods
Sixty-day-old broiler chicks (Vencob, India) received from a commercial hatchery were randomly divided into six replicates of ten chicks each and housed in well-ventilated clean cages with optimum, continuous lighting. The chicks were provided with feed and water, ad libitum.
2.1. Standard Toxin
Pure ochratoxin A obtained from M/s. Sigma chemicals, USA was used as the standard for the experiment.
2.2. Fungal Culture
Aspergillus ochraceus NRRL 3174 obtained from S. W. Peterson, Microbiologist, Microbial properties research, United States Department of Agriculture, Agricultural Research Service, 1815, North University Street, Peoria, Illinois 61604, USA, in lyophilized form was used as the seed culture.
The fungus was maintained by subculturing at 10 days interval in Czapek Dox agar supplemented with 20 per cent sucrose and 0.7 per cent yeast culture powder, as slant culture [ 5 ].
2.3. Production of Ochratoxin A
Ochratoxin A was produced on broken wheat [ 6 ].
One hundred g of the substrate( broken wheat) was taken in 500 ml conical flasks and soaked in 80 ml tap water for 2 hours. The flasks were autoclaved at 15 psi for 15 min and cooled. Five ml of distilled water was added to slant cultures of the fungi which was already prepared, and one ml of this was inoculated into the conical flasks. The flasks were kept at room temperature in slanting position, and hands taken vigorously six to ten times a day to avoid clump formation. On the fourteenth day, the mouldy substrate was briefly steamed for five minutes to kill the spores and dried overnight at 60°C in a hot air oven. The mouldy substrate was powdered and the ochratoxin content was estimated by the method of Manning and Wyatt [ 7 ].
2.4. Estimation of Toxin
Quantity of ochratoxin A in mouldy substrate was estimated by the method followed by Manning and Wyatt [ 7 ]. Ten grams of mouldy feed was blended with 150 ml of chloroform methanol mixture (1 : 1) for 10 minutes and filtered. The filtrate was extracted twice using 75 ml of 0.5 M sodium bicarbonate in a separating funnel. The sodium bicarbonate fractions were pooled and acidified to pH 1.5 with concentrated HCl and extracted twice with 75 ml chloroform. The chloroform extracts were combined, orated to dryness, and dissolved in 2 ml benzene. An aliquot of the benzene solution and an ochratoxin A standard solution were serially diluted and spotted on a precoated silica gel 60 thin layer chromatography plates (0.2 mm thickness). The plates were developed in benzene-acetic acid (9 : 1), allowed to dry and examined under longwave ultraviolet light. The OA content was quantified by both spot comparison and dilution to extinction methods.
The experimental design consisted of three dietary levels of OA at 0, 1 and 2 ppm for 28 days from hatch. OA produced in broken wheat [ 6 ]. Known amount of powdered wheat culture containing OA were incorporated into OA free broiler starter mash to yield 1 ppm and 2 ppm OA. One control diet was also prepared. The diet contained 23 per cent crude protein.
Feed consumption, feed conversion, and body weight were worked out for each group. Blood samples collected by cardiac puncture were allowed to clot and centrifuged for 20 minutes at 1500 rpm to separate the sera. Pooled serum samples were used to determine the total serum protein and albumin [ 8 ].
2.5. Statistical Analysis
The data generated from different parameters of the experimental study were subjected to one way analysis of variance(ANOVA) by using SPSS software. | 3. Result and Discussion
3.1. Body Weight
Feeding OA significantly reduced the growth rate of broiler chickens ( Table 1 ). The reduction was observed from the first week onwards in OA treated groups. However, reduction observed between the control and 1 ppm level at first week was insignificant.
Similar observations of growth depression with varying levels of dietary ochratoxin A ranging from 0.8 to 10 μ g per gram were also studied by Huff et al. [ 9 ], Prior et al. [ 10 ], Dwivedi and Burns [ 11 ], Kubena et al. [ 12 ], Manning and Wyatt [ 7 ], and Huff et al. [ 4 ].
The reduced weight gain induced by OA could be attributed to the reduced feed intake as observed in this study. Impaired protein metabolism was implicated as the main cause [ 13 ]. It was observed that OA competes with phenylalanine for binding sites on the Phenylalanyl transfer-RNA-synthetase enzyme, thus inhibiting protein synthesis [ 4 ].
3.2. Feed Consumption
Feeding OA to broiler chickens resulted in reduction in feed consumption ( Table 2 ). Total feed consumed were 1295.76, 1241.06, and 1227.03 g for 0, 1, and 2 ppm, respectively, at the end of 4th week. Though there was reduction in feed consumption, it was not statistically significant ( P < .05).
Similar observations of reduced feed consumption were also made by Hamilton et al. [ 14 ], Prior et al. [ 10 ], and Hamilton et al. [ 3 ].
3.3. Feed Conversion
Feed conversion was decreased in OA fed birds ( Table 3 ). However, it was not statistically significant ( P > .05). Hamilton et al. [ 3 ], Kubena et al. [ 12 ], and Gibson et al. [ 13 ] also reported decreased feed conversion in ochratoxicosis.
3.4. Total Protein and Albumin
Feeding OA significantly reduced the total serum protein and albumin levels in broiler chickens (Tables 4 and 5 ). Similar observations were also made by Manning and Wyatt [ 7 ], Huff et al. [ 4 ], and Kubena et al. [ 12 ].
Huff et al. [ 4 ] reported that the total protein and albumin were the sensitive indicator of ochratoxicosis. The mechanism by which OA produced hypoproteinaemia and hypoalbuminaemia is due to inhibition of phenylalanyl transfer-RNA-synthetase with phenylalanine. [ 15 , 16 ] and renal leakage of albumin resulting from kidney lesions induced by OA [ 4 ].
An analysis of the experimental data generated in this investigation clearly point to the adverse effect of OA on feed consumption and growth rate in young broiler chicks. The impairment of protein metabolism would lead to increased susceptibility to various infections and tell upon their production performance. An in-depth study of the immunological alterations evoked by OA in chicks would help to throw more light on the pathogenesis of ochratoxicosis. | 3. Result and Discussion
3.1. Body Weight
Feeding OA significantly reduced the growth rate of broiler chickens ( Table 1 ). The reduction was observed from the first week onwards in OA treated groups. However, reduction observed between the control and 1 ppm level at first week was insignificant.
Similar observations of growth depression with varying levels of dietary ochratoxin A ranging from 0.8 to 10 μ g per gram were also studied by Huff et al. [ 9 ], Prior et al. [ 10 ], Dwivedi and Burns [ 11 ], Kubena et al. [ 12 ], Manning and Wyatt [ 7 ], and Huff et al. [ 4 ].
The reduced weight gain induced by OA could be attributed to the reduced feed intake as observed in this study. Impaired protein metabolism was implicated as the main cause [ 13 ]. It was observed that OA competes with phenylalanine for binding sites on the Phenylalanyl transfer-RNA-synthetase enzyme, thus inhibiting protein synthesis [ 4 ].
3.2. Feed Consumption
Feeding OA to broiler chickens resulted in reduction in feed consumption ( Table 2 ). Total feed consumed were 1295.76, 1241.06, and 1227.03 g for 0, 1, and 2 ppm, respectively, at the end of 4th week. Though there was reduction in feed consumption, it was not statistically significant ( P < .05).
Similar observations of reduced feed consumption were also made by Hamilton et al. [ 14 ], Prior et al. [ 10 ], and Hamilton et al. [ 3 ].
3.3. Feed Conversion
Feed conversion was decreased in OA fed birds ( Table 3 ). However, it was not statistically significant ( P > .05). Hamilton et al. [ 3 ], Kubena et al. [ 12 ], and Gibson et al. [ 13 ] also reported decreased feed conversion in ochratoxicosis.
3.4. Total Protein and Albumin
Feeding OA significantly reduced the total serum protein and albumin levels in broiler chickens (Tables 4 and 5 ). Similar observations were also made by Manning and Wyatt [ 7 ], Huff et al. [ 4 ], and Kubena et al. [ 12 ].
Huff et al. [ 4 ] reported that the total protein and albumin were the sensitive indicator of ochratoxicosis. The mechanism by which OA produced hypoproteinaemia and hypoalbuminaemia is due to inhibition of phenylalanyl transfer-RNA-synthetase with phenylalanine. [ 15 , 16 ] and renal leakage of albumin resulting from kidney lesions induced by OA [ 4 ].
An analysis of the experimental data generated in this investigation clearly point to the adverse effect of OA on feed consumption and growth rate in young broiler chicks. The impairment of protein metabolism would lead to increased susceptibility to various infections and tell upon their production performance. An in-depth study of the immunological alterations evoked by OA in chicks would help to throw more light on the pathogenesis of ochratoxicosis. | Academic Editor: Sagar M. Goyal
The effect of ochratoxin A (OA) on the body weight, feed intake, and feed conversion was investigated in broiler chicken fed dietary levels of OA at 0, 1, and 2 ppm for 28 days from hatch. Feeding OA significantly reduced the growth rate of broiler chicken. The reduction was observed from the first week onwards in OA-treated groups. Feed consumption and feed conversion also showed a diminishing trend from the first week of feeding toxin. Its implication on the performance of broiler chicken is discussed. | CC BY | no | 2022-01-13 03:35:57 | Vet Med Int. 2010 Dec 27; 2010:590432 | oa_package/db/7c/PMC3014719.tar.gz |
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PMC3014722 | 20578888 | Introduction
Venom apparatuses are common structures of hymenopterans and are involved in the production of active compounds to be delivered through an ovipositor or sting. Many hymenopterans have stings, which, apart from being used to subdue their prey, can be used effectively for defense. In some ants, the sting is used for colony defense, and some people can develop serious anaphylactic reactions to ant venoms ( Brown and Heddle 2003 ).
Some ants of the genus Solenopsis Westwood (Hymenoptera: Formicidae) are known as fire ants ( Vinson 1986 ) because of their painful stings. They aggressively attack in swarms when their fragile, earthen nests are disturbed. Fire ants are native to the Americas and most diverse in South America, but some species of this group have been shipped and introduced into other world regions inadvertently. At least one species, Solenopsis invicta Buren, has become a major public concern, mainly in the United States, because of its marked adaptability to human environments and the allergenicity of its sting ( Rhoades et al. 1989 ; deShazo and Banks 1994 ; deShazo and Williams 1995 ; deShazo et al. 1999 ; Kemp et al. 2000 ). One species, Solenopsis saevissima Smith, is still restricted to South America and common in Brazil ( Rossi and Fowler 2004 ). It has not been studied as extensively as S. invicta.
Both species belong to a particularly problematic ant group, in terms of taxonomy and systematic, known as the “ Solenopsis saevissima group of species” ( Pitts et al. 2005 ). It includes 13 fire ant species that exhibit marked morphological similarity and intraspecific variability. Some species are capable of hybridization, rendering most morphological characters for species separation unreliable ( Vander Meer 1985 ; Pitts et al. 2005 ). There is still some ongoing discussion about the validity of these species and the best characters to be used in defining each species ( Ross and Trager 1990 ; Ross and Shoemaker 2005 ).
The venom apparatus of Solenopsis richteri was thoroughly described, including histological aspects, by Callahan et al. ( 1959 ). Later, the venom apparatus of S. invicta, a similar species with which S. richteri can hybridize ( Vander Meer 1985 ), was briefly described by Billen ( 1990 ), who also analyzed some ultrastructural aspects of it. No other venom apparatuses of any species of this group have been described, but it is well known that the venoms of the different species of fire ants have distinct chemical composition ( Jones and Blum 1982 ; Fox, Palma and Bueno, unpublished data). The different compositions might reflect differences in the internal organization of the structures of the venom apparatus, and some of these differences might help elucidate the systematics for this group.
The present investigation about the morphological and cellular organization of the venom apparatus of S. saevissima was carried out, pointing out specific differences through comparison of the observed structures with what has been done with other species in the genus. | Materials and Methods
The ants were obtained from a house garden in the outskirts of Pedro do Rio, RJ (22°20′30.45′′S; 43°07′44.51′′W), following the methods for collecting, handling and rearing fire ants in the laboratory as described by Banks et al. ( 1981 ).
The venom apparatuses were dissected under a stereomicroscope with fine tweezers from cold-anesthetized ants into a droplet of 0.09% saline solution and were transferred into an eppendorf tube with Dietrich's solution (900 ml distilled water, 450 ml 95% ethanol, 150 ml 40% formaldehyde, 30 ml acetic acid). Some venom apparatuses were dissected and placed in a droplet of saline to be analyzed directly under a stereomicroscope without fixing. Digital pictures of these were taken with a Sony Cybershot digital camera directly attached to the ocular lens. The following procedures were completed about 24h later. | Results
The venom apparatus of S. saevissima was a sac-like reservoir with two tubular filaments located at the distal end of the gaster ( Figure 1A ). The whitish venom reservoir (about 754 μm long × 362 μ m wide) was slightly transparent with a rugous surface. The convoluted gland had a faint yellowish hue that could be seen in the interior. The free filaments were delicate, semi-transparent and about 435 μm long ( Figure 1A ). The basal end of each filament was attached to the reservoir, and the apical end was situated freely in the body cavity. The free filaments were internally continuous with the convoluted gland ( Figure 1B ).
At the base of the filaments on the venom reservoir, there were abundant intruding trachea ( Figure 1B , 2A ). The ultrastructure of the reservoir wall is shown in Figure 2B . The ultrastructure consisted of a soft tissue of sparse irregular cells with small ovoid nuclei, some endoplasmic reticulum, and a few vesicles. This tissue was surrounded on both sides by a tunica propria of variable width completely lined with a continuous 1 μm-thick cuticle ( Figure 2B ). In Figure 2A , the outer cuticle has been torn in some regions during the processing of the sample, revealing the rugous surface of the tunica propria lying underneath.
The convoluted gland was a delicate, semi-transparent, yellowish mass inside the venom reservoir. Interestingly, when some portion of the gland was gently pulled with a fine forceps, it continuously uncurled as a long, apparently unbranched, sinuous, semi-transparent thread (not shown). In Figure 3A , it has been completely removed from the venom reservoir, showing its irregular surface that was more transparent and delicate at the base of the free filaments. This particular region will be here referred to as the “intermediary zone.”
The convoluted gland was roughly shaped like a brain and occupied much of the internal volume of the venom reservoir ( Figure 3B, Figure 3C ). The convoluted gland is a prolongation of the free filaments.
Through ultrastructure, the convoluted gland was composed of an intertwined mass of class III gland cells (cell complexes described by Noirot and Quennedey ( 1974 ) as bicellular units of closely associated secretory and duct cells), sinuous internal ducts, and tracheoles of various diameters ( Figure 4 ). It was also lined with a continuous dark cuticle, and there were big vesicles with secretion ( Figure 4A, B ). It was difficult to discern between the two cell types of the cell complex because they were similar and the limits were irregular, but the duct cells were typically abundant in mitochondria, and irregularly shaped with roughly spherical nuclei ranging 1–3 μm in diameter ( Figure 4B, C ). The secretory cells were larger and more-regularly shaped, with nuclei of various shapes ranging 3– 8 μ m in size, often having markedly darker cytoplasm ( Figure 4D ). Both cell types frequently contained dark vesicles of various sizes ( Figure 4C, D ), within some of which traces of organelles could be seen (not shown), suggesting that some of these vesicles were some type of lysosome. Both cell types presented nuclei with different degrees of cromatin condensation, and they usually contained a few smaller vesicles and endoplasmic reticulum (not shown). Neither golgi complexes nor rugous enoplasmic reticula were observed. Inside the convoluted gland, duct cells were more abundant than secretory cells. Secretory cells presented end apparatuses (invaginated spaces lined with microvilli linking ductules to secretory gland cells as defined by Noirot and Quennedey ( 1974 )) ( Figure 4A, B, C ). Tracheoles of various diameters were sporadically observed ( Figure 4D ), and the sinuous ducts (of irregular shape and calibres) were abundant in the convoluted gland ( Figure 4C, D ).
Some ducts had electro-dense material inside ( Figure 4D) .
The intermediary zone was the delicate semitransparent zone between the convoluted gland and the free filaments; it was positioned externally to the venom reservoir, and it was generally similar in cellular organization to the convoluted gland (compare Figure 3C with Figure 5A, B ). In this intermediary region, the ducts were much more abundant, but neither end apparatuses nor tracheoles were observed. This suggests that it is mainly composed of duct cells. The duct cells of this region were markedly abundant in mitochondria and dark vesicula, which tended to form clusters ( Figure 5C, D ). Myellinic bodies in the cells were occasionally seen (not shown) and some lysosomes were observed ( Figure 5C ).
The free filaments were of continuous width and had a smooth surface. They were also externally lined with a thin cuticle ( Figure 6A, B ). There was a gradual change of cellular organization from the intermediary zone to a more organized cubic epithelium surrounding a central collecting duct ( Figure 6B ). At the proximal region of the filaments, some mitochondria and vesicles were present inside the duct cells, and multilamellar inclusions ( Figure 6C, D ) and a few end apparatuses (not shown) were observed. Toward the distal portion of the filaments ( Figure 7A ) the cubic cells of the epithelium became gradually larger and more abundant. They had clearer cytoplasm, few small mitochondria and large round nuclei with well-defined borders ( Figure 7B ). Again, no ribosomes or golgi complexes were observed. Ducts were less abundant, and, consequently, few duct cells were observed ( Figure 7A, B ). No tracheoles or end apparatuses were found in this region. At the tip of the free filaments, these cubic cells were predominant. The detail of a nucleus of one of these cells is presented in Figure 7C , where a vesicle of endoplasmic reticulum can be seen. | Discussion
The general aspect of the venom apparatus of this species is similar to what was described for S. invicta and S. richteri (Callahan et al. 1956; Billen 1990 ), but markedly different from those described for ants of other genera (Schoeters and Billen 1995; Ortiz and Camargo-Mathias 2003; Nunes and Camargo-Mathias 2005 ; Ortiz and Camargo-Mathias 2005). The lack of muscle fibers associated with the venom reservoir indicates that the propelling force for the venom to be injected must be provided by a strong contraction of the gaster. As a consequence, the venom reservoir would have to be a relatively resistant structure because of the soft internal tissue and tunica propria within the continuous outer cuticle.
The fact that the convoluted gland is formed by a single, greatly-coiled, long duct forming a mass inside the venom reservoir agrees with the description of some other ants by Schoeters and Billen ( 1998 ), but it is radically different from the proposed model of this gland as illustrated in Billen ( 1990 ). The proposed model in Billen ( 1990 ) suggests that the venom gland of S. invicta is strikingly different from that of S. saevissima. The convoluted glands in the S. saevissima specimens were never immersed completely in the venom reservoir, as was shown in Billen ( 1990 ), where the proposed model entirely lacked an external intermediary zone. Some glands of S. invicta were dissected and observed directly confirming that the general disposition of the apparatus was similar to that of S. saevissima and to what was described for S. richteri by Callahan et al. ( 1959 ). The convoluted gland was composed of a single, long convoluted tube, without the side ramifications of the collecting duct proposed by the model in Billen ( 1990 ).
There were differences between these results and the findings of Callahan et al. ( 1959 ). These authors repeatedly illustrated the convoluted gland inside the venom reservoir of S. richteri as roughly elliptical, while the shape of this gland in these sections resembled that of a brain or mushroom. In their illustrations of the venom gland, Callahan et al. ( 1959 ) described and illustrated, in detail, the internal organization of the various parts of the venom apparatus. The cellular disposition in the free filament cells was similar to the present observations, but the cellular nuclei in the free filaments of S. saevissima appeared to be much bigger than the nuclei of the secretory cells of the convoluted gland and intermediary zone. The drawings of S. richteri in Callahan et al. ( 1959 ) indicate the opposite. Moreover, the main collecting duct in the free filaments was represented in the drawings of Callahan et al. ( 1959 ) as a clear and continuous tube inside the free filaments, while the same duct inside the filaments of S. saevissima seemed markedly narrow and sinuous, even difficult to detect in some sections. Lastly, the free filaments of the venom gland of S. richten were much longer than those observed for S. saevissima, although they had roughly the same diameter. As these traits were repeatedly illustrated by Callahan et al. ( 1959 ), these differences should be directly verified. For this study, there were no readily obtainable S. richten workers. If these differences prove to be discernible among different fire ant species, they may be of some utility to systematics and taxonomy. It should be noted that cellular differences in size might reflect differences in physiological status; thus these should be considered with caution in comparative studies.
As mentioned by Billen ( 1990 ), the venom of these ants is composed generally of piperidine alkaloids (see also Brown and Heddle 2003 ) and has very low protein content. This was reflected in the absence of granular endoplasmic reticulum in the cells of the venom apparatus. Mitochondria, however, were abundant ( Figures 4B , 5B , 6B ; Billen 1990 ), as were vesicles ( Figure 4A ), thus indicating the intense production of compounds and metabolism within this organ.
The fine cellular structure of the venom apparatus and the distinct differences in tissue organization of the various parts, e.g. the intermediary zone and the free filaments, likely reflects specialization of the secretory activity of each region. Most of the passage of synthesized substances into the convoluted duct probably takes place inside the convoluted gland, where end apparatuses were markedly abundant. Most of the synthesis was observed in the intermediary zone, and some was observed in the convoluted gland. The tightly intertwined duct was described by Callahan et al. ( 1959 ) as presenting only one discharging exit to the venom reservoir. Therefore, some changes should occur with the collected products before entering the venom sac.
The semi-obstructed ducts observed may be correlated with the observations made by Callahan et al. ( 1959 ), in which the venom had crystallized inside the ducts in some regions, possibly clogging the final exit duct. The multilamellar inclusions observed ( Figure 6C ) could be correlated with the observations of Callahan et al. ( 1959 ) where some cells plasmolyzed in the venom gland, possibly as a consequence of this duct obstruction in the convoluted gland. This may have something to do with possible biochemical changes occurring inside the long duct. The multilamellar inclusions ( Figure 6C ) were found inside the duct cells, suggesting that such cells may be short lived, possibly because of the intensity and nature of their metabolic activities and the toxic nature of their secretions. Multilamellar inclusions were also observed previously by Billen ( 1991 ) in ant secretory glands and end apparatus, and the author suggested that those could be products of secretion, possibly in association with lipidic compounds. These inclusions may be correlated with the function of the long convoluted duct and possibly with extracellular alterations to the venom secretions, thus their true nature would credit deeper investigation.
The results suggest that the venom apparatus is composed of simple partitioned structures that produce different compounds. The composition of the electron-dense vesicles inside the duct cells of the convoluted gland and intermediary region is unclear, but some remains of cellular materials were noticed inside some of them (e.g. membranes), thus some could actually be lysosomes. Those vesicles probably do not carry venom secretions, because they are much more eletron-dense than the contents of the ducts, the venom reservoir and the end apparatuses. Additional histochemical studies are necessary to help understand those structures and more clearly elucidate the function of the apparatus as a whole.
The results suggest that most secretions are produced directly by the venom duct cells, especially those of the intermediary zone and those in the convoluted gland. There seems to be little metabolism in the free filaments, and no substances seem to be produced by the reservoir at all.
The differences (i.e., general aspect and length of the free filaments) observed between the venom apparatus of S. saevissima and that of the other fire ant species were only slight; therefore, they will likely be of little use in taxonomy. | A group of 13 species of the genus Solenopsis is markedly difficult to assess taxonomically, although they are of considerable economical and medical importance in some countries where some of them were introduced. These ants are aggressive and their venomous stings can be very allergenic. The venom apparatus has been described in fine detail for only two of these species, and differences in this structure among the different species might prove useful as taxonomic characters. The venom apparatus of Solenopsis saevissima Smith (Hymenoptera: Formicidae) is herein described with the aid of light and electron microscopy techniques, and compared to that of S. invicta and S. richteri. The cellular organization of the different parts present differences that suggest functional specialization. In general, the different tissues were abundant in vesiculae and mitochondria, but presented little endoplasmic reticulum and few ribosomes, probably because they produce little protein. The length of the free filaments of the venom gland and the width of their internal ducts seems to vary from what was described for S. richteri, but this may be of little use to taxonomy.
Keywords | Samples for optical microscopy
Ten venom apparatuses were dehydrated with a graded ethanol series and placed in paraffin blocks, which were cut into 7 μm sections and later stained with haematoxylin and eosin for analysis under an optical microscope (Zeiss Axiostar, www.zeiss.com ). Digital pictures of the cuts were taken with a Sony Cybershot digital camera directly placed over an ocular lens.
Samples for scanning electronic microscopy (SEM)
Ten venom apparatuses were rinsed thrice with 0.1 M sodium cacodylate buffer (pH 7.2), post-fixed with 1.0% osmium tetroxide for one hour and dehydrated in a graded series of ethanol, then submitted to critical-point drying with CO2. After this, the dried samples were mounted over aluminium stubs with double-faced adhesive tape and gold-covered with a Balzers MED 010 ‘sputterer’ device. These were analyzed under the Zeiss LEO 435 VP microscope at 20 kv as soon as possible.
Samples for transmission electronic microscopy (TEM)
Some Ten venom apparatuses were rinsed thrice with 0.1 M sodium cacodylate buffer (pH 7.2), post-fixed with 1.0% osmium tetroxide for two hours, and then dehydrated in a graded acetone series, embedded in ‘Spur’ resin. Once solidified, these blocks were cut alternately with a microtome in 120 nm / 60–90 nm-thick sections. The semi-thin sections were mounted over glass slides and stained by briefly heating with toluidine blue, while the thinner sections were mounted over prepared copper grids and stained with 2.5% uranyle acetate (40 min) and lead citrate (20 min) ( Reynolds 1963 ). The semi-thin sections were used for locating the areas of interest in the blocks, and then thin sections were taken and observed under a Zeiss EM-900 electron microscope at 50 kv. | Acknowledgements
We would like to thank Dr. Elliot Watanabe Kitajima from NAP/MEPA ESALQ-USP for access to his microscope facility and Dr. Maria Izabel Camargo Mathias for the help provided. We are also thankful for the constructive comments of two anonymous reviewers and the suggestions of the editors that greatly improved the manuscript. The first author was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant #2006/02115-6. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by CEIS or UNESP of any product or service to the exclusion of others that may be suitable.
Abreviations
scanning electron microscopy;
transmission electron microscopy | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Mar 23; 10:24 | oa_package/81/8b/PMC3014722.tar.gz |
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PMC3014723 | 20578889 | Introduction
A common problem facing entomologists and ecologists working with invertebrate communities is dealing with the sheer number of invertebrates usually associated with most invertebrate sampling techniques. Most field sampling techniques generate samples with hundreds to thousands of invertebrates ( New 1998 ), and investigators are faced with the daunting task of processing samples in the laboratory. This process usually includes sorting invertebrates from debris, and then counting and identifying them to the desired taxonomic level. Thus, the laboratory processing of invertebrate samples associated with community ecology and biodiversity studies is costly and time consuming. One solution to this problem is to subsample, whereby investigators process and identify a random portion of the sample ( Vinson and Hawkins 1996 ).
Most research on subsampling techniques for invertebrates has been conducted in the context of aquatic biomonitoring studies, which use macroinvertebrates to assess the health or biological integrity of aquatic ecosystems (e.g., Courtemanch 1996 ; Walsh 1997 ; Doberstein et al. 2000 ; Ostermiller and Hawkins 2004 ). Because of the extensive use of aquatic macroinvertebrates as bioindicators of stream quality, and the large numbers of invertebrates associated with these samples, the use of subsampling techniques in this field is widespread. For example, a survey conducted by Carter and Resh ( 2001 ) showed that 74% of the methods used by U.S. state agencies employed subsampling techniques in the laboratory, and the standard operating procedure within the US Environmental Protection Agency's Rapid Bioassessment Protocols includes laboratory subsampling ( Barbour et al. 1999 ).
In contrast to research on subsampling techniques for aquatic invertebrates, few studies have examined subsampling techniques for terrestrial invertebrates (see Corbet 1966 , for an exception). This is true, even though terrestrial field techniques, like aquatic ones, can collect large numbers of invertebrates ( Corbet 1966 ; New 1998 ). Yet with the growth of fields such as conservation biology and applied ecology, the number of studies examining terrestrial invertebrate biodiversity has increased rapidly. Recent studies in ecosystems ranging from forests to grasslands have involved collecting thousands to tens of thousands of invertebrates, even with relatively little sampling effort in the field (e.g., DeBano 2006 ; Brosi et al. 2007 ; Hilt et al. 2007 ; Wenninger and Inouye 2008 ; Kennedy et al. 2009 ). Laboratory processing of such samples is costly and time-consuming, and the common practice of counting all terrestrial invertebrates collected in samples limits the number of ecological and biodiversity studies that can be undertaken, which, in turn, effectively limits knowledge in these areas. Therefore it is crucial to develop a standard subsampling strategy for terrestrial invertebrates. In this study, the effectiveness of a standardized subsampling technique that would be simple, efficient, and effective in describing basic attributes of terrestrial invertebrate communities was investigated. The specific objectives of this study were to: 1) develop an apparatus specially designed for subsampling invertebrate samples collected with two common terrestrial field techniques, pitfall traps and pan traps; 2) investigate the accuracy of a fixed area subsampling method in estimating the total abundance of all invertebrates in a sample; and 3) determine the method's accuracy in estimating total taxonomic richness at the order or family level. | Materials and Methods
Terrestrial invertebrate samples from plastic pan traps (55 × 37 × 15 cm) were collected in the summers of 2006 and 2007 in riparian areas of northeastern Oregon, and samples from 550 ml pitfall traps were collected in the summer of 2007 from grassland sites in the Zumwalt Prairie in northeastern Oregon. Both types of traps were filled with soapy water and left open for one week The contents of traps were poured through a sieve with a 500 μm mesh size in the field and samples were stored in 75% alcohol until processed in the laboratory.
A subsampling apparatus was constructed using a plastic plate with a metal frame ( Figure 1 ). The plastic plate formed the subsampling arena and consisted of a turntable or “Lazy Susan” plate (MadeSmart Housewares Inc., www.madesmart.com ). A divided metal frame that fit inside the turntable was built from thin, scrap metal strips (6 × 1 mm). The outer diameter of the sampling arena was 25.4 cm and the inner diameter was 22.9 cm. The metal frame was built to fit inside the subsampling arena, and had 45 complete cells, with each cell measuring 2.54 × 2.54 cm (6.45 cm2). The total area inside of the plate was 412 cm2, or the equivalent to 63.6 subsampling cells. For simplicity, subsampling was limited to complete cells. The sampling plate was placed on a three-wheel dolly (Shepherd Hardware Products LLC, www.shepherdhardware.com ) to facilitate easy movement of the plate under a stereomicroscope during the subsampling process.
To subsample, the following technique was used. Each sample was poured into a sieve with a 4 mm mesh size to remove large specimens, which were retained in the sieve. The portion of the sample that passed through the 4 mm mesh sieve was retained by a 0.02 mm mesh sieve. Large specimens were counted and identified, and the time taken for this process was recorded. The remaining sample was poured into the subsampling plate and dispersed with a small brush to provide an even distribution of invertebrates inside the subsampling plate. The subsampling frame was then placed inside the plate and invertebrates in 16 randomly selected cells (25% of the total area of the plate) were counted and identified, typically to order or family. The amount of time taken to count and identify invertebrates in each cell was also recorded. After 16 subsamples were taken, a quick scan was conducted of the remaining sample on the plate for individual taxa (to the level of order or family) that had not been found during sorting of the large invertebrates or in any of the 16 subsamples. The presence or absence of these taxa was recorded and used to calculate taxa richness. Each of the remaining individuals in the sample were then counted and identified to obtain the true number of individuals and taxa richness in the sample. The time necessary to complete processing of the entire sample was recorded. Three individuals, each with extensive experience in processing samples from the two studies, were involved in processing both types of samples. There were no obvious biases in the time taken or accuracy of identification among individuals.
To obtain an estimate of the total number of individuals in a sample, and the number in each taxon based on the subsampling effort, the average number of individuals per cell was calculated for 1–16 subsamples. That number was multiplied by 63.6 cells per plate. That estimate was divided by the actual number in the sample (minus the number of large specimens removed in the first phase) to obtain a “percent accuracy” score. Percents under or over 100%) indicate underestimates and overestimates, respectively.
To investigate whether the effectiveness of the subsampling method varied with the total number of individuals in the sample, samples were classified into three general categories based on overall abundance of individuals: low abundance samples had < 250 individuals, moderate abundance samples had 250–500 individuals, and high abundance samples had > 500 individuals. In each abundance category (low, moderate, and high), 10 samples were examined for each type of sampling method (pitfall and pan traps).
To compare the means of abundance and richness, 95% confidence intervals were used for estimates derived using from 1 to 16 subsamples and the means of those variables after processing the entire sample. Nonoverlapping confidence intervals indicated statistically significant differences. The mean time spent processing samples with 10 and 16 subsamples was compared with the mean time spent processing the entire sample using analysis of variance (ANOVA). Separate analyses were conducted for low, moderate, and high treatments for pitfall and pan traps. Means that were significantly different at α = 0.05 were compared using a least significant difference (LSD) test. Means in the text are reported ± one standard error. | Results
Pan traps
A total of 27,663 invertebrates were counted in the 30 pan trap samples. The number of individuals found in low abundance samples ranged from 122 to 237 invertebrates, with a mean of 178 ± 12; moderate abundance samples ranged from 286 to 375 invertebrates, with a mean of 336 ± 10; and high abundance samples ranged from 676 to 5,337 invertebrates, with a mean of 2,193 ± 514 individuals. After taking 16 subsamples, the number of invertebrates in low and moderate abundance samples was overestimated by less than 10% ( Figure 2a, b ). The number of invertebrates in high abundance samples was estimated even more accurately; after 16 subsamples, accuracy was 101% ( Figure 2c ). There was no appreciable improvement in the accuracy or precision of abundance estimates for low, moderate, or high abundance pan trap samples associated with subsampling more than 10 cells (or 16% of the area in the plate) ( Figure 2a, b, c ).
The taxa found in pan traps are listed in Table 1 . Taxa richness of pan trap samples corresponded to the size of the sample; mean taxa richness in low, moderate, and high abundance samples was 13.6 ± 0.9, 15.5 ± 0.9, and 18.7 ± 0.6, respectively. Initially, the percent taxa richness detected rapidly increased with increasing number of subsamples, but the rate of increase declined after taking approximately eight subsamples ( Figure 3a, b, c ); on average, less than two additional taxa were detected in samples after processing subsamples 9–16. The quick scanning procedure detected one or two more taxa than found after subsampling all 16 cells. On average, using the three-phase protocol and subsampling all 16 cells detected 82% of the taxa in low abundance samples, 90% of the taxa for moderate abundance samples, and 93% of the taxa for high abundance samples ( Figure 3a, b, c ).
Of the 30 taxa identified in pan traps, 14 taxa were common (found in more than 50% of all 30 samples, Table 1 ). Only two of these common taxa, Formicidae and adult Trichoptera, were missed in more than 15% of the samples. Only four relatively rare taxa were not detected by the subsampling technique in 50% or more of the samples in which they were present ( Table 1 ).
Pitfall traps
A total of 12,195 invertebrates were counted in the 30 pitfall trap samples. Although low and moderate pitfall trap samples had similar numbers of invertebrates compared to pan traps, high abundance pitfall samples contained fewer invertebrates than high abundance pan trap samples. Number of invertebrates ranged from 93 to 164 for low abundance samples, with a mean of 131 ± 8; from 314 to 384 for moderate abundance samples, with a mean of 354 ± 8; and from 504 to 813 for high abundance samples, with a mean of 662 ± 33. The number of invertebrates in low abundance pitfall traps was overestimated by the subsampling procedure by almost 20% ( Figure 4a ).
However, estimates of the number of individuals in moderate and high abundance samples were highly accurate; the accuracy of estimation for both types of samples was approximately 100% after taking 10 subsamples ( Figure 4b, c ). There was no appreciable improvement in the accuracy or precision of abundance estimates for low, moderate, or high abundance samples associated with sampling more than 10 cells ( Figure 4a, b, c ).
Similar to pan trap results, taxa richness for pitfall samples increased with increasing numbers of subsamples, but the rate of increase declined after taking approximately eight subsamples ( Figure 5a, b, c ). On average, processing subsamples 9–16, quick scanning, and complete processing of the samples each added approximately two additional taxa to the total taxa richness. On average, using the three-phase protocol and subsampling all 16 cells detected 91% of the taxa in low abundance samples, 87% of the taxa for moderate abundance samples, and 89%) of the taxa for high abundance samples ( Figure 5a, b, c ). Taxa richness found after complete processing corresponded to the size of the sample; mean taxa richness in low, moderate, and high abundance samples of pitfall traps was 15.4 ± 0.9, 18.6 ± 1.2, and 23.7 ± 1.0, respectively.
Of the 43 taxa identified in pitfall traps, 21 taxa were common (i.e., found in more than 50% of all 30 samples, Table 1 ). Six of these taxa (Cercopidae, Aphidae, spiderlings, Biphylidae, Machillidae, and Lepidoptera larvae) were missed in more than 15% of the samples. Three taxa, a relatively rare Auchenorrhyncha taxon, and Collembola and Curculionidae, were not detected by the subsampling technique in more than 50% of samples.
Time savings associated with subsampling
The first and third phases of the subsampling procedure are the least time consuming ( Table 2 ).
The first phase (separating, sorting, and identifying large invertebrates from samples) took, on average, less than 10 min for pan trap samples and less than 12 min for pitfall trap samples, with larger samples taking more time for this phase ( Table 2 ). The third phase (quick scanning) took, on average, 6–7 min for pan trap samples and 4 min for pitfall trap samples, and showed little to no variation with respect to sample size ( Table 2 ).
The second phase (subsampling individual cells) was the most time-consuming step and was more variable with respect to sample type and size. For pan trap samples, the second phase for low and moderate abundance samples required approximately the same amount of time to be processed (16–38 min for sampling 10–16 cells; Table 2 ). However, the second phase for high abundance pan trap samples required approximately a four-fold increase in time (58–93 min for 10–16 cells) compared to low and moderate abundance samples. The entire three-phase subsampling procedure took 38–109 min for 16 cell counts and 28–74 min for 10 cell counts ( Table 2 ). This is compared to 94–383 min for counting the entire sample. The time required to count the entire sample was significantly greater than the time required to process samples using 10 or 16 subsamples for all size categories ( Table 2 ). Taking 16 subsamples saved, on average, approximately 1 hour per sample for low and moderate abundance pan trap samples and more than 4 hrs per sample for high abundance pan trap samples, compared to complete counting of the entire sample. An additional 10–35 min were saved per sample by taking 10 subsamples instead of 16 ( Table 2 ).
For pitfall traps, the first phase for low and moderate abundance samples required 9–22 min and high abundance samples required 22–35 min for sampling 10–16 cells ( Table 2 ). The entire three-phase subsampling procedure took 22–52 min for 16 cell counts and 17–39 min for 10 cell counts ( Table 2 ). This is compared to 37–117 min for counting the entire sample. As with pan traps, the time required to count the entire sample was significantly greater than the time required to process samples using 10 or 16 subsamples for all size categories ( Table 2 ). Taking 16 subsamples saved, on average, approximately 15–30 min per sample for low and moderate abundance samples and over an hour per sample for high abundance pitfall traps compared to complete counting of the entire sample. An additional 5–13 min were saved per sample by taking 10 subsamples instead of 16 ( Table 2 ). | Discussion
A formidable challenge faced by investigators of terrestrial invertebrate ecology and biodiversity is processing dozens to hundreds of samples, each with potentially hundreds to thousands of individuals. The time involved in processing these samples makes many largescale studies of terrestrial invertebrate communities cost-prohibitive. Aquatic invertebrate ecologists face the same challenge and have developed subsampling techniques designed to reduce the time required to process large samples of invertebrates while maintaining accuracy in estimates of abundance and taxa richness ( Vinson and Hawkins 1996 ; Walsh 1997 ; Somers et al. 1998 ; Doberstein et al. 2000 ). In contrast, little information is available relative to the effectiveness of subsampling techniques for terrestrial invertebrate samples including descriptions of an effective subsampling apparatus and laboratory technique and data on the precision, accuracy, and time-savings associated with such a technique. We are aware of only one study that examined a form of laboratory subsampling for terrestrial invertebrates; Corbet ( 1966 ) described a technique used to estimate abundance of large samples of Trichoptera adults collected with light traps. His technique was aimed primarily at estimating changes in abundance in common Trichoptera species. He made no comparisons of how well his technique estimated the true abundance or taxa richness of the larger sample, and he presented no data on time savings of subsampling.
The results of this study illustrate how a threephase subsampling technique that involves (1) retaining and sorting large specimens, (2) taking random subsamples using a specially designed subsampling apparatus, and (3) quick scanning of the remainder of the sample can be effectively used to address research questions primarily concerned with terrestrial invertebrate abundance (number of individuals) and/or questions of broad taxa richness. Importantly, this subsampling method resulted in significant time savings with little compromise in the accuracy of abundance and taxa richness estimates for moderate and high abundance samples. In this study, 60 samples, which varied in abundance from 93-5,337 individuals each, were examined from pan and pitfall traps. Complete counting of high abundance samples from pan traps took approximately 6.4 h, and the largest samples required more than 12 h to process the entire sample. On average, more than 4.5 h of processing time per sample was saved when the subsampling method was used on these samples. Also, significant time savings of 1–1.5 h were associated with subsampling low and moderate abundance pan trap samples. Subsampling pitfall traps also resulted in time savings, although the amount of time saved for low and moderate pitfall samples was less than it was for pan traps. Nevertheless, subsampling high abundance pitfall trap samples resulted in a substantial decrease (> 1 hour per sample) in processing times.
It is important to note that these are time savings associated with the processing of individual samples, and thus must be interpreted in the context of the average number of samples associated with a typical study. Frequently, studies examining questions of ecological and conservation interest can easily involve hundreds of pitfall and/or pan traps, making the potential for indepth studies virtually impossible. For example, in this study, pan trap samples were taken from a two-year study that involved 14 riparian areas sampled eight times each year. Each site had four pan traps, resulting in a total of 896 pan trap samples. If one-third of these samples were low abundance, one-third were moderate abundance, and one-third were high abundance and the entire samples were processed, it would take 1.4 work years to process these samples to order or common families. This estimate does not include other time-consuming components of processing such as initial sample preparation, recording, labeling, and further identification. Using the subsampling technique suggested here for moderate and high abundance samples (and using whole counts for low abundance samples) would take only 0.43 work years (or 31%) as long) for the example given above. These time savings will change proportionally to the ratio of moderate and high abundance samples.
An important factor to weigh against time savings associated with a subsampling procedure is its accuracy. This study showed that the subsampling technique estimated abundance relatively accurately and precisely for moderate and high abundance samples. However, the abundance of invertebrates in low abundance pitfall trap samples was overestimated by approximately 20%. This margin of error is fairly large and the time savings were relatively small for low abundance samples; therefore, subsampling low abundance samples is not recommended.
The subsampling technique also appeared to provide a good estimate of broad scale taxa richness. All three-phases of the sorting process -- retaining and sorting large specimens, taking random subsamples, and quick scanning of the remainder of the sample -- provide information for taxa richness estimates. The first phase is important in detecting large, sometimes rare, taxa, and it also aids in the uniform distribution of the remaining invertebrates inside of the subsampling tray. Many aquatic macroinvertebrate protocols have a similar step (often called a “large-rare search”) for the purpose of improving estimates of taxa richness (e.g., Gerritsen et al. 2000 ; Carter and Resh 2001 ; King and Richardson 2002 ). The third phase, quick scanning, aids in identifying small, relatively rare taxa that are an important component of taxa richness. This step is not used in aquatic macroinvertebrate subsampling techniques because the amount of substrate associated with the typical benthic macroinvertebrate sample is large, making a visual scan of this type unproductive. In contrast, samples from pan and pitfall traps have relatively little substrate, and taxa not found in the second phase can be detected in the third phase and used to improve taxa richness estimates. The combination of these three phases resulted in, on average, less than two taxa being missed using the 16 cell subsampling procedure compared to the whole counting process. Except for low abundance pan trap samples, in which only 82% of the taxa were detected, the three-phase sampling technique detected 87–93%) of the taxa present in a sample.
Another objective of this study was to determine how many subsamples maximize the information obtained from each sample while minimizing the time involved in processing. High accuracy for moderate and high abundance samples was achieved after even as few as six subsamples. In general, the accuracy of abundance estimation did not change substantially after 8–10 subsamples were taken for both pan and pitfall trap samples. On average, taxa richness increased rapidly during the first 8 subsamples but the rate of increase slowed when taking 9–16 subsamples. Reducing the number of cells subsampled may result in a less accurate estimate of taxa richness; on average, two additional taxa were detected when taking 9–16 subsamples. However, it is likely that the missing taxa would be detected during the quick scanning procedure. Nevertheless, in studies where detecting small differences in taxa richness are important, taking up to 16 subsamples is recommended. The need for accuracy must be weighed against the potential time-savings. In this study, taking 10 subsamples instead of 16 saved between 8–36 min for pan trap samples and 3–9 min for pitfall trap samples ( Table 2 ).
This research also aimed to determine whether the technique was associated with any biases in taxa detection, such that certain taxa were more prone to be missed in the subsampling process than others. In general, taxa that were relatively rare tended to be missed more often than common taxa. For example, although Trichoptera larvae were not detected with the subsampling technique in any of the pan trap samples, they were only present in three of the 30 samples. However, a few taxa were fairly common and were frequently missed in pan trap samples, including Cercopidae, Aphidae, and spiderlings, which were not detected in 29–36% of the samples ( Table 1 ). Two factors probably contributed to the tendency to miss these taxa — size and body color. Individuals of these taxa are not only small, but are also light colored, and thus were difficult to see against the white background of the sampling tray. Thus, when using subsampling techniques, particular care should be taken when dealing with small specimens that blend into the background. If these taxa are common or of particular interest, more effort can be taken to develop a search image for these taxa, or a different colored sorting tray (e.g., black) can be used so that the taxa are more noticeable.
Another question of interest is whether the effectiveness of the subsampling method varied depending on whether the sample was collected with pan traps or pitfall traps. There were several important differences between the two types of samples. Pitfall trap samples generally had fewer invertebrates than pan trap samples, especially for high abundance samples. There were also differences in the size and condition of invertebrates in the two types of samples. Pitfall traps contained, on average, larger and better preserved invertebrates than pan traps. In addition, because the pan traps were fairly large and received a relatively high amount of sunlight, many samples had algal growth, which tended to entangle invertebrate specimens. All of these factors resulted in longer processing times for pan traps compared to pitfall traps, and thus, the time savings associated with subsampling pitfall samples was reduced compared to pan traps. In general, then, longer processing times may be needed when a collecting method results in smaller, more fragile, and/or algal entangled invertebrates, and time savings associated with subsampling in those cases can be substantial. Another difference between the two types of samples was that more taxa were missed during subsampling of pitfall trap samples as compared to pan trap samples. This pattern may be, in large part, due to the fact that taxa in pitfall samples were identified to a higher taxonomic resolution than taxa in pan trap samples. Pitfall traps also contained larger amounts of substrate (e.g., sand, silt, and other debris) which might obscure small taxa.
There are limitations to the use of this technique. The method was only applied to common forms of terrestrial sampling that result in collections with specimens preserved in liquids. Liquid facilitated the even distribution of invertebrates inside of the sampling plate. Even distribution of samples inside of the sampling tray was very important for accurate abundance estimation. Further tests are needed to examine how the method might be modified to accommodate samples that are not preserved in liquid. The size of the tray could also be adjusted, depending on the typical sample size. For example, a larger sampling tray and divided metal frame could be used to hold extremely large samples. | Significant progress has been made in developing subsampling techniques to process large samples of aquatic invertebrates. However, limited information is available regarding subsampling techniques for terrestrial invertebrate samples. Therefore a novel subsampling procedure was evaluated for processing samples of terrestrial invertebrates collected using two common field techniques: pitfall and pan traps. A three-phase sorting protocol was developed for estimating abundance and taxa richness of invertebrates. First, large invertebrates and plant material were removed from the sample using a sieve with a 4 mm mesh size. Second, the sample was poured into a specially designed, gridded sampling tray, and 16 cells, comprising 25% of the sampling tray, were randomly subsampled and processed. Third, the remainder of the sample was scanned for 4–7 min to record rare taxa missed in the second phase. To compare estimated abundance and taxa richness with the true values of these variables for the samples, the remainder of each sample was processed completely. The results were analyzed relative to three sample size categories: samples with less than 250 invertebrates (low abundance samples), samples with 250–500 invertebrates (moderate abundance samples), and samples with more than 500 invertebrates (high abundance samples). The number of invertebrates estimated after subsampling eight or more cells was highly precise for all sizes and types of samples. High accuracy for moderate and high abundance samples was achieved after even as few as six subsamples. However, estimates of the number of invertebrates for low abundance samples were less reliable. The subsampling technique also adequately estimated taxa richness; on average, subsampling detected 89% of taxa found in samples. Thus, the subsampling technique provided accurate data on both the abundance and taxa richness of terrestrial invertebrate samples. Importantly, subsampling greatly decreased the time required to process samples, cutting the time per sample by up to 80%. Based on these data, this subsampling technique is recommended to minimize the time and cost of processing moderate to large samples without compromising the integrity of the data and to maximize the information extracted from large terrestrial invertebrate samples. For samples with a relatively low number of invertebrates, complete counting is preferred.
Keywords | Recommendations
In general, the cost and impracticality of processing samples that contain several thousand invertebrates leads to the need of using some type of subsampling procedure to provide an unbiased representation of a larger sample ( Barbour and Gerritsen 1996 ). Using a subsampling apparatus, as described here, is recommended to divide the entire sample into equal subsamples. Subsamples should be randomly selected. After large invertebrates and plant material are removed, the sample should be evenly distributed inside of the sampling tray by agitating and detaching entangled invertebrates using a small brush. This step is particularly important in order to assure uniform distribution of invertebrates in the subsampling tray. Counting the invertebrates in only 10 cells (i.e., ∼16% of the entire sample) provided accurate estimates of abundance and taxa richness; counting additional cells did not appear to increase precision or accuracy of abundance estimates. Whether this level of subsampling provides accurate estimates of abundance and taxa richness for terrestrial invertebrate samples collected using other techniques or collected in other locations still needs to be tested. Because abundance estimates of low abundance samples were not very accurate, subsampling samples that contain <250 invertebrates is not recommended. Thus, if the average number of invertebrates per cell is < 4 after sampling 10 cells, counting the entire sample is recommended. For samples with average densities > 4 invertebrates per cell, the total abundance of invertebrates can be estimated by multiplying by the average number of invertebrate per cell by the number of cells per sampling tray (for this apparatus it is 63.6 cells/per plate). The number of large invertebrates separated from the sample during the first phase is added to this estimate to obtain an abundance estimate for the entire sample. Taxa richness is simply calculated by adding the number of taxa in all three phases (large invertebrate separation, subsampling, and the quick scan). | Acknowledgements
We thank Abigail Arnspiger, Anne Madsen, Kimberly Tanner and Sarah Carlson for their help in field collection and laboratory processing. This research was supported by two USDA National Research Initiative grants (# 2005-35102-16305 and # 2006-35101-16572) and a grant from the Agricultural Research Foundation, a corporate affiliate of Oregon State University. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Mar 25; 10:25 | oa_package/09/6f/PMC3014723.tar.gz |
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PMC3014725 | 21234370 | 1. Introduction
Cryptosporidiosis is a zoonosis caused by the apicomplexan intracellular, extracytoplasmic coccidian parasite of the genus Cryptosporidium that can infect a wide range of animals, including man [ 1 , 2 ]. Cryptosporidium hominis (formerly C. parvum genotype 1) is human specific and maintained in human-to-human transmission cycles, while C. parvum (formerly C. parvum genotype 2) is maintained by a number of different animal reservoir host species including bovines [ 3 ]. C. parvum also causes disease in humans and is, therefore, a zoonosis transmitted from cattle to humans but in immunocompromised individuals such as children and patients with Acquired Immunodeficiency Syndrome (AIDS), the infection fulminates and might be life-threatening [ 4 ].
In cattle Cryptosporidium infections are transmitted by the faeco-oral route, and the disease is readily transmissible: oocysts persist for long periods in suitable environment [ 5 ], and low numbers of oocysts may produce infection in susceptible hosts [ 6 ]. Clinical illness and diarrhoea caused by Cryptosporidium spp. have been reported in several species of young animals including bovine calves as young as 4 days [ 7 ].
In Tanzania, Cryptosporidium spp. infections in human and livestock appear common [ 8 – 12 ]; however, systematic studies targeting young calves reared under different production system are patchy and not well documented. As a result information on disease prevalence, distribution, and impact on these future replacement stocks is inadequate and not precisely known.
This study investigated the prevalence of Cryptosporidium spp. infections amongst bovine calves in two contrasting cattle production systems of Tanga region, Tanzania. An attempt was made to investigate potential risk factors for infection in these calves that may point to methods by which infections may be controlled and/or transmission to humans limited. | 2. Material and Methods
2.1. Study Area and Population
The study was conducted on both smallholder dairy herds (≤10 graded animals of all ages, breed, and sex and intensively managed) and traditional herds (≥30 indigenous cattle of all ages and sex and extensively managed) in and around Tanga municipality. Characteristics of the study area are described in detail elsewhere [ 13 ]. The type of animals kept under smallholder units includes taurine breed (Friesian, Ayrshire, Jersey, Simmental) and crosses of these breeds with bos indicus breeds (Tanzania shorthorn Zebu, Boran, and Sahiwal). The level of taurine blood varies from 50 to 85%. Traditional herds comprise mainly Tanzania shorthorn zebu (TSHZ). Only calves up to 3 months of age were sampled. Calves are known to be susceptible and suffer the severe form infections compared to other age group [ 5 , 7 ].
2.2. Study Design and Herds Selection
This study was part of the broader cross-sectional study on zoonotic diseases and relative impact on public health conducted on the same farms between May 2003 and January 2004. During the cross-sectional study, 130 farms (105 smallholder dairies and 25 traditional herds) were selected randomly (using Epitable calculator of the Epi-info, version 6.04b) [ 14 ] from a sampling frame of 1,730 farms, comprising 12,500 animals that belonged to the Tanga dairy development programme and district livestock department databases. Recruitment of study calves was based on inclusion criteria of calves on the study farms with an age of less than three months as at 1st May 2003 through 1st January 2004. This selection criterion resulted in the recruitment of 28 smallholder dairy and 16 traditional herds comprising 117 calves. Consistent to other studies, cryptosporidiosis is considered one of the most common causes of neonatal diarrhea in cattle [ 7 , 15 ]. All calves were sampled only once (cross-sectional) during the period of May 2003 and January 2004.
2.3. Questionnaire Design and Data Collection
Information about each herd and the calves kept was collected by means of a structured questionnaire, which was completed at all the selected herds on a single visit. The questionnaire was designed to comprise mostly closed ended (categorical) questions to ease data processing, minimize variation, and improve precision of responses [ 16 ]. The questionnaire was administered using the national Swahili dialect by a veterinary department staff member, who was trained in participatory research methodologies. Important herd-, calf- and area-level data recorded included calf location (urban, periurban, or rural), age determined from birth records, type of floor in the calf house (concrete, kraal/stone, wooden) as well as level of floor hygiene or cleanness subjectively assessed as highly, moderate, or mild dirty, and source of drinking water (tap, rain water, shallow well, river, pond). At the same time, information on diarrhoea and other ill health signs (coughing, dullness, ocular and nasal discharges) in calves was as well collected.
2.4. Sample Collection and Handling
During the visit to each herd, a fresh rectal faecal sample was collected from each calf into a sterile, airtight, 10 mL plastic tube. Collected faecal samples were labelled and transported in a cool-box to local laboratories in Tanga (at least within 6 hours of collection) prior to despatch in refrigerated containers to Sokoine University of Agriculture (SUA), Morogoro for analysis.
2.5. Laboratory Analysis of Fecal Samples
Presence of Cryptosporidium spp. oocysts in faeces samples was detected using the modified Ziehl-Neelsen staining technique as described by Clarke and McIntyre [ 17 ]. Briefly, faecal smears were prepared on a microscope slide, air dried at room temperature, and fixed with absolute alcohol (methanol) for 5 minutes. Fixed smears were stained with dilute carbol fuchsin (1 : 10) for 3–5 minutes and washed with tap water. Smears were decolourised using 3% acid alcohol (3% HCL in ethanol) for 10–15 minutes then counterstained with 0.5% malachite green solution for one minute. Smear slides were washed with tap water, air dried, and then examined under the microscope at x400 magnification. Cryptosporidium spp. oocysts appear as pink to red, spherical to ovoid bodies against a green to purple background. Samples were considered positive if at least one morphologically distinct Cryptosporidium spp. oocyst was observed. Intensities of the oocysts detected based at x400 magnification were expressed as 1+ (<5 oocyst per slide), 2+ (1 to 10 oocysts per field per view) and 3+ (>10 oocysts per field per view) [ 18 ].
2.6. Biostatistical Analysis
The data were stored using Microsoft Access and analyzed by using Epi-Info version 6.04d software (CDC, 1996) [ 14 ] statistical packages. Descriptive statistics like rate, frequency, and proportions were used. Risk factors thought to be associated with the prevalence of Cryptosporidium spp. oocyst shedding (age, location, production system, source of water, house flooring system, and level of cleanness) were evaluated using Chi-square analysis (Epi-Info version 6.04d software, CDC, 1996), with P < .05 as the indicative of significance level. | 3. Results
3.1. Distribution of Risk Factors
Descriptions of herd-level and calf-level for the 117 calves sampled in the area under study are given in Table 1 . Fifty seven percent of the sampled calves consisted of the indigenous breeds. Majority of the herds (63.6%) visited were smallholders dairy managed under exclusive zero grazing. Housing flooring systems was classed as made up of stones/kraal and were often found dirty (68%) at a time of visit. Approximately equal proportions of calves were sampled from the age category of one to two months. The mean age of the 117 calves investigated was 49 days (range 7–84 days), with 70 (60%) of the calves being rural residents. None of the examined calves were diarrheic or showing any obvious ill health signs. Further details are shown in Table 1 .
3.2. Estimating Cryptosporidium spp Oocysts Infection Prevalence
The overall individual calf- and herd-level cryptosporidiosis prevalence and their respective 95% Confidence intervals [CIs] are shown in Table 2 . Calves in traditional herds had a significant lower prevalence compared to calves in smallholder herds ( P < .05). The cryptosporidiosis prevalence profiles (with 95% CI for the exact binomial proportions) by age category are shown in Figure 1 . Calves of ≥1 to ≤2 months of age had a higher prevalence than younger (≤1 month) and older calves (≤3 months) ( P < .05). Based on oocysts detection intensities, 7.3% (4/41) of the Cryptosporidium spp positives were categorized as heavy, 9.7% (4/41) as moderate, and 83% (34/41) as mild infection.
3.3. Relationship between Variables Investigated and Cryptosporidium Oocyst Infection
One variable was significantly associated with variation in prevalence to Cryptosporidium spp infection in the univariable analysis. Calves were associated with a higher risk of exposure to Cryptosporidium spp infection if they slept/or stayed at dirty floors compared to those slept at moderate to clean floors (odd ratio [OR] = 1.82, 95%CI 1.15–3.02, P = .01 for dirty floors). At herd level, however, the type of flooring in the calf housing, either concrete or dirt flooring, did not influence the cryptosporidiosis herd prevalence. Also the water source, urban tap water as compared to water from shallow wells or surface water, was not associated with the cryptosporidiosis prevalence at herd level. None of the other investigated variables were associated with Cryptosporidium spp oocyst shedding. | 4. Discussion
In this study, there was evidence that, in and around Tanga municipality, calves are infected by Cryptosporidium spp, indicating that bovine cryptosporidiosis is endemic and locally widespread. The overall herd-level prevalence of 54.5% implies the presence of the disease in majority of the herds in the study area. Regarding production systems, 63% of herds in the traditional and 50% of the herds tested in smallholder dairying system had at least one Cryptosporidium spp. oocyst shedding calf per farm. The herd-level prevalence difference observed in the two production systems was not statistically significant ( P > .05). This demonstrates the disease being important in both production systems. The recorded prevalence indicated, however, that certain herd-level factors, such as those involving close contact with unhygienic or dirty floor houses, were relatively high-risk in terms of Cryptosporidium spp infection. Some studies have shown that parasite oocysts are able to survive for extended periods in faeces and environment, and very low dose of viable oocysts can cause an infection [ 2 ]. The warm humid coastal environment of Tanga region may also favour the survival and spread of infective oocysts.
The detected prevalence (35%) of infection in the calves was relatively higher than the 16.5% reported in diarrhoeic calves under 3 months of age in Morogoro, Tanzania [ 10 ], but generally lower than the prevalence observed in other studies of similar age range in Tanzania and elsewhere, for example, up to 62% [ 19 ] and 80% in calves in Britain [ 20 ].
The relevant data from North and East Africa, America, and from other environments similar to those found in Tanzania also indicate that bovine cryptosporidiosis is prevalent and widespread among calves populations. For example in other modified ZN screening-based investigations, prevalences of 86.7%; 38%; 18%; 17.1%, and 25% have been reported in Tunisia, Uganda, Kenya, Argentina, and Mexico, respectively [ 7 , 21 – 24 ]. The apparent variability of prevalence between geographical localities and reports may reflect differences in the levels of calf management practices employed at farm level, housing-related factors (i.e., single housed calves, cleanness of the calf sleeping places), calf-related factors at a time of sampling (diarrhoeic versus nondiarrhoeic), nature of the study (cross-sectional versus prospective longitudinal studies), and fecal screening technique used [ 25 – 27 ].
Although modified ZN remains the widely used screening test for cryptosporidiosis, the test does have limitations [ 28 – 30 ]. Therefore, our prevalence estimates are likely to be at variance with the true prevalence in calves. A wider use of ZN under field conditions is constrained by the low sensitivity, time consuming (about 30 to 45 minutes), necessitating intensive training and experience to interpret the results. Resource constraints affecting logistics and laboratory capacity were the main reasons that prohibited utilization of tests of higher sensitivities in this study. However, consistent with the results of studies by [ 9 , 19 ], the findings of this study indicate evidence of exposure to the Cryptosporidium spp. and suggest that there is a need for strict adherence to hygienic and good calf husbandry practice at farm level. It can also be recommended that other tests of higher sensitivities, that is, PCR, ELISA, should be employed in order to improve test sensitivity [ 27 , 30 – 33 ].
Sex of the calf was not associated with Cryptosporidium spp. oocyst shedding in this study, and this is in agreement with the report of [ 34 , 35 ]. The prevalence rate in middle age group (43%) showed statistically significant difference from young (27%) and older calves (31%) ( P < .05). This finding was at variance with earlier reports [ 24 , 36 ] where such age range was concluded to have no role in cryptosporidiosis epidemiology. Perhaps this assumption needs further investigation. | 5. Conclusion
In conclusion, overall animal and herd prevalence was high, suggesting that cryptosporidiosis is endemic and locally widespread disease. Calves were more likely to shed Cryptosporidium spp. positive oocysts if they were raised at dirty floor houses most likely due to the increased microenvironment for Cryptosporidium spp. oocyst survival and persistency. A further study prospective in nature, capturing seasonal variations to elucidate the magnitude of the disease (mortalities and reduced production), is desirable. Moreover, studies to understand the dynamics of transmission cycles and the genetic diversity of Cryptosporidium spp. on the farms, and to identify and if possible alter management practices that are risk factors for human infections, should be initiated and undertaken. | Academic Editor: David W. Horohov
A cross-sectional study was conducted to determine prevalence and risk factors of cryptosporidiosis in bovine from two contrasting production system in and around Tanga municipality between May 2003 and January 2004. The study populations comprised 117 calves aged ≤3 months, randomly selected from 44 smallholders dairy and traditional managed herds, respectively. Individual calf and herd-level information was collected using a structured questionnaire and feacal samples were screened for Cryptosporidium spp oocysts using the modified Ziehl-Neelsen method. Overall, 35% of the calves in the study were shedding Cryptosporidium spp oocysts, with at least one positive calf detected in 54.5% of herds. Independent risk factors for cryptosporidiosis were: age ≥1 to ≤2 months and level of cleanness of calf house floor categorized as dirty ( P < .05). Similarly an increases risk of Cryptosporidium spp infection was found in calves from smallholder dairy units compared to traditional herds ( P < .05). The finding highlights that Cryptosporidium spp is prevalent among calves in the area under study. The high prevalence of cryptosporidiosis detected in this study suggests that it may have a significant impact on livestock industry and that the close interaction between cattle and human may play a role in zoonotic transmission to humans. | Conflict of Interests
The authors declare that they have no competing interests. | Acknowledgments
The authors thank all the stockmen who participated and the livestock field staff for their very considerable support and assistance. Thanks are extended to the Director of Veterinary Service, Tanzania for permission to publish this paper. | CC BY | no | 2022-01-13 03:35:57 | Vet Med Int. 2010 Dec 22; 2010:676451 | oa_package/99/96/PMC3014725.tar.gz |
PMC3014732 | 20575740 | Introduction
Fire is one of the most common disturbances that can cause dramatic changes in species diversity ( Whelan 1995 ). Its effects depend on spatial heterogeneity, vegetation types, abiotic conditions, and human activities ( Veblen et al. 1999 ; Mermoz et al. 2005 ). The ecological impact of fires depends on their frequency, intensity, and extent, but always causes simplification of the burned habitat ( Bulan and Barrett 1971 ). Habitats that differ in vegetation structure may also suffer this simplification in different ways. Intense fires reduce the litter layer to mineral ash, and they kill all the above-ground vegetation. This produces changes in microclimatic conditions by increasing temperature and reducing humidity above ground ( Bond and van Wilgen 1996 ; Paritsis et al. 2006 ) and below ground ( Alauzis et al. 2004 ). This initial habitat simplification leads to high similarity among post-fire habitats in sites that differed structurally before fire. However, in different pre-fire habitat types, surviving organisms dictate much of the initial successional pattern and influence the competitive environment encountered by colonizers ( Turner et al. 1998 ).
In northwestern Patagonia, wildfire is a major disturbance that affects the dynamics of forests and shrublands. Fires are very frequent; it has been estimated that for the last 100 years, fire intervals have ranged from 3 to 17 years ( Veblen et al. 1999 ). Additionally, wildfires were often of high intensity and burned large areas ( Veblen et al. 1999 ). Qualitative historical descriptions from the 18 th and 19 th centuries, as well as historical photographs from the late 19 th and early 20 th centuries, indicate that extensive, severe fires occurred in all the woodland and forest types ( Veblen et al. 2003 ). The widespread occurrence of fires in this region in the late 1990s provided a rare opportunity to examine the effects of large wildfires on different communities. Numerous studies have documented post-fire vegetation changes in the Andean forest ( Veblen and Lorenz 1987 , 1988 ; Gobbi et al. 1995 ; Raffaele and Veblen 1998 , 2001 ; Ghermandi et al. 2004 ) and fire history ( Kitzberger et al. 1997 ; Veblen et al. 1999 ). However, few studies have addressed the effects of fire on insects and other arthropods, such as beetles, that contribute substantially to faunal biodiversity and play key roles in the ecosystem's dynamic ( Farji-Brener et al. 2002 ; Sackmann and Farji-Brener 2006 ). Moreover, in this region, beetles are poorly known, are highly diverse in species number and trophic functions, and present high endemism ( Morrone and Roig-Juñent 1995 ). Beetles have also proven to be useful bioindicators for environment monitoring and assessment, with their high diversity and sensitivity to environmental conditions providing a fine-grained view of ecological change ( Orgeas and Andersen 2001 ). Coleoptera are known to respond to factors such as vegetation complexity and microclimate and to conditions in the soil and litter layers ( Niemelä et al. 1993 ; Baker 2006 ), and these factors are likely to vary in post-fire succession. In addition, beetles are good colonizers, as many species can fly and others are active foragers that become abundant in post-fire sites ( Borror et al. 1992 ; Swengel 2001 ). These reasons make beetle assemblage appropriate for studying the effects of fire disturbance on insect community structures in different habitat types.
In this study case the initial response of beetle diversity to a wildfire was examined. An intense and widespread fire occurred in 1999 that affected adjacent, but different, habitat types, allowing the study of changes over time in coleopteran assemblages under different pre-fire conditions. In northern Patagonia, following the burning of shrublands and xeric open forests, resprouting shrubs often dominate post-fire regeneration, while vegetative regeneration of tree species is rare. The establishment of tree species is often facilitated by already established shrubs that operate as nurse plants ( Raffaele and Veblen 1998 ; Kitzberger et al. 2000 ). Shrubland communities are particularly well-adapted to regenerate by resprouting after fire ( Veblen et al. 2003 ). This strategy allows this system to reach its pre-fire biomass and composition in a few years, giving it an extraordinary resilience to this kind of disturbance. Because different habitats often support different beetle assemblages ( Lövei and Sunderland 1996 ), we hypothesized that the effect of habitat type, defined by pre-fire conditions, would determine distinct post-fire communities because beetle diversity is different among different habitats and years and because their successional patterns will be related to vegetation structure.
The specific aims of the study were: 1) to compare beetle abundance, richness, and assemblage composition in three adjacent habitats for three years after the fire, 2) to describe the relationship between beetle diversity and vegetation structure in these habitats after the fire, and 3) to determine whether beetle abundance and richness were associated with different early post-fire mosaics of vegetation and which of the vegetation variables (e.g. plant species richness, vegetation cover, and height) could explain successional beetle assemblages of each habitat type. | Materials and Methods
Study area
The study was conducted in the Andean forest in northern Patagonia, Argentina, in the Nahuel Huapi National Park (40° 38′ S - 72° 42′ W; 850 m above sea level)( Figure 1a ), during three growing seasons (2002, 2003, and 2004). In this region the mean minimum temperature is -2° C (July), the mean maximum is 23° C (January), and the mean annual temperature is 8° C. Precipitation is concentrated mainly in autumn and winter, and it occurs as snow, with an annual rainfall of 1700 mm ( Barros et al. 1983 ), resulting in an asynchrony between the wet season and the growing season, and there are strong summer water deficits ( Paruelo et al. 1998 ). At this latitude, mean precipitation decreases abruptly from about 4000 mm/yr on the western side of the Andes to less than 500 mm/yr, only 80 km to the east ( De Fina 1972 ). As a result, forest species composition from northwestern Patagonia changes along the precipitation gradient east to west and also along the temperature gradient associated with an increase in elevation ( Veblen et al. 1992 ). In the wetter area, the lowland rain forests are mainly dominated by the evergreen Nothofagus dombeyi (Mirb.) Oerst. (Fagales: Nothofagaceae). In the intermediate of the precipitation gradient, at low elevations, N. dombeyi forms monospecific mesic forests or mixed stands with the conifer Austrocedrus chilensis (D. Don) Pic. Serm. & Bizzarri (Pinales: Cupressaceae) at drier sites, and in the eastern region, this conifer forms relatively open woodlands. In the western and central areas, forest understory is typically dominated by dense and tall (> 2 m) populations of Chusquea culeou Desvaux (Poales: Poaceae). Tall and dense shrublands occur throughout the western to eastern precipitation gradient at sites that are not edaphically suitable for development of tall forests or that are successional communities that develop after burning of tall forests ( Mermoz et al. 2005 ). Extensive post-fire stands of tall Nothofagus and/or Austrocedrus forests are characterized by low regeneration due to the obligate seed reproduction being able to be replaced by shrubland with vigorous post-fire resprouting ( Kitzberger and Veblen 1999 ; Veblen et al. 2003 ).
In February 1999, an extensive (3821 ha) and severe wildfire affected the study site ( Figure 1b ) (“Administración de Parques Nacionales,” unpublished report “PN Nahuel Huapi Argentina: Incendios temporada 1998–1999”). The intense fire consumed all forest floor litter ( Alauzis et al. 2004 ) and killed the aboveground vegetation, leaving all standing trees killed. Three adjacent habitat types were studied, each one defined by pre-fire dominating vegetation: 1) evergreen forest dominated by N. dombeyi ( N. dombeyi forest) with a typically dense understory of C. culeou, 2) a mixed forest of the evergreen conifer A. chilensis ( A. chilensis forest) and N. dombeyi , and 3) a shrubland (shrubland community) where a diverse community of shrub species such as Schinus patagonicus , Discaria articulata and Lomatia hirsuta coexist. Replication fires were not possible because no fires with the same characteristics of severity and spread (3821 ha) were found. The study area was 7 kilometres from unburned shrubland, 10 kilometres from unburned N. dombeyi forest, and 14 kilometres from unburned A. chilensis forest. It was not possible to get unburned controls because of these distances and the heterogeneity of the landscape. Also it was not possible to get prefire controls because this fire was natural and not prescribed.
Experimental design
In the three adjacent habitat types noted above, areas were selected that were severely and homogeneously burnt in 1999. Within each habitat type, five replicate areas of 1600 m2 (40 × 40 m plots) positioned at least 100 m apart ( Figure 1c ) were delimited. These separation distances are within the range reported in previous studies ( Rykken et al. 1997 ; Koivula et al. 1999 ), so it can be assumed to provide independent information for ecological analysis of ground-dwelling arthropods avoiding spatial autocorrelation ( Moretti et al. 2004 ). Beetles may move up to 60 m by foot ( Koivula et al. 1999 ), thus the scale we used in this study was appropriate for detecting habitat selection. Sampling of the beetles and vegetation was carried out during the austral summers of 2002, 2003, and 2004 because spring and summer seasons correspond to the major activity period of arthropods in this temperate region.
Beetle sampling
Ground beetles were collected by 8 pitfall traps within each 40 × 40 m plot. Traps were placed along two transects (4 traps per transect) at 5 m intervals. Transects were placed no closer than 10 m. Each trap consisted of a 500 ml plastic cup partially filled with preservative solution (ethylene glycol, water 5:95) and buried in the soil. Pitfall traps were left open for 5 days during each January and February; this corresponded to the summer season period of thermal activity of insects ( Niemelä 1990 ). The contents of the 8 traps of two intervals (January and February) were pooled into one sample per plot per year. Samples were sorted in the laboratory. Whenever possible beetles were identified to species, otherwise they were assigned to morphospecies. The identifications were checked and modified by appropiate specialists (see Acknowledgements). Voucher specimens are held at the Laboratory Ecotono, Universidad Nacional del Comahue, Río Negro, Argentina.
Three beetle assemblage traits were analysed: abundance, species richness and composition. Beetle abundance was expressed as the total number of individuals per plot, habitat, and year. Richness was calculated by the randomization process using EcoSim ® ( Gotelli and Entsminger 2001 ). Rarefaction eliminated variation in species richness due to differences in sample size (e.g. number of beetles captured at each collecting station) by re-sampling a pool of individuals repeatedly, at random, on each habitat type ( Gotelli and Colwell 2001 ).
Vegetation sampling
To determine post-fire vegetation structure on the three habitat types over years, on each 40 × 40 m plot, 40 circular sub-plots of 80 cm radius (2 m 2 ) were installed systematically on a 1 × 1 m grid. On each sub-plot the following data were recorded: 1) total number of vascular species (richness), 2) the cover of all vascular plant species using a scale from 1–100%, and 3) maximum heights of the woody species (vertical distance above the ground). Mean vegetation cover was calculated on average of 40 subplots per plot. Plant richness was not calculated by randomization because sample size was the same for all plots and sub-plots.
Data analysis
Repeated-measures analysis of variance (ANOVA) was used to examine whether ground-active beetles responded differently over time under different habitat types ( Mead 1988 ). Two measures of beetle response were examined: total beetle abundance and rarefied beetle species richness. The analysis included years and habitat types ( N. dombeyi forest, A. chilensis forest and shrubland community) as fixed factors. Tukey tests were used for posteriori comparisons ( Sokal and Rohlf 1995 ). Analysis of similarity (ANOSIM) with Bray-Curtis similarity index was used to determine if there were significant differences in beetle assemblages during the years since the fire (2002, 2003, and 2004) and among habitat types ( N. dombeyi forest, A. chilensis forest and shrubland community). ANOSIM is a non-parametric permutation procedure applied to rank similarity matrices underlying sample ordinations ( Clarke and Warwick 2001 ), that produces a global R-statistic, which is an absolute measure of distance between groups. An R-value approaching 1 indicates strongly distinct assemblages, whereas an R-value close to zero indicates that the assemblages are barely separable. To illustrate patterns in beetle assemblage composition in relation to habitat types and years, non-metric multidimensional scaling (NMDS) ordination with the Bray-Curtis similarity index ( Clarke 1993 ) was used. Beetle species characteristics of the three habitat types and years were identified using the Indicator Value method ( Dufrêne and Legendre 1997 ). This method assesses the degree to which a species fulfills the criteria of specificity (uniqueness to a particular habitat) and fidelity (frequency of occurrence). A high indicator value (IndVal, expressed as percentage) indicates that a species can be considered characteristic of a particular habitat or site. This method can derive indicators for hierarchical and non-hierarchical site classifications and is robust to differences in the numbers of sites among site groups ( McGeoch and Chown 1998 ). Indicator values for each species were calculated based on a species abundance matrix, and Dufrêne and Legendre's ( 1997 ) random reallocation procedure of sites among site groups was used to test for the significance of IndVal measures for each species. Dufrêne and Legendre ( 1997 ) was followed in assuming a species is characteristic of a habitat if the species IndVal is > 25% and significant at p < 0.05.
Overall, to test differences in vegetation structure (plant species richness, vegetation cover and height) among habitats over time, repeated-measures ANOVA (Mead 1998) were performed including habitat types and years as fixed factors. Tukey tests were used for posteriori comparisons ( Sokal and Rohlf 1995 ). Analysis of similarity (ANOSIM) with Bray-Curtis similarity index was used to determine if there were significant differences in vegetation assemblages between years since fire (2002, 2003, and 2004) and among habitat types ( N. dombeyi forest, A. chilensis forest and shrubland community). To illustrate patterns in vegetation assemblage composition in relation to habitat types and years, non-metric multidimensional scaling (NMDS) ordination was used with the Bray-Curtis similarity index ( Clarke 1993 ).
To describe the relationship between beetles and vegetation, correlations were performed between beetle abundance and total species richness with vegetation structure parameters (richness, cover, and height). Because different vegetation types could recover at different rates and because beetles could respond to different characteristics of these habitats, the relationship between beetles and vegetation was calculated with data of the last year, 2004. Additionally, to describe the relationship between beetle and vegetation composition, a Mantel permutation test was performed with 10,000 randomizations ( Mantel 1967 ). The beetle and vegetation similarity matrixes were constructed using Bray-Curtis similarity measures per habitat type only for the last year, 2004 ( Legendre and Legendre 1998 ). To explain the successional relationship between beetle assemblages with vegetation variables over years, the BIOENV procedure was used on each habitat ( Clarke and Ainsworth 1993 ). BIOENV produces a rank-correlation coefficient among measures for the biological distance among years on each habitat (calculated by using assemblage composition data for each habitat over years) plotted against measures for environmental distance among habitats (calculated by using vegetation data for each habitat over years). The beetle and vegetation similarity matrix were constructed using Bray-Curtis similarity measures per habitat type over years ( Legendre and Legendre 1998 ). Spearman's rank correlation coefficients (p) were then calculated for the species matrix and each of the vegetation variables (richness, vegetation cover and vegetation height per habitat type). The variable or set of variables that have the highest ρ-value are those that best explain the beetle species data ( Clarke and Gorley 2001 ). | Results
Beetle succession
Over the three years following the wildfire, a total of 2,734 adult beetles were collected from 57 species belonging to 24 families. The presence and location of beetle species caught are shown in the Appendix. The dominant families were Carabidae with 11 species, comprising 70% of all individuals collected, followed by Staphylinidae with 7 species (17%), Coccinelidae with 6 species (1.5%), Curculionidae with 4 species (0.3%) and Leiodidae with 4 species (2.5%) (Appendix). The majority of beetles collected were predators like Carabidae, Staphylinidae and Coccinelidae, but some families were herbivorous like Curculionidae or scavengers like Leiodidae.
The total beetle abundance showed differences in habitat types over years, between habitat types and among years ( Table I ). In the habitat types over years, the total beetle abundance in the shrubland community was lowest in 2002, reached a maximum in 2003, and decreased to intermediate values in 2004 (Tukey, p = 0.004)( Figure 2a ). However, in the N. dombeyi and A. chilensis forest, the abundance increased over years and the difference between years was not significant ( Figure 2a ).
Beetle rarefied species richness was similar between habitat types but varied over years ( Table 1 ). On the three habitat types, species richness was intermediate in 2002, highest in 2003, and in 2004 it was significantly lower than in 2002 and 2003 ( Figure 2b ).
Beetle assemblage composition was similar among habitats and presented different patterns over post-fire years ( Table 2 ). Ordination of beetle assemblages in relation to differences in habitat types and years ( Figure 3a ) illustrates that years is the stronger influence on beetle assemblages. Beetle assemblage presented a transition in composition from 2002 in the right, 2003 in the middle, and 2004 to the left of the figure ( Figure 3a ). Although there was some overlap in beetle assemblage composition in years 2003 and 2004, there was a distinct cluster for year 2002. Furthermore, beetle assemblages within years did not differ among habitat types ( Figure 3a ).
Indicator species analysis to identified species characteristics of habitat types was low (60%); however, the indicator value for years was higher with a maximum of 80%. For habitat types, indicator species analysis identified two species characteristic of shrubland community, one species characteristic of N dombeyi forest, and two species of A. chilensis forest ( Table 4 ). Four species were characteristic of 2002, five species of 2003, and three species of 2004 ( Table 4 ).
Vegetation structure and beetle assemblages
Early post-fire vegetation succession was different between habitat types and years. Plant species richness differed between habitats and years ( Table 3 ). The A. chilensis forest (47.4 ± 1.8) had more species than the shrubland community (38.7 ± 1.8; Tukey, p = 0.02) and the N. dombeyi forest (37.7 ± 1.8; Tukey, p = 0.005). Plant species richness was very low in 2002 (38.5 ± 1.2) compared with 2003 (42.6 ± 0.9; Tukey, p = 0.0002), and 2004 was similar to 2003 (42.2 ± 1.4; Tukey, p = 0.0005). Vegetation cover and height were similar among habitats but differed among years ( Table 3 ). Total vegetation cover was very low in 2002 (70.5% ± 3.3) and differed from 2003 and 2004. In 2003, it increased to 101.7% ± 2.6 (Tukey, p = 0.0001), and in 2004, it was similar to 2003 (118.91% ± 3.32; Tukey, p =0.0001). On the other hand, vegetation height increased over years and differed between them (Tukey, p < 0.05). In 2002, vegetation reached 80.0 cm ± 1.9, and, in 2003, it reached 101.7 cm ± 2.6. It was 118.9 cm ± 3.3 in 2004. Plant assemblage composition differed between habitats and years ( Table 2 ). Ordination of plant assemblages in relation to habitat types and years differences ( Figure 3b ) illustrates that both influence vegetation assemblages. On the top, N. dombeyi forest was separated from the shrubland and A. chilensis forest, which were overlapped at the bottom ( Table 2 , Figure 3b ). Among years, plant species composition presented a transition from 2002 in the left, to 2003 in the middle, and 2004 to the right ( Table 2 , Figure 3b ); however, years 2003 and 2004 overlapped ( Figure 3b ).
Only one significant relationship was found between beetle diversity and structural vegetation characteristics (richness, height, and cover) including the three vegetation types together. Beetle richness increased with vegetation cover (r = 0.54, p = 0.04) ( Figure 4 ). There was no significant relationship between beetle abundance and plant richness (r = 0.3, p = 0.2), vegetation cover (r = 0.2, p = 0.5) or vegetation height (r = 0.2, p = 0.5), nor between beetle abundance and vegetation richness (r = 0.03, p = 0.9), and vegetation height (r = 0.3, p = 0.3). Mantel test detected a positive association between coleopteran assemblage and vegetation assemblage for 2004 data (r = 0.48, p = 0.004). In addition, BIOENV analysis revealed that beetle species composition over years was best explained by vegetation cover (ρ = 0.8) in shrubland, by plant richness (ρ = 0.6) in N. dombeyi forest, and by vegetation cover (ρ = 0.4) in A. chilensis forest. | Discussion
Beetle succession
Although these results arise from a case study (one large fire), this research offers strong evidence over time (three consecutive years) of post-fire beetle succession in Patagonia. The assumption was that habitat pre-fire conditions would determine different post-fire beetle communities. However, the results indicated that beetle communities varied more over years than among habitats during the early regeneration after fire. Different beetle abundance patterns were found over years on each habitat type. Beetle abundance in shrubland peaked in 2003, but, beetle abundance increased over three years in N. dombeyi and A. chilensis forest. Beetles rarefied species richness for all habitats also varied among years, with a highest number of species in 2003. Despite the number of beetle species found - it was relatively low compared to previous results ( Sackmann and Farji-Brener 2006 ) - differences were found between years. Shrubland abundance and rarefied richness both showed the highest value for 2003. This could be due to several non-exclusive causes. One possible explanation could be beetle species survival and arrival after the wildfire ( Moretti et al. 2006 ). Some species could have benefited from recent post-fire conditions and immigrated to the burned area and, also, some species could survive wildfire, varying abundance and richness over years. Another explanation could be due to climatic variations among years and extreme climatic events (e.g. droughts). These events are known to alter life history traits such as breeding phenology ( Ayres and Lombardero 2000 ). During the 3-yr study period, environmental conditions varied markedly (INTA Bariloche), particularly precipitation. The 2001–2002 growing season was dry (October-March: 27.16 mm/month). In contrast, the 2002–2003 growing season was extremely wet (55.33 mm/month), and the 2003–2004 growing season was the driest (22.16 mm/month). Blanch et al. ( 2001 ) showed that the number of individuals, families, and species was strongly determined by seasonal and yearly patterns of rainfall. When substantial rain fell before the time of sampling, total beetle abundance and richness increased markedly ( Blanche et al. 2001 ). Moreover, shrubland plant communities are well adapted to regenerate by resprouting after fire ( Veblen et al. 2003 ). This plant community could have taken advantage of this limiting resource (rain), increasing growth and indirectly influencing the ability of beetles to obtain food and shelter, as well as directly affecting microclimatic conditions necessary for their survival ( Niwa and Peck 2002 ). Overall, these variations in precipitation could explain the pattern found in beetle abundance in shrubland and species richness over years in all habitats, which were highest in 2003.
Parallel to the results of beetle abundance and richness, differences in beetle species composition were found between years rather than between habitats. There was a shift in beetle assemblage composition with time since fire in all habitat types. The year 2002 was very different from the years 2003 and 2004, and all habitats showed similar beetle composition over the years. This successional beetle pattern occurred concurrently with the vegetation successional pattern, although vegetation also changed among habitats. As a result, beetle succession could be more influenced by recolonization and survivorship, accompanied by climatic conditions and recovery rate of plants communities over years ( Bess et al. 2002 ) than by pre-fire habitat conditions. All habitats in 2002 supported species that may have colonized the recently burned habitat and/or species that may have survived the disturbance ( Baker 2006 ), such as Eriopsis conexa , Calosoma vagans , Adonia variegata and Euspilotus bisignatus . These species are from the families Histeridae, Tenebrionidae and Coccinelidae, which are associated with open habitats that seemed to have benefited from habitat alteration induced by fire ( Sackmann and Farji-Brener 2006 ). However, in 2003 and 2004, characteristic species were present in both years, but their abundance changed. In 2003, Tetragonoderus viridis (Carabidae) and morphospecies of Staphilinidae and Bruchidae were the characteristic species. The families Carabidae and Staphilinidae are predators, and Bruchidae are seed predators ( Borror et al. 1992 ). Staphilinidae were associated with decaying materials and moist microsites, such as under stones and other objects on the ground. In 2004, Trirammatus uniestratus and Anisostichus laevis , both Carabidae (predators), and Epicauta sp. (Meloidae) (herbivorous) were the characteristic species. Assemblage composition appeared to shift rapidly 3–5 years after the fire as was previously found by Bess et al. ( 2002 ). These results suggested that wildfire in NW Patagonia can have a substantial, short-term impact on beetle abundance and species composition, but further research following the post-fire succession over time would be worthwhile.
On the other hand, vegetation diversity varied with both habitat types and years since the fire. Post-fire plant species richness and composition differed among habitat types, and there were changes in vegetation cover, height, plant richness and composition over years. Austrucedrus chilensis forest had the highest plant richness, probably because it was the driest ( Veblen et al. 2003 ), and it also had the most heterogeneous habitat in terms of light and soils. This environmental variability and heterogeneity led to the establishment of more plant species. Conversely, N. dombeyi forest differed in species composition from shrubland and A. chilensis forest. Those two habitats shared species and species abundance, despite differing in species richness. Plant richness, vegetation cover, and composition increased greatly from 2002 to 2003. This plant recolonization was probably due to the climatic variations and extreme events discussed above. Overall, pre-fire conditions determined different post-fire plant communities and successions in each habitat type.
It was predicted that beetle successional patterns would be related to vegetation structure, and the results partly support this prediction because there was succession over time in plants and beetles, but vegetation communities were also different between prefire habitat types. Beetle richness increased with vegetation cover, taking into account the three habitats together in 2004. Areas with more vegetation cover could be associated with habitat availability for many arthropods ( Abensperg-Traun et al. 1996 ; Suominen et al. 1999 ), whereas this relationship was similar for the three habitats and consistent with the findings of previous studies conducted in the region ( Sackmann and Farji-Brener 2006 ). Additionally, there was a positive correlation between beetle and vegetation assemblages in 2004. Beetle and vegetation assemblage succession patterns were parallel over years but not among habitats. There were differences in vegetation assemblages among habitats. Over years, beetle assemblage was related with vegetation cover in shrubland and the A. chilensis forest, however, beetle assemblage was related to plant richness in the N. dombeyi forest.
Pre-fire conditions of each habitat type determined different post-fire communities of plants, but not of beetles. The pattern of beetle succession was more influenced by the time since the fire than by habitat conditions prefire. Wildfires produce simplification and homogenization of habitat types, and this was reflected by beetle diversity. These findings suggest that beetles might not provide effective indicators of pre-fire habitat types. | Wildfires are one of the major disturbances in the dynamics of forests and shrublands. However, little is known about their effects on insect communities that contribute to faunal biodiversity and play key roles in the ecosystem's dynamics. An intense and widespread fire occurred in 1999 in the Nahuel Huapi National Park in the Andean forest in northern Patagonia, Argentina. This fire affected adjacent, but different, habitat types. After the fire, beetle abundance, species richness and assemblage composition were compared among three habitats that were structurally different before the fire. These habitats were: 1) evergreen forest dominated by Nothofagus dombeyi (Mirb.) Oerst. (Fagales: Nothofagaceae), 2) a mixed forest of the evergreen conifer Austrocedrus chilensis (D. Don) Pic. Serm. and Bizzarri (Pinales: Cupressaceae) and N. dombeyi and 3) a shrubland with a diverse community of shrub species. The relationship between beetle diversity and vegetation structure was investigated over three consecutive years. Ground beetles were collected by pitfall traps, and plant species richness, vegetation cover, and height were measured. Beetle communities varied more over years between habitats during the early regeneration after fire. There was a shift in beetle assemblage composition with time after the fire in all habitat types, probably due to similar colonization rates and microclimatic conditions. Therefore, beetle succession was more influenced by recolonization and survivorship, accompanied by climatic conditions and recovery rate of plant communities over time, than it was influenced by pre-fire habitat conditions. These results suggest that in NW Patagonia, wildfire can have a substantial, short-term impact on beetle abundance and species composition. The pre-fire conditions of each habitat type determined the structure of post-fire communities of plants but not beetle assemblages. Wildfires produce simplification and homogenization of habitat types, and this was reflected by beetle diversity.
Keywords | Acknowledgements
We thank Emiliano and Daniela Sasal, María Noelia Barrios-Garcia-Moar, Norlan Tercero and Juan Paritsis for their field assistance. We thank Maria Laura Suarez for her help with figures. We also thank Sergio Roig-Juñet (Carabidae), Mariana Chani (Staphylinidae), Eider Ruíz-Manzanos (Scarabeidae), Gustavo E. Flores (Tenebrionidae), and Alfred Newton (Leiodidae) for their assistance with beetle identifications. Administración de Parques Nacionales allowed access to the study site. This study receives the support of the National Science Foundation (NSF 9421881), “Agenda de Promoción Científica y Tecnológica (PICT 01-07320BID OC-AR) and “Universidad Nacional del Comahue” (B 103). | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 17; 10:37 | oa_package/20/29/PMC3014732.tar.gz |
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PMC3014733 | 20575739 | Introduction
The fall armyworm, Spodoptera frugiperda Smith (Lepidoptera: Noctuidae), is an important pest of many crops, causing important yield losses in different regions of the Americas ( Sparks 1979 ). In northern Argentina, S. frugiperda infestations may result in maize yield losses between 17% and 72% ( Perdiguero et al. 1967 ). To date, control of this pest relied mainly on the use of synthetic pesticides ( Hruska and Gould 1997 ), and as a result, individuals resistant to insecticides have been selected ( Yu et al. 2003 ). Resistance of S. frugiperda to various carbamate, organophophorus, and pyrethroid insecticides has also been observed in field strains collected from corn in north, central and south Florida ( Yu 1991 , 1992 ).
S. frugiperda is a native pest in Argentina with a diverse complex of natural enemies. The impact of parasitoids on these populations has been well studied ( Virla et al. 1999 ; Berta et al. 2000 ; Murua et al. 2003 ). In turn, some work has focused on entomopathogenic organisms such as nuclear polyhedrosis virus, Nomuraea rileyi , Metahrizium anisopliae and Beauveria bassiana (see references in Vera et al. 1995 ; Berreta et al. 1998 ; Lecuona and Diaz 2001 ). However, there is a remarkable lack of information on the role of natural predators. A few exceptions are technical reports on the action against this pest by some carabid beetles and other Coleoptera, lacewings (Neuroptera), and some bugs (Heteroptera) ( Saini 2005 ).
Dermaptera are omnivorous insects that may be considered either damaging or helpful organisms within agroecosystems ( Van Huis 1981 ; Jones et al. 1988 ; Gravena and Da Cunha 1991 ; Mariani et al. 1996 ). The beneficial actions of Dermaptera in many crops of economical relevance have been described previously by Buxton ( 1974 ) and Cañellas et al. ( 2005 ). Most adult earwigs hide during the day in dark, but some species, like Chelisoches morio , are diurnal and extremely active, running over the leaves during the hottest part of the day in search of food ( Zimmerman 1948 , as cited by Hudson 1974 ).
Doru taeniatum has been reported as an effective predator of S. frugiperda in Central America ( Jones et al. 1988 ; Lastres 1990 ). In Brazil, the importance of two species of Dermaptera in annual crops is well known: Doru luteipes seems to be the most abundant earwig and has been mentioned as an efficient predator of S. frugiperda and Helicoverpa zea in soybean ( Lanza Reis et al. 1988 ; Cruz 1992 , 1995 ); Doru lineare (Dermaptera: Forficulidae) also has been mentioned as a beneficial agent in soybean (Otero and Belarmino 1993, Belarmino and Gati 1993) and for controlling Alabama argillacea in cotton ( Gravena and Da Cuhna 1991 ), Diatraea saccharalis in sugar cane ( Soussa-Silva et al. 1992 ), and Sitotroga cerealella in stored grains.
Both D. luteipes and D. lineare species have been found in maize crops in northern Argentina, although their role in the agroecosystem is still unknown. In Tucumán province, during summertime, D. lineare show evidence of foraging activities through the day. This earwig previously was reported preying on S. frugiperda egg masses in corn crops ( Mariani et al. 1996 ). Although some bionomic studies on D. lineare have been conducted under laboratory conditions ( Romero Sueldo and Dode 2001 ), there is a lack of information about its predation-capability on S. frugiperda eggs and newly emerged larvae.
Recently, functional response of parasitoids and predators in relation to prey density has received increasing attention in the entomological literature ( Houck and Strauss 1985 ; Mohaghegh et al. 2001 ; Fernández-Arhex and Corley 2003 , 2004 ). However, there are no reports on the functional response of any Dermaptera species.
Because S. frugiperda is considered a key pest of corn in northwestern Argentina and because D. lineare populations frequently occur in the field, it needs to be determined if this species acts as a predator and should be considered a potential biological control agent. The aim of this study was to investigate the predation efficiency of this earwig through its functional response to S. frugiperda newly emerged larvae in the laboratory. | Materials and Methods
Origin and maintenance of insect colonies
Both S. frugiperda and D. lineare colonies were established with specimens collected during December 2004 from a subsistence cornfield located near El Manantial (Dpto. Lules, Tucuman province, Argentina) (26°49′50.2′′S, 65°16′59.4′′W, elevation: 495 m).
S. frugiperda larvae were placed individually in glass tubes (12 cm high × 1.5 cm diameter) with host leaves and carried to the laboratory. Adults of S. frugiperda were maintained in polyethylene-terephthalate cylindrical cages (30 cm high × 10 cm diameter). For aeration, the top was covered with a nylon mesh cloth. These cages contained pieces of paper that allowed females to rest and to lay eggs. Food was provided via a cotton wick saturated with a honey and water solution (1:1 vol/vol). Cages were checked daily for egg masses, and these were collected and deposited in glass tubes as above. Upon eclosion the neonate larvae were placed in 250 cc plastic pots containing artificial diet ( Ozores et al. 1982 ). Pots were covered with a nylon mesh cloth until the larva reached the 3 rd instar, at which time they were isolated in glass tubes to prevent cannibalism.
Earwig colonies were maintained in plastic cages (30 × 25 × 8cm) containing pieces of corrugated cardboard as refuge. Commercial cat food and a cotton wick saturated with a honey + water solution (1:1 vol/vol) were provided as food. In each cage, a maximum of 20 couples were maintained together to prevent cannibalism. Cages were examined daily, and eggs were transferred carefully with the female to a 250 cc plastic pot and provided with a plastic soda cup filled with wet cotton (1.5 cm high - 3.0 cm diameter). Normally, females transported their offspring into the soda cups. Ten days after nymphal eclosion, they were transferred to larger plastic cages (as described above) until they completed development.
Insect cultures were conducted in the laboratory at 26 ± 2°C, 14:10 (L:D) photoperiod, and 70 ± 10% RH. All predatory individuals used in the experiments were reproductively active females of D. lineare that were two to three weeks old. Females were starved for 48 h before trials and were randomly collected from the breeding cages.
Handling time trials
Handling time of newly emerged S. frugiperda larvae is defined in this study as the time interval starting with the piercing of larval tegument until the complete consumption of the prey item, excluding the cephalic capsule. Preliminary observations showed that sometimes earwigs do not consume the cephalic capsule.
Handling time was assessed by direct observation under stereoscopic microscope and measured with chronometer. Each female of D. lineare ( n = 100) was placed with 5 to 10 larvae in a 6 cm (diameter) by 0.5 cm (depth) Petri dish. The trials were run at 26 ± 2°C.
Potted corn plants (2 nd vegetative stage) covered with a polyethylene-terephthalate cylindrical cages (35 cm long × 18 cm diameter) were used as the experimental arenas. Each cage was covered with a fine nylon mesh allowing air exchange.
Functional response trials
Predation rate was determined by releasing a single female earwig on potted corn plants that contained newly emerged S. frugiperda larvae at different densities: 1, 7, 10, 20, 40, 70, 100, 115, 130, 160, 190, 250, 360, 420 and 500 larvae. Larvae were placed using a paintbrush in the whorl region, and usually most of them spread over the entire plant. So, at the release time, the prey were randomly distributed on the corn plant. After two hours, the predators were removed and the number of remaining intact larvae alive was recorded. Six replicates were done for each prey density, and consumed prey were not replaced.
Predator searching efficiency was obtained from the quantity of dead and available prey using the formula: where, Pc = searching efficiency, Na = number of consumed prey, and Nt = number of offered prey.
Data analysis
Following Trexler et al. ( 1988 ) and Fernández-Arhex and Corley ( 2004 ), a stepwise logistic regression ( Legendre and Legendre 1998 ) was used to fit the best curve, where the functional responses of type II and III are differentiated by the presence of a different number of significant components in the z term of the equation:
Where P is the proportion of prey killed, z is the function of the prey abundance x , and b 0–2 are the parameters of function z . By this method, three different results are expected: (1) if none of the parameters, b 0 , b 1 , b 2 or only b 0 is significant, there is functional response type I in which the asymptote is not reached in the densities used in the experiment; (2) if b 1 is significantly negative, a type II functional response exists; and (3) if b 1 and b 2 are significantly positive and negative respectively, a type III functional response exists. Functional response type 1 was not considered since it does not make biological sense for insect predators.
The logistic model was fit to the data by stepwise nonlinear regression, using the maximum likelihood criterion and the Wald statistic ( Legendre and Legendre 1998 ). The significance was calculated by comparing the Wald statistic with a X 2 distribution. The parameters were fitted iteratively using a program written in the Python programming language version 2.3.5 (P ython Software Foundation 2005 , www.python.org/psf/ ), with the numarray module for statistical analysis ( Greenfield et al. 2003 ).
After selecting the type of functional response, the data were fit by nonlinear regression to one of the following equations: Type II “Disc Equation” or Type III ( Hassell 2000 ). The regression was performed with the GNUPLOT version 4.0.0 program ( Williams et al. 2004 ), using the weighted least squares method and statistical significance estimated by a t -test.
Voucher specimens
Insect voucher specimens were deposited in the Instituto Fundación “Miguel Lillo” collection, San Miguel de Tucumán, Argentina. | Results and Discussion
Handling time trials
The observed time required for an earwig to consume a single, newly emerged S. frugiperda larvae ranged from 10.1 to 55.2 seconds (x = 24.3; SE = 8.8). Based on this mean handling time alone, maximum consumption was predicted to not exceed 296 S. frugiperda larvae in the 2 h functional response experiments.
Functional response trials
Logistic regression analyses revealed a type II functional response by D. lineare to S. frugiperda larval density ( Figure 1 ). The Type II: Holling's “disc equation” was used: where N a is the number of prey consumed, a is the searching efficiency (proportion of successfully attacked prey per unit time), T is the total time in the patch (here, the length of the experiment was two hours or 7200 s), N t is the prey density and T h is the handling time. The parameters estimated for this equation were a = 0.374 ± 0.055, and T h = 182.9 ± 14.7 s (0.0253979 * 7200 s).
At the lowest prey density (1 larvae/plant), 66.6 % of D. lineare individuals failed to attack the prey; although, facing up to 10 larvae/plant, all earwigs ate at least 2 of them. In the type II model, as prey density increases, searching for prey becomes a less important limit on the rate of predation. Prey items are easy to locate and rate of consumption is more affected by handling time (i.e., the time it takes a predator to subdue, consume, and digest its prey). As searching becomes less important and handling becomes more important, the rate of consumption shows a decelerating rate of increase. Eventually, search is not limiting at all and the rate of consumption levels off at an upper limit determined by handling time alone ( Table 1 ). Earwig satiation (the estimated asymptotic maximum in the model) occurred at 39.37 larvae. The observed maximum number of larvae consumed by a single female during the 2 h experiment was 42 individuals.
Many arthropod predators exhibit a type II functional response as described by Holling's disk equation; this type of response is characterized by a predation rate that is limited only by handling time. The search efficiency estimate ( a = 0.374) obtained in this study was well within the range of others obtained for predators of Noctuidae (Lepidoptera) pest. For example, Parajulee et al. ( 2006 ) obtained 0.489 for the big-eyed bug ( Geocoris punctipes ) and 0.220 for the green lacewing ( Chrysopa oculata ) against bollworm, Helicoverpa zea , eggs, and Mohaghegh et al. ( 2001 ) obtained 0.067 for the pentatomid Podisus maculiventris preying on fourth-instar larvae of the beet armyworm, Spodoptera exigua. Similarly, Morales et al. ( 2001 ) reported a searching efficiency of 0.79 for the egg parasitoid Telenomus remus against S. frugiperda . Search efficiency decreases with increasing density because the predator spends more time searching for prey at lower densities ( Hassel et al. 2000 ). Saini et al. ( 1997 ) registered a similar decrease in search efficiency when evaluating the functional response of Podisus conexivus attacking Anticarsia gemmatalis larvae.
Functional response studies in small laboratory arenas have been criticized ( O'Neil 1989 ; Wiedenmann and O'Neil 1991 ) because factors such as large searching areas, host plants, and weather under field conditions may influence the effectiveness of predators. Generally, the density levels used in laboratory studies are substantially higher that those occurring in the field. However, in this study, densities were realistic, taking into consideration the biology of both the predator and the pest. S. frugiperda eggs are deposited in layers and covered with scales from the female's body ( Beserra et al. 2002 ). Each egg cluster has an average of 109 ± 98.6 eggs ( Murua and Virla 2004 ), the eclosion rate is over 95 %, and the larvae remain aggregated on the host plant during the first hours after emergence. According to the findings, a single D. lineare female may be able to consume almost half the offspring of a single egg cluster.
An estimation of the potential impact of D. lineare predation on the population of S. frugiperda larvae may be generated by combining the results of experiments described here with estimates of earwigs and pest larvae densities in situ . Although the dynamics of generalist predators are not tightly coupled to those of any one of their prey, such predators can have dramatic effects on prey populations ( Murdoch et al. 2002 ). Clearly, predation by D. lineare on S. frugiperda larvae may significantly influence survival to the larval stage in this pest. However, in functional response studies, field data are an essential complement for the laboratory results because in natural conditions other variables can interfere in predator behavior. The performance of this earwig as a potential biocontrol agent can only be appreciated when considering all relevant aspects of its biology, including development and reproduction. The present study has improved understanding of the role of D. lineare and its potential value in maize agroecosystems. | Results and Discussion
Handling time trials
The observed time required for an earwig to consume a single, newly emerged S. frugiperda larvae ranged from 10.1 to 55.2 seconds (x = 24.3; SE = 8.8). Based on this mean handling time alone, maximum consumption was predicted to not exceed 296 S. frugiperda larvae in the 2 h functional response experiments.
Functional response trials
Logistic regression analyses revealed a type II functional response by D. lineare to S. frugiperda larval density ( Figure 1 ). The Type II: Holling's “disc equation” was used: where N a is the number of prey consumed, a is the searching efficiency (proportion of successfully attacked prey per unit time), T is the total time in the patch (here, the length of the experiment was two hours or 7200 s), N t is the prey density and T h is the handling time. The parameters estimated for this equation were a = 0.374 ± 0.055, and T h = 182.9 ± 14.7 s (0.0253979 * 7200 s).
At the lowest prey density (1 larvae/plant), 66.6 % of D. lineare individuals failed to attack the prey; although, facing up to 10 larvae/plant, all earwigs ate at least 2 of them. In the type II model, as prey density increases, searching for prey becomes a less important limit on the rate of predation. Prey items are easy to locate and rate of consumption is more affected by handling time (i.e., the time it takes a predator to subdue, consume, and digest its prey). As searching becomes less important and handling becomes more important, the rate of consumption shows a decelerating rate of increase. Eventually, search is not limiting at all and the rate of consumption levels off at an upper limit determined by handling time alone ( Table 1 ). Earwig satiation (the estimated asymptotic maximum in the model) occurred at 39.37 larvae. The observed maximum number of larvae consumed by a single female during the 2 h experiment was 42 individuals.
Many arthropod predators exhibit a type II functional response as described by Holling's disk equation; this type of response is characterized by a predation rate that is limited only by handling time. The search efficiency estimate ( a = 0.374) obtained in this study was well within the range of others obtained for predators of Noctuidae (Lepidoptera) pest. For example, Parajulee et al. ( 2006 ) obtained 0.489 for the big-eyed bug ( Geocoris punctipes ) and 0.220 for the green lacewing ( Chrysopa oculata ) against bollworm, Helicoverpa zea , eggs, and Mohaghegh et al. ( 2001 ) obtained 0.067 for the pentatomid Podisus maculiventris preying on fourth-instar larvae of the beet armyworm, Spodoptera exigua. Similarly, Morales et al. ( 2001 ) reported a searching efficiency of 0.79 for the egg parasitoid Telenomus remus against S. frugiperda . Search efficiency decreases with increasing density because the predator spends more time searching for prey at lower densities ( Hassel et al. 2000 ). Saini et al. ( 1997 ) registered a similar decrease in search efficiency when evaluating the functional response of Podisus conexivus attacking Anticarsia gemmatalis larvae.
Functional response studies in small laboratory arenas have been criticized ( O'Neil 1989 ; Wiedenmann and O'Neil 1991 ) because factors such as large searching areas, host plants, and weather under field conditions may influence the effectiveness of predators. Generally, the density levels used in laboratory studies are substantially higher that those occurring in the field. However, in this study, densities were realistic, taking into consideration the biology of both the predator and the pest. S. frugiperda eggs are deposited in layers and covered with scales from the female's body ( Beserra et al. 2002 ). Each egg cluster has an average of 109 ± 98.6 eggs ( Murua and Virla 2004 ), the eclosion rate is over 95 %, and the larvae remain aggregated on the host plant during the first hours after emergence. According to the findings, a single D. lineare female may be able to consume almost half the offspring of a single egg cluster.
An estimation of the potential impact of D. lineare predation on the population of S. frugiperda larvae may be generated by combining the results of experiments described here with estimates of earwigs and pest larvae densities in situ . Although the dynamics of generalist predators are not tightly coupled to those of any one of their prey, such predators can have dramatic effects on prey populations ( Murdoch et al. 2002 ). Clearly, predation by D. lineare on S. frugiperda larvae may significantly influence survival to the larval stage in this pest. However, in functional response studies, field data are an essential complement for the laboratory results because in natural conditions other variables can interfere in predator behavior. The performance of this earwig as a potential biocontrol agent can only be appreciated when considering all relevant aspects of its biology, including development and reproduction. The present study has improved understanding of the role of D. lineare and its potential value in maize agroecosystems. | Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) is considered as the most important pest of maize in almost all tropical America. In Argentina, the earwig Doru lineare Eschscholtz (Dermaptera: Forficulidae) has been observed preying on S. frugiperda egg masses in corn crops, but no data about its potential role as a biocontrol agent of this pest have been provided. The predation efficiency of D. lineare on newly emerged S. frugiperda larva was evaluated through a laboratory functional response study. D. lineare showed type II functional response to S. frugiperda larval density, and disc equation estimations of searching efficiency and handling time were (a) = 0.374 and (t) = 182.9 s, respectively. Earwig satiation occurred at 39.4 S. frugiperda larvae.
Keywords | Acknowledgements
We would like to thank Eduardo Frias and Esther Mercado (PROIMI-Biotechnology) for technical assistance and Roxana Mariani (Natural Sciences Museum at La Plata, Buenos Aires) for earwig species taxonomic confirmation. We are grateful to the anonymous reviewers for critically going through the manuscript and providing valuable suggestions. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 27; 10:38 | oa_package/e9/e5/PMC3014733.tar.gz |
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PMC3014735 | 20578949 | Introduction
Hilltopping in insects, as initially defined by Shields ( 1967 ), is the behaviour of males aggregating on a peak to wait for the females that fly there and mate with them. In a paper dealing with some Conopidae from South Africa, Smith ( 1967 ) reported an observation made by Stuckenberg on hilltopping behaviour in a species of Pleurocerinella. Stuckenberg described a small swarm of these flies, including both males and females, that were found clustering tightly together on a leaf “on the flat summit of a mountain”. He suggested that this behaviour could have had a sexual function. Gathering of Conopidae at a particular landmark, though not on a hilltop, also has been reported by Kröber ( 1927 ). Hundreds of Leopoldius coronatus (Rondani) of both sexes were observed consecutively for a few days in Lautembach, a site near Gernsbach (Schwarzwald, Germany), on a forest path that was in the sun in the late afternoon. The L. coronatus were flying together along a small ditch filled with ferns and brambles but otherwise devoid of flowers: “a place where I could have never expected to find conopids” ( Kröber 1927 , in translation). Although matings were not described, a sexual function of the gathering is possible.
The above mentioned observations, both relevant to species of the subfamily Conopinae, seem to be the only published evidence of hilltopping and swarming behaviour in this family of flies. Despite this, the phenomenon has been recognized for some time. Label data on specimens in collections clearly indicates that some entomologists have noticed and collected these insects on hilltops for decades. Recently, Skevington ( 2008 ), reviewing the available knowledge, suggested that hilltop collecting may be an effective way to survey many groups of insects, particularly parasitoids such as Conopidae, Pipunculidae, and Tachinidae.
This paper provides the first evidence of hilltopping in species belonging to the subfamilies Myopinae and Dalmanniinae. Field observations and museum specimen examinations were conducted in Italy and Canada. This research adds new observational data on hilltopping and mating behaviour in these poorly understood parasitoid Diptera, and will be of interest to those studying insect mating systems, parasitoid-host interactions, and hilltopping. | Materials and Methods
Colle Vescovo observational data
Observations by the senior author took place at the summit of a hill named Colle Vescovo, (448 MASL), at the southwest edge of the Lucretili Mountains near the town of Tivoli (Latium, Italy) (41° 58′ 39′′ N, 12° 48′ 30′′ E). The hill was covered by very degraded xerophilic bushes, characterized by Ampelodesma mauritanica Durand and Schinz (Poales: Poaceae), Styrax officinalis L. (Ericales: Styracace), Paliurus spina-christi Miller (Rosales: Rhamnaceae), Pistacia terebinthus L. (Sapindales: Anacardiaceae), Cercis siliquastrum L. (Fabales: Fabaceae), Phillyrea latifolia L. (Oleales: Oleaceae), and Spartium junceum L. (Fabales: Fabaceae) (Montelucci 1947). There were abandoned olive groves at the foot of the hill and a cork oak thicket on the northeast slope. The summit of the hill was almost bare, with a low rock outcropping, oriented along a northeastsouthwest direction and supporting a large olive bush, Olea europeea var. oleaster L. (Oleales: Oleaceae) about 2.5 m tall, two much smaller terebrinth, P. terebinthus, bushes, and a Spanish broom, S. junceum bush. At the time of the observations, a dead, contorted trunk of a small P. terebinthus tree, about 3 m tall, was standing in the middle of the larger of the P. terebinthus bushes. The hilltop area was approximately 40 square meters ( Figure 1 ). The vegetation of Colle Vescovo and the neighbouring hills was degraded by fires that occur in the surroundings of Tivoli almost every summer, and from overgrazing by cattle and horses. A fire destroyed the vegetation of the northwest slope almost up to the summit in the late summer of 2001.
Observations on Colle Vescovo were carried out from 1999 to 2003. Throughout this period, the site was sampled at irregular intervals from February to August. In all, 30 visits were made to the hilltop for a total of about 100 hours of observation. Research was concentrated in the spring, and most of the observations were carried out in the months of March, April and May ( n =20 visits). Observations were made primarily in the morning ( n ≈ 80 hours), from 09:00 until 13:30, when all insects, and conopids in particular, were most active. About half of the total field work was carried out in 2001, when the site was sampled 2–3 times every month from February to May, then monthly until August ( n =14 visits). The prevailing winds on the summit, as recorded during the course of the observations, were either from the west or southeast.
None of the conopids observed on the hilltop were marked. Due to the relatively sporadic nature of their presence and to the sparse vegetation of the hilltop, however, it was often possible to record the activity of individuals without interruption for many minutes. Specimens were collected with a hand net and were deposited in the senior author's collection. Voucher specimens are in the Museum of Zoology of the University of Rome “Sapienza”.
Mount Rigaud observational data
Mount Rigaud is a well-defined hill south of the Ottawa River in southwestern Quebec, Canada (45° 27′ 59′′ N, 74° 19′ 35′′ W). The hill is in a 5440 ha forested area that was dominated by sugar maple, Acer saccharum Marshall (Sapindales: Sapindaceae) and American beech, Fagus grandifolia Ehrh. (Fagales: Fagaceae). This area of Quebec has a mosaic of farmland and forest, but the location of the hilltop in a large tract of accessible forest made it one of the most entomologically diverse hilltops in eastern North America ( Skevington 2008 ). The summit of Mount Rigaud is rocky and largely open, with two thickets of stunted northern red oak, Quercus rubra L. (Fagales: Fagaceae) (maximum height about 5 m) and shorter thickets of shadbush, Amelanchier humilis Wiegand (Rosales: Rosaceae), pin cherry, Prunus pensylvanica L., and chokecherry, Prunus virginiana Pardee. A viewing stand and a large cross have been built on the summit, but apart from these structures the vegetation is still intact ( Figure 2 ). Skevington visited this site 18 times between May and August from 2001 to 2008. Behaviour of individual flies was observed directly and captured specimens were collected with a hand net and deposited in the Canadian National Collection.
Data from insect collections
All data from Conopidae collected within 120 km of Ottawa, ON, Canada and represented in the Canadian National Collection of Insects, Arachnids and Nematodes are summarized. Ottawa was chosen as a focal point because three hilltops in the vicinity have received annual collecting attention since 1961. The hilltops involved are King Mountain, QC (49° 29′ 20′′ N, 75° 51′ 45′′ W), Duncan Lake vicinity, Masham, QC (45° 40′ 53′′ N, 76° 3′ 1′′ W) and Mount Rigaud, QC. The collections in the Museum of Zoology of the University of Rome “Sapienza” and in the Civic Museum of Zoology of Rome, were examined as well, to summarize data on any conopids collected within 1.5 km of the summit of Colle Vescovo. | Results
Colle Vescovo collection records
Fifteen species of Conopidae were collected on the summit and/or the slopes of Colle Vescovo ( Table 1 ), but only four of these were clearly hilltoppers as they were recorded regularly at the summit over the course of the study: Dalmannia aculeata L., Myopa pellucida RobineauDesvoidy (= M. extricata, see Stuke and Clements 2005 ), Myopa picta (Panzer), and Zodion cinereum F. (= Z. notatum , see Mei and Stuke 2008 ). Four species were represented by isolated specimens on the summit, and the remaining seven species were not collected on the hilltop ( Table 1 ).
Ottawa area collection records
Nineteen species of Conopidae were collected in the Ottawa area ( Table 2 ). Three of these are regularly found on hilltops and are rarely found away from hilltop sites: Myopa clausa Loew, Physocephala marginata (Say), and Physocephala sagittaria (Say). A further four species have been collected regularly on hilltops, in addition to non-hilltops: Dalmannia nigriceps Loew, Myopa vesiculosa Say, Zodion fulvifrons Say, and Zodion intermedium Banks. Six species have been collected on hilltops, but more evidence is needed to confirm hilltopping as a mating strategy for these species: Dalmannia vitiosa Coquillett, Myopa virginica Banks, Physocephala furcillata (Williston), Physocephala texana (Williston), Thecophora occidensis (Walker), and Thecophora nigripes (Camras). The remaining six species have no records of males collected on hilltops and likely are not hilltoppers: Myopa vicaria Walker, Thecophora abbreviata (Loew), Thecophora longicornis (Say), Thecophora propinqua (Adams), Zodion abitus Adams, and Zodion americanum (Wiedemann).
Phenology
Conopids were found at Colle Vescovo on 11 out of 30 visits, and in general their occurrence on the summit was rather sporadic. The two species of Myopa appeared on the hilltop by the end of March ( M. picta, 24 March 2001, the earliest record) and were recorded at the study site through the first half of May, being most frequent in late April. D. aculeata was recorded from the second half of April to the second half of May, although it was particularly abundant around the first week of the latter month. The relatively few available records for Z. cinereum were concentrated between the last days of April and the first week of May. By the end of May, none of the four species was still active on the hilltop. Conopids were never observed at the study site in the afternoon. D. aculeata was by far the most abundant and frequently recorded species; more than one hundred individuals have been observed, and 75 males and 2 females were collected. About 35 specimens of M. picta were observed, and 26 collected, all males. In all, 3 females and 12 males of M. pellucida, and 10 Z. cinereum males were observed and collected. Many D. aculeata and nearly all the Z. cinereum males collected were newly emerged individuals, with the ptilinum still partly everted.
Conopids were found at Mount Rigaud on most visits. The earliest specimens collected were a male Z. fulvifrons on 13 May 1987 and a male P. texana on 13 May 2008. The latest specimen taken was a male Z. americanum on 25 September 1985. The peak of the diversity on Rigaud was from late May to the end of June. Only two species seem to be common later in the season: P. furcillata and P. sagittaria typically occur in July and August.
Perch site preferences
The four hilltopping species at Colle Vescovo showed marked preferences in the choice of perch site, and the preferences were strongly consistent from year to year.
D. aculeata individuals were invariably observed perched on exposed leaves or on outer twigs of the O. oleaster and especially on the Spartium bushes, about 0.6 to 1.5 m above ground. Particular (often more prominent) twigs were clearly preferred as perching sites and were regularly occupied over the course of the day(s). On a single occasion a male was collected feeding on a dandelion flower, Taraxacum sp. (Asterales: Asteraceae), at the base of the Spartium bush.
Most of the Myopa individuals were observed in a sheltered spot by the rock, between the O. oleaster and one of the P. terebinthus bushes, where they chose the lowest twigs of the bushes, the grass stems, and sometimes the flowers of Taraxacum and Bellis (Asterales: Asteraceae) as perching sites. Such perches were situated from a few centimeters to 0.4 m above ground. Only 2 of the 35 M. picta males observed and 3 of the 12 M. pellucida were found perching on twigs at the top of the Spartium bush, and only once a male of M. pellucida was observed feeding on the Taraxacum flowers.
All of the Zodion males were collected perching and feeding at ground level, on Taraxacum, with the single exception of a male that was observed perching on a twig at the top of the Spartium bush.
Most of the conopids were invariably found on the sides of the bushes and of the summit, directly exposed to the sun. The sun appeared to be the single most important condition to insect activity on the hilltop. Insects, including conopids, were still active in moderately strong winds, provided there was sunlight on the summit; however, activity decreased dramatically and suddenly as soon as the sun was obscured by clouds. In persistently cloudy weather, conopid activity was always nearly absent, and a specimen was only occasionally collected on the summit in such conditions. Sunlight appears to be important limiting factor for all hilltopping Diptera ( McAlpine and Munroe 1968 ; O'Hara 2003 ; Skevington 2008 ).
Notes were made on only a few of the species that occured at Mount Rigaud. P. marginata individuals ( Figure 3 ) were almost restricted to perching on a single P. virginiana bush (and, in fact, to a small area of exposed leaves about 1.0 m above the ground). This shrub was at the summit of the hill between the viewing platform and a patch of Q. rubra. The site was in full sun for most of the day and was one of the most protected spots (i.e., lowest wind) on the summit. Specimens collected were generally replaced within five minutes by another specimen. M. clausa ( Figure 4 ) males seemed to use a larger variety of perching sites and were typically closer to the ground. They were often on the A. humilis leaves and appeared to readily use sites in both direct sun and dappled light. Individuals of D. nigriceps have been collected at Rigaud by Monty Wood of the Canadian National Collection (not all specimens were available to us). He noted that this species was collected by sweeping the topmost branches of the Q. rubra (about 5 m above the ground) (Wood, personal communication). During a visit to Rigaud in June 2008, it was found that the largest Q. rubra had been pruned of nearly all branches and that males of D. nigriceps were now found perching on leaves of a nearby P. virginiana shrub at a height of 1.5 m.
Male behaviour at perch site (observations from Colle Vescovo only)
Waiting D. aculeata males ( Figure 5 ) were extremely alert, often adjusting their position on the perch or moving from one perch to another nearby at irregular intervals. It was possible to see their heads frequently tilting to follow the flight of insects, passing by within a distance of about 20–30 cm. Males alternated periods of perching with brief patrolling flights about the bush in which the perch was located, at a distance of a few centimeters from the bush itself. Some of these patrolling flights, however, were much longer with the insect leaving the bush to an unknown location. Sometimes such reconnaissance flights appeared spontaneous, but often they were triggered by the approach of a flying, similar-sized insect (e.g., tachinid fly or small bee). In such a case, the conopid would suddenly follow and closely inspect the insect, returning soon after to the bush.
The approach of a conspecific male initiated more complex interactions, involving apparently ritualized flight contests or actual contact among the opponents, often in the form of pseudocopulation. A perching D. aculeata male would dart toward an approaching conspecific and a frantic skirmish would follow, with both flies flying, making loops and spirals in the air while keeping within a few centimeters of each other, and always facing the bush at a short distance from it. This kind of interaction could last for 20 s to more than 1 min at a time ( n = 6 interactions observed). The chase could stop abruptly, the two combatants going to rest on nearby perches only to resume the contest after a while. During such a flight one of the flies would grasp the other, and they would fall together almost to the ground before parting again. In one occasion, three D. aculeata males were involved at the same time in such a chase.
Pseudo-copulation was observed quite frequently between males ( n = 10). Most of these instances consisted of a male approaching a perching conspecific male, facing it briefly, and then pouncing on it, apparently trying to copulate. Mounted on its dorsum, the attacker extended the abdomen downwards in an attempt to clasp its genitalia. Most often the attacked males initially did not even try to dislodge the intruder; instead, on two instances, the attacked male was observed raising its abdomen like a female during copulation (see below). On one occasion, however, the perched male reacted to the attack, and both males fell together toward the ground. Pseudo-copulation could also end an aerial chase, with the clutched individuals suddenly landing on the bush, where one of them tried to copulate with the other. At irregular intervals, all perching males would perform self-grooming (see below), most often soon after a patrolling flight or a chase.
There were fewer observations of M. picta and M. pellucida. Males of both species were observed alertly perching on convenient perch sites, chasing conspecifics and other insects, like hoverflies, tachinids and small bees. Pseudocopulation was observed once for M. picta: what seemed to be a mating pair perched on a grass stem, was netted and confined in a vial, where it turned out that two males were involved. The flies remained attached for more than 30 min.
Z. cinereum males were observed waiting on flowers and on the S. junceum bush, but the few encounters with this species on the summit did not allow for more detailed observations.
Observations on mating behaviour (observations from Colle Vescovo only)
A pair of D. aculeata was observed at 09:45 on 12 May 2003, perched on a twig of the S. junceum bush. The male, a newly emerged individual, was standing on the dorsum of the female, but the genitalia were not engaged: it was assumed that the copulation had already occurred (see below). After being carefully placed into a glass vial, the pair eventually separated and afterwards the two individuals did not interact. On a second occasion an actual copulation of this species was observed under artificial conditions: a male and a female separately collected on the hilltop during the morning of 1 May 2002, were put together (in the afternoon) in a large glass vial stopped with cotton wool that contained a strip of paper as support. As soon as the male came in contact with the female, it grasped the female from behind and extended its genitalia in an effort to clasp the tip of the female's abdomen. The female did not react at first, but after about 15 s and several attempts by the male, it raised its abdomen and extended the tip backwards. The male moved to the copulatory position, and the genitalia were engaged. During copulation, the pair was motionless except for a slow pulsation in the male abdomen. The forelegs of the male rested on the sides of the thorax of the female, behind the wing bases, while the mid and hind legs grasped the abdomen ( Figure 5 ). Copulation lasted for about 5 min, then the male genitalia were retracted, and the very long aedeagus was slowly pulled out, with repeated tractions of the whole abdomen. Still mounted on the motionless female, the male groomed its abdomen with the posterior legs, except for the genitalia,. During this time, the genitalia, with the still everted aedeagus, moved rhythmically back and forth. Slowly, over the course of about 2 min, the aedeagus was completely withdrawn to its resting position. The male groomed itself for several more minutes, still atop the female, then very suddenly the pair separated and the flies started running quickly around the vial.
A pair of M. pellucida was observed in copula, at 09:30 on 25 May 2001, perched on a Taraxacum flower. The male arrived suddenly and immediately grasped the female that was standing on the flower head. After a few seconds, the female raised her abdomen and extended her genitalia; at the same time, the male tilted backwards, lowering his abdomen engaging the female's genitalia; then the male resumed the horizontal position on the female's dorsum ( Figure 5 ). The fore and mid legs were in contact with the eyes and the thorax, respectively, of the female, while the hind legs grasped the base of the female's abdomen. At intervals, the male moved his mid legs quickly and rhythmically back and forth on the female's thorax. After about 4 min, copulation was interrupted and genitalia were retracted.
Observation was terminated at this time, and the pair was collected.
As indicated above, no females of M. picta or Z. cinereum were observed at the site during the course of the study.
Other observations
Observations of male activity at perch sites included the grooming behaviour of D. aculeata. Males ( n = 12) were observed grooming the head, the wings, and the abdomen, genitalia included. Eyes, antennae and the extended proboscis, were repeatedly rubbed in sequence with the patch of pubescence on the inner side of both fore tibiae. As soon as the grooming was done, the tibiae were cleaned with both of the fore tarsi. The wings and abdomen were groomed by the hind tibiae, usually both wings together, then the abdomen, upper and under surfaces, and finally the everted genitalia. Also in this case, the tibiae were then cleaned by the hind tarsi. There was some degree of flexibility in the grooming movements, as a male was once observed grooming simultaneously one side of the abdomen with the respective hind leg and the opposite wing with the other leg, then alternating the sides. Grooming of the head and of wings plus abdomen, could be performed in sequence or, as in several instances, separately.
Netted M. picta assumed a characteristic posture ( Figure 5 ), remaining stiff and motionless for many seconds. This posture, coupled with the disruptive patterning of the body and wings of this conopid, is very effective in concealing the fly from view. This behaviour appears to be typical of this species and has never been observed by the authors in other Myopa.
On Colle Vescovo, two D. aculeata males were found on 5 May 2003 with triungulines of a blister beetle, Meloe ( Eurymeloe ) sp. (M. Bologna del), attached at the base of the proboscis (see Freeman 1966 ). | Discussion
Hilltopping is considered to be a strategy that facilitates mate-finding in insect species that are either rare and widely dispersed or dependent on sparsely distributed resources (Thornhill and Alcock 1983, 1987 ; Skevington 2008 ). Many species of Conopidae have very sparse populations and are very rarely seen, and more research may show that all but the most common conopids are engaged at least to some extent in hilltopping activity.
D. aculeata is one of the most rarely encountered conopids in Italy. In the past 14 years only five isolated specimens were collected away from hilltops in Central Italy, and, in all, only 16 records are available from Italy for the last 150 years (Mei, unpublished data). The present data indicates, however, that this species can be readily, and consistently, observed on Colle Vescovo. The two hilltopping species of Myopa in Italy, though being by no means “common” species, are found more often than D. aculeata. M. pellucida, in particular, is one of the most frequently recorded species of the genus in Italy ( Mei 2000 ; and unpublished data) and in Europe ( Stuke and Clements 2008 ).
Of the 19 conopid species found in the Ottawa area, only two might be considered common ( Table 2 ). The Canadian National Collection contains 59 specimens of P. furcillata from over 30 sites in the region. Interestingly, only five specimens of this species have been taken on local hilltops, suggesting that it is either not a hilltopper or that it may use hilltopping only as a mating strategy in rare instances, perhaps when population densities are low. Z. intermedium follows a similar pattern, although another possible explanation for this species is given below. Despite being commonly encountered on hilltops, M. clausa, P. marginata, and P. sagittaria have rarely been collected away from hilltops.
The data presented above demonstrate, for the first time, the occurrence of hilltopping behaviour in 22 species of conopids. Although hilltopping in some species is represented by only a few specimens, the behaviour was pronounced and could be clearly demonstrated in D. aculeata, M. picta, M. pellucida, M. clausa, P. marginata, P. sagittaria, and Z. cinereum. The species D. nigriceps, M. vesiculosa, Z. fulvifrons, and Z. intermedium have been collected on hilltops in the past, but not exclusively so. Also, it is almost entirely males of these species that have been collected on hilltops. These specimens may represent instances of faculative hilltopping in years of low population density ( Skevington 2008 ).
On the hilltops, males aggregated at locations that were species-specific and were occupied year after year with great consistency (e.g. Wood 1987 , 1996 ; Alcock and Kemp 2006 ; Skevington 2008 ). On the summit of Colle Vescovo, the four species had clearly separate perch sites, i.e., the S. junceum and Olea bushes for D. aculeata and the relatively sheltered spot by the rock for the Myopa species. Species of Myopa on Mount Rigaud were likewise found to choose perches close to the ground. The males were almost invariably found at their specific “mating stations” ( Chapman 1954 ; Wood 1987 ) during the 5-year period of the study. Similarly, the Nearctic P. marginata was very specific in its site choice and was rarely encountered away from a single bush. D. nigriceps, on the other hand, may represent an instance of aggregation location shifting due to human interference.
Males of D. aculeata and of M. picta pursued almost every flying insect of suitable size and chased conspecific males, often starting elaborate flying “contests” (e.g., D. aculeata ). Pseudo-copulation between males was also frequently observed. These interactions could be interpreted as aggressive, territorial behaviour such as that seen in the tachinid species, Leschenaultia adusta (Loew) ( Alcock and Kemp 2006 ). However, considering the whole of the available evidence, it is more convincing to exclude the occurrence of true male territoriality, according to the interpretation given by Wood ( 1987 ) for the Tachinidae. Several males (up to five in D. aculeata ) often perched on the same bush at a very short distance from one another and did not defend their perches from conspecifics. They were never observed to actually drive away a conspecific, instead, after most of the contests, both individuals returned to their waiting stations on the same bush. Chases and pseudo-copulations may be explained as a product of heightened physical response and little discriminating ability, in the context of a strategy aiming to intercept any and all potential mates, before all other waiting males. This can be effectively done by chasing indiscriminately each and every passing insect (Thornhill and Alcock 1983; Wood 1987 ).
Among truly hilltopping insects, the females have been rarely encountered on hilltops, probably because they frequent the summit singly and only for the time necessary to select a mate. As a consequence, copulations are rarely observed (e.g., Chapman 1954 ; Dodge and Seago 1954 ; Alcock 1987 ; Wood 1987 ; Hansen 2003 ; Alcock and Kemp 2006 ). The present study is no exception, with females of 13 species never observed on hilltops.
The mating behaviours of D. aculeata and M. pellucida are described and illustrated here for the first time. In the latter species, the behaviour of the mating pair appeared to be almost identical to that of Myopa buccata (see de Meijere 1912 ).
Hilltopping appears to be an important mating strategy in the conopids. More research is required to understand why some species hilltop and other species do not. It may be that nonhilltoppers are more likely to attack colonial Hymenoptera. Remaining near such a colony thus is more likely to ensure mating success than hilltopping. Species of conopids that have rare or less predictably distributed hosts may be more prone to using hilltopping as a mating strategy. For example, among the European species of Dalmannia, D. punctata is known to mate near the colonies of their host bee ( Knerer 1973 ) and has not been found on hilltops.
At least in some insect species, hilltopping is only one of the mating strategies available to males ( Alcock 1987 ), and surely this is the case for the species considered here. The senior author (unpublished data) has collected M. pellucida pairs in copula on flowers in a meadow and also observed a D. aculeata male chasing a megachilid bee (Megachilidae) on flowers of Silybum marianum (Asterales: Asteraceae), in the very place and circumstances (Rome, Tenuta della Cervelletta, in the first week of May) where two females of the same species were found in preceding years. This suggests that a mating strategy alternative to hilltopping in D. aculeata may be used in some circumstances.
The synonymy of Zodion notatum with Z. cinereum has been proposed on morphological grounds ( Mei and Stuke 2008 ). They stated that the problem of the identity of Z. notatum deserves a more thorough study, with a full analysis of the variability and ecology of both forms in all parts of their range. In this respect, it is interesting to note that all of the hilltopping Italian Zodion observed in the present study were of the “notatum” form, while the “cinereum” form was collected mainly on the slopes of Colle Vescovo and was observed on the summit only once.
Z. fulvifrons and Z. intermedium have many variant forms and ranges covering the entire southern half of the Nearctic region ( Camras and Hurd 1957 ). The present data indicates that both species are often, but not exclusively, collected on hilltops in the Ottawa area ( Table 2 ). These two may represent species complexes. Further study may help to unravel the taxonomy of these taxa by showing that some of these forms hilltop while others do not. Research on the systematics and ecology of conopids will benefit from future efforts that focus on hilltop collecting and observation.
Eight species of conopids were collected on Colle Vescovo ( Table 1 ), while 13 species were collected on the summit of Mount Rigaud ( Table 2 ). This partly emphasizes the significance of these hilltops but also suggests that many more species from other regions will be found hilltopping if the effort is made to document them. It also illustrates how important hilltops are when conducting a survey of parasitoid flies in a region. Furthermore, a loss of hilltop habitats to human development will likely endanger many rare insect species. | Associate Editor: Tugrul Giray was editor of this paper.
Direct observations of hilltopping behaviour in the thick-headed flies (Diptera: Conopidae) have only been mentioned once in the literature. Hilltop collecting, however, may be an effective way to survey these endparasitoids. The first evidence of hilltopping in species belonging to the subfamilies Myopinae and Dalmanniinae is presented and discussed. Field observations were conducted on Colle Vescovo, Italy and Mount Rigaud, Canada, and museum specimens were examined. Observations and records indicate that four species in the genera Dalmannia, Myopa, and Zodion are hilltoppers on Colle Vescovo, while three species in the genera Myopa and Physocephala are hilltoppers on three hilltops near Ottawa, Canada. Fifteen additional species of conopids have been collected on hilltops and could possibly utilize hilltops in some years as a part of their mating strategy. Detailed phenologies and observations of mating and perching behaviours are given for species in the genera Dalmannia, Myopa, Physocephala, and Zodion. The importance of hilltop habitat preservation is stressed.
Keywords | Acknowledgements
Jens-Hermann Stuke (Leer, Germany) and Niccolò Falchi (Roma), respectively, gave useful suggestions and improved the English text of an early draft of this paper; Giuseppe Lo Giudice (Rome) took the picture of the Colle Vescovo hilltop. Jim O'Hara, as well as two anonymous reviewers, provided helpful reviews of a later draft of the paper. Funding to JHS was provided by Agriculture and Agri-Food Canada and an NSERC Discovery Grant. JFG is supported by an NSERC Postgraduate Scholarship. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Mar 29; 10:27 | oa_package/7a/c5/PMC3014735.tar.gz |
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PMC3014736 | 20575738 | Introduction
Mature Lepidoptera larvae seek diverse, species-specific sites suitable for pupation, such as in the soil, between leaves, under bark, and on tree trunks. This behavior may be favorable for them, by avoiding enemies or the adverse microclimate ( Odebiyi et al. 2003 ; Veldtman et al. 2007 ). As many lepidopteran species pupate near the ground, including in soil or leaf litter, arboreal caterpillars likely descend from their food plants to the ground for pupation. They usually crawl, descend on silk threads, or occasionally fall to the ground (e.g., Shirota et al. 1976 ; Sun et al. 2000 ; Wang et al. 2000 ). However, these methods of descent may not be safe or involve energy expenditure. Wandering long distances could expose caterpillars to many predators and parasitoids. Producing silk threads requires protein and energy ( Berenbaum et al. 1993 ), and falling from tree canopies directly to the ground may inflict mechanical damage to larvae (cf. Yanoviak et al. 2008 ). In this paper, an efficient descent behavior of the nettle caterpillar, Scopelodes contracta Walker (Lepidoptera: Limacodidae), and its interaction with important natural enemies are described.
S. contracta is widely distributed and is found in Japan, China, and India and infests various tree species such as persimmon, bayberry, and cherries ( Sakagami et al. 2003 ). Although the population density of S. contracta is usually relatively low, this moth sometimes becomes epidemic in urban areas and orchards. Eggs are laid in batches on the underside of host leaves, and hatched larvae feed on the leaves in groups as early instars and solitarily as late instars. The larvae, which are slug-like and up to ∼25 mm long, emerge twice a year (June to July and August to October) in central Japan ( Sakagami et al. 2003 ; Yamazaki personal observations). Miyatake ( 1977 ) briefly noted that mature S. contracta larvae riding singly on leaves fell to the ground from a hackberry tree, Celtis sinensis Persoon (Rosales: Ulmaceae), to pupate in the soil. However, this bizarre behavior has yet to be examined in more detail, and other aspects of the larval ecology of this species have rarely been studied. In the present study, the descent behavior of mature S. contracta larvae and the associated attacks by ants were examined to improve the understanding of the natural history of this moth. | Materials and Methods
Study sites
Field studies were conducted at three sites: Tsurumi-ryokuchi Park (34° 43′ N, 135° 35′ E, ∼5 MASL), Shotenyama Park (34° 38′ N, 135° 30′ E, ∼7 MASL), and the bank of the Yamato River (34° 35′ N, 135° 30′ E, ∼5 MASL) in Osaka Prefecture, central Japan. These sites were located in the urban area of Osaka. At the two parks, many trees, including those examined in the study, had been planted, while naturally occurring willows ( Salix spp.) and C. sinensis grew sparsely along the riverbank.
Descent behavior
The descent behavior of second-generation S. contracta larvae was examined along the bank of the Yamato River and in the Tsurumiryokuchi Park in autumn 2008. On the riverbank, the number of leaves cut by larvae and found on the ground, the number of larvae on the ground, and the number of descending larvae in a 30 min period were counted in a 16 m 2 (4 m × 4 m) quadrat under a C. sinensis tree (tree height: ∼8 m, diameter at breast height: 336 mm) that was clearly suffering from severe herbivory by S. contracta larvae. This monitoring occurred during the day (15:00 – 17:00) at 1 to 6-day intervals from late September to mid-October. Larvae on tree foliage were carefully observed in order to witness the cutting of the basal part of the leaves for parachuting. In addition, an S. contracta larva was returned to an arboreal leaf (∼1.3 m above the ground) soon after it parachuted to the ground, and its behavior was observed at 16:25 on 5 October. To survey S. contracta individuals in the soil, ten 0.09 m 2 (0.3 m × 0.3 m) quadrats were established in arbitrarily chosen locations under the C. sinensis tree, and they were dug to a depth of 50 mm using a hoe to inspect moth cocoons. New cocoons could be discriminated from older ones because of the light color and lack of emergence holes.
At Tsurumi-ryokuchi Park, the number of larval-cut leaves found on the ground, the number of larvae on the ground, and the number of larvae descending in a 30 min period were counted under a tree of heaven, Ailanthus altissima Swingle (Sapindales: Simaroubaceae), (tree height: ∼15 m) on 2 October. In addition, one yoshino cherry, Prunus × yedoensis Matsumura (Rosales: Rosaceae) (∼7 m tall) and three keyaki Zelkova serrata (Thunb.) Makino (Rosales: Ulmaceae) (∼7 m tall) were inspected for descent behavior by S. contracta larvae. Arboreal larvae were observed carefully for 5 h, as described above, to document their leafcutting behavior.
Ant predation
Predation by ants on first-generation S. contracta larvae was observed at the Tsurumi-ryokuchi and Shotenyama Parks during the day (14:00 – 17:00) on 10 July 1998 and 5 August 2007, respectively. At Tsurumiryokuchi Park, many S. contracta larvae were inspected under one poplar, Populus nigra var. italica L. (Salicales: Salicaceae), (∼10 m tall), that was heavily damaged by the larvae. At Shotenyama Park, six S. contracta larvae were observed under a C. sinensis (∼5 m tall). Some of the ants attacking the larvae were collected for identification. Attacking ants were also sought during the examination of larval descent behavior. | Results
Descent behavior
Along the riverbank, the C. sinensis tree suffered from severe herbivory by a number of S. contracta larvae, with a visual estimate of leaf area loss of ∼90%. On 23 September, no descent behavior was observed, while on 28 September, many larvae were seen exhibiting descent behavior ( Table 1 ). On the ground, there were 804 leaves with the basal parts consumed, 35 crawling or motionless larvae, and three dead larvae, possibly killed by predators, were observed. In a 30 min period, 10 larvae were observed singly on leaves falling to the ground ( Table 1 ). All parachuting larvae were on the upper leaf surface. Most leaves lacked most basal parts of the lamina, but some lacked only the petiole ( Figure 1 ). The heads of the parachuting larvae faced the base of the leaf. The larvae began burrowing into the soil to spin their cocoons 1 to 5 min after reaching the ground. Descent behavior was also observed on 4 and 5 October, although it was past its peak. On 11 October, no larvae were found on the ground; only one larva was observed on a twig, and only seven cut leaves were present on the ground ( Table 1 ).
Although more than 50 S. contracta larvae were carefully inspected on arboreal leaves, none were observed cutting the leaf bases. However, when a larva that had just parachuted to the ground was returned to a twig of the study tree, the larva repeated its parachuting behavior ( Figures 2 , 3 , 4 ). First, the larva spun a few silk threads on the basal part of a leaf and then positioned itself with its head facing the petiole. The larva subsequently fed on the apical part of the petiole ( Figure 2 ). After 11 min, the connection of the petiole to the twig became unstable and the leaf swung from side to side. After 14 min, the larva parachuted to the ground on the falling leaf ( Figures 3 and 4 ).
A number of spherical S. contracta cocoons were found at a soil depth of 0 to 30 mm under the study tree ( Figure 6 ). Examination of these cocoons revealed a density of new cocoons, excluding old empty ones, of 98.9 ± 16.9 (mean ± SE) cocoons/m 2 ( n = 10 quadrats examined). This value was relatively close to the density of parachuting larvae of 70.9 larvae/m 2 estimated based on the cumulative number of cut leaves found under the tree (1135 leaves in 16 m 2 ).
At Tsurumi-ryokuchi Park, leaves cut by S. contracta larvae and larval parachuting behavior were observed under an A. altissima tree ( Table 1 , Figure 5 ). In addition, a parachuting larva was found under a P. × yedoensis tree, and many cut leaves were found under three Z. serrata trees. Most leaves cut by larvae were missing basal parts, as in C sinensis. No larvae were actually observed cutting leaves on these trees.
Ant predation
At Tsurumi-ryokuchi Park in July 1998, ∼50 still or crawling S. contracta larvae that had just parachuted down to the ground were observed under a P. nigra tree. All larvae were attacked, and some were carried away by numerous worker ants, Tetramorium tsushimae Emery (Hymenoptera: Formicidae).
At Shotenyama Park, six S. contracta larvae were attacked by worker ants, Pristomyrmex punctatus Mayr (Hymenoptera: Formicidae), under a C. sinensis tree. These trees harbored many S. contracta larvae. Along the riverbank, and in autumn 2008 at Tsurumi-ryokuchi Park, no ants were observed attacking S. contracta larvae. | Discussion
The presence of fallen leaves without basal parts and crawling larvae, together with the observations of individual larvae on leaves that were falling to the ground ( Table 1 ) of four tree species and the high density of cocoons in the soil ( Figure 6 ), indicate that mature S. contracta larvae parachute from tree crowns to the ground to spin their cocoons in the soil. Although the descent behavior has not been sufficiently observed for larvae, the larvae that were preyed on by ants under trees at Tsurumi-ryokuchi and Shotenyama Parks were those that had just descended. Therefore, this study, together with that of Miyatake ( 1977 ), suggests that this behavior is an inherent trait of S. contracta larvae. This descent behavior can be classified as ‘parachuting,’ because it involves an undirected aerial descent (see Dudley et al. 2007 ).
However, the consumption of the basal parts of leaves by S. contracta larvae was not observed naturally on tree crowns. The low population density of S. contracta larvae, their arboreal habits in high tree crowns, and the difficulty in observing limacodid leaf-feeding (due to concealment of the head and mouthparts) lessened the chances of detecting leaf consumption. In addition, leaf cutting may have been completed relatively quickly. In fact, when a larva that had just parachuted to the ground was returned to a twig, the larva cut the apical part of the petiole of another leaf and parachuted back to the ground in 14 min. Under natural conditions, the fall of leaves and larvae may be interrupted midway to the ground by twigs or other leaves; thus, this repeated parachuting behavior may be favorable to the larvae.
In many cases, larvae consumed basal parts of the leaf lamina, and other larvae cut only petioles. Consumption of basal leaf laminas may be more laborious than that of petioles, although the former may contribute more to food intake than the latter. Because C. sinensis leaves, a preferred food for the larvae, have a palmate venation with three or four main veins at the basal part of the leaf lamina ( Kitamura and Murata 1979 , Figures 1 , 2 , 4 ), larvae must cut the main veins in order to parachute. However, when a larva cuts a basal leaf lamina in a semicircular manner, the larva can locate itself at the center of the leaf, near the leaf's center of gravity, thereby enabling a more stable descent.
The parachuting behavior of S. contracta larvae may be favorable for their cocoon spinning in the soil because both crawling distance and the chance of encountering arboreal enemies may be diminished, and the larvae can descend safely to the ground. This behavior of S. contracta may be unique among the speciose Lepidoptera. Stigmella sp. (Lepidoptera: Nepticulidae) larvae are leaf miners and induce leaf abscission in their host tree, Quercus gilva . They can complete development in the fallen leaves on the ground and pupate in the soil; although, together with the leaves, most are preyed on by deer ( Yamazaki and Sugiura 2008 ). This example is analogous to the present study because both species descend safely from crowns to the ground using leaves. Furthermore, herbivore-caused “greenfall” (non-consumptive leaf loss by herbivores such as petiole clipping, leaf fragment discarding, and leaf abscission) is a widespread phenomenon in forests and may be an important resource for decomposers ( Risley and Crossley 1988 ). Since leaves cut by S. contracta contribute to the greenfall, this behavior may affect various biological communities in forests during S. contracta outbreaks.
The evolution of this parachuting behavior remains unknown. However, the descent behavior appears to be associated with cocoon spinning in the soil. Limacodids have diverse cocoon-spinning sites, such as among leaves and on twigs or trunks ( Young 1986 ; Sugi 1987 ; Yamazaki et al. 2007 ). Limacodid species that make cocoons on twigs or trunks are vulnerable to parasitoid or bird attacks ( Yamada 1992 ; Yamazaki et al. 2007 ). Although cocoon spinning in soil is not common among limacodids, this habit seems favorable to escape from pupal or prepupal parasitoid and bird attacks. The natural enemies of S. contracta have not been intensively examined, except for nuclear polyhedrosis during the larval period ( Aratake and Watanabe 1973 ). In the present study, however, the parachuting S. contracta were occasionally, but severely, attacked by numerous ants just after landing. If ant colonies are located under host trees, upon descent, most larvae may be preyed on by ants, as in July 1998 at Tsurumi-ryokuchi and in August 2007 at Shotenyama Park. As the C. sinensis tree along the riverbank was located within 5 m of flowing river water, most larvae there may successfully spin cocoons in the soil because of the ant-poor environment. The relationship between the unique parachuting behavior and ant predation of S. contracta larvae may account for the fluctuating population dynamics of this species, although further studies on its population ecology, including other developmental stages and mortality factors, are required. | This paper documents the bizarre descending behavior from the tree crown to the ground of the larvae of the moth, Scopelodes contracta Walker (Lepidoptera: Limacodidae) and the interaction of the larva with predatory ants. S. contracta larvae infest leaves of many tree species in urban areas and orchards in Japan. Mature larvae and leaves without basal leaf parts were found under trees of four species infested with S. contracta larvae in Osaka, Japan. Individual larvae riding on leaves were observed falling from tree crowns to the ground. Many S. contracta cocoons were found in the soil below the trees two weeks after the observed parachuting. These observations indicate that S. contracta larvae parachuted to the ground where they spin their cocoons in the soil. When a larva that had just parachuted down was returned to an arboreal twig, the larva repeated the parachuting behavior. This parachuting behavior appears to be adaptive, because larvae can descend to the ground safely and with low energy cost. Worker ants of Tetramorium tsushimae Emery (Hymenoptera: Formicidae) and Pristomyrmex punctatus Mayr (Hymenoptera: Formicidae) occasionally attacked larvae on the ground before they had a chance to burrow in the soil.
Keywords | Acknowledgments
I thank Yorio Miyatake for helpful advice on this study. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 27; 10:39 | oa_package/c9/ac/PMC3014736.tar.gz |
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PMC3014737 | 20578950 | Introduction
The sweet potato whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, is a major pest and plant virus vector attacking a wide variety of food crops worldwide ( Perring 2001 ). The expulsive increase in B. tabaci population has been attributed in part, to the heavy application of pesticides to control it that in turn has resulted in the development of pesticide resistance and in the decline of the whitefly's natural enemies ( Van Lenteren 2000 ). Therefore, it became important to promote the use of effective biological control agents that can efficiently control the pest, are safe to the environment and are acceptable to the farmers and greenhouse growers ( Heinz 1995 ).
Parasitoids of the genera, Amitus, Eretmocerus and Encarsia are among the most important natural enemies of the B. tabaci complex (Cock 1996, Hoelmer 1995 ). Heinz and Parella ( 1998 ) showed that the percentage of parasitoids successfully developing to adults was greater for Encarsia formosa than for Eretmocerus sp., independent of the host plant. Collection records and descriptions of parasitism of Bemisia spp. worldwide indicate differences in guild structure and parasitism intensity depending on the species of host plant sampled and host plant age and size ( Hu and Vinson 2000 ; Hu et al. 2002 ).
A study on the effects of two different host plant species, each infested with B. tabaci, on the fitness of two populations of Eretmocerus sp. was conducted by Powell and Bellows ( 1992 ). The study showed significant differences in preimaginal (development during the egg stage) developmental rate, survival and fertility between host plant species. Also, Liu and Stansly ( 1996 ) described the effect of B. tabaci age at the time of parasitization on the number of egg oviposited, egg to adult development and survivorship of Encarsia pargandiella.
The objectives of this study were to compare reproduction of Encarsia bimacalata attacking various stages of B. tabaci infesting soybean, Glycine max L. (Merr), cowpea, Vigna unguiculata L. and garden bean, Phaseolus vulgaris L. (Fabeles: Fabaceae) and to see how best host plant resistance and biological control can be integrated to suppress whitefly population. | Materials and Methods
Plant materials
Seeds of G. max, P. vulgaris and V. unguiulata were obtained from Guangdong Agricultural Institute in Guangzhou, South China. The seeds were placed in Petri dishes with water to initiate germination. The partially germinated seeds were grown individually in 12 cm diameter plastic pots and used in the experiment at the 4–6 leaf stage. These pots were placed into cages (60 × 60 × 60 cm).
Host and parasitoid culture
Adult B. tabaci were collected from cucumber plants in the field and released into cages (60 cm × 60 cm × 60 cm) containing cucumber as the source host. These cages were placed in a temperature controlled room maintained at 25° C with a relative humidity of 70 % under a photoperiod of 14:10 (L:D).
Encarsia bimaculata cultures were established by collecting parasite wasp pupae from fields located in South China Agricultural University. These were brought to the laboratory and placed into Petri dishes with 10% honey solution as food following eclosion. Adult parasitoids were released on caged cucumber, Cucumis sativus L. (Malvales: Malvaceae) plants infested with with B. tabaci nymphs. Pupae of E. bimaculata turn black inside of parasitized B. tabaci. These black pupae were sexed as female or male by using the criterion that females produce two meconial pellets while males produce four. Virgin female parasitoids were obtained by confining female pupae in vials. Upon emergence, female parasitoids were placed on detached cucumber leaf with abundant whitefly nymphs for approximately six hours. The detached cucumber leaf was confined in a Petri dish, and the leaf petiole was covered with moist cotton wool to retain leaf turgidity. Parasitoids obtained sufficient nutrients for egg lying by feeding on honeydew and the body fluid of whitefly nymphs, which oozed out of ovipositor punctures. Mated female parasitoids were obtained by confining a newly emerged female with two males in a Petri dish for six hours. Two-day-old females were used in all experiments.
Age specific developmental time and parasitism of E. bimaculata
Small confinement cages were made from transparent reagent bottle caps (2.5 cm diameter), into which a capsule-sized hole had been punched for ventilation. Approximately 15–20 unsexed B. tabaci adults were released into each clip cage placed on soy bean leaves. After t24 hours, all adult whiteflies were aspirated from the clip cages and the area of the leaf covered by the clip cages was marked with indelible ink. The egg-infested plants were confined in cages (60 cm × 60 cm × 60 cm) and host stages were allowed to develop to the desired instars (sedentary first, second, third, fourth and pharate adults). When the desired host stage was reached, 40–50 different stage nymphs (sedentary first, second, third, fourth and pharate adults) were independently exposed to 3 mated E. bimaculata parasitoids held in a clip cage. Bemisia tabaci nymphal instars were identified by measuring the body length of the whitefly, and young instars were selected based on their relatively flat appearance ( Gelman et al. 2002b ). All whitefly nymphs that were not of the desired instar were removed from the leaf before the parasitoids were released. This was done to ensure that all the exposed instars were of the same age. Clip cages with parasitoids were removed after 24 hours and the soybean leaf was left in the cage to trap emerging adult flies. On each subsequent day, several randomly selected B. tabaci nymphs were dissected until the egg, first, second and third instars of E. bimaculata were obtained. Parasitoid developmental time was counted from the day the female parasitoid was released. Egg to first instar developmental time was determined when the first instar was observed during dissection. Developmental times for second instar, third instar, prepupa (yellowish-white instar preceding the appearance of black wasp pupa, with two large meconia on either side of the host puparia), and pupa were determined in the same way. For each instar parasitized, at least 10 parasitized whitefly nymphs were dissected each day after parasitization until parasitoid adults emerged. When a wasp pupa was formed, it was removed and placed in a petri dish. The developmental time from egg to pre-pupa, pre-pupa to pupa and pupa to adult E. bimaculata eclosion was recorded separately on all whitefly nymphs (sedentary first, second, third, fourth and pharate adults). Parasitoid total developmental time (egg to adult) for each instar was also recorded from the same observations. Percent parasitism was estimated for all B. tabaci instars based on the number of nymphs in which parasitoid eggs were found following dissection. Percentage of successful adult parasitoid eclosion were also estimated based on the number of adult parasitoids that eclosed and the total number of whitefly nymphs that were parasitized (which was only an estimate of total parasitism) separately for each host stage of B. tabaci. All experiments were repeated six times for each B. tabaci host stage.
Developmental time and parasitism with respect to host plant
Developmental time and parasitism of E. bimaculata on the three test plants was achieved using the same procedure as in the age specific and developmental time experiment except that B. tabaci third instar was used as host for the parasitoid on the three test plants. Approximately 40–50 unsexed B. tabaci adults collected from the culture were released into each clip cage placed on the leaves of the test plants, soybean, cowpea and garden bean. After 24 hours, all adult whiteflies were aspirated from the clip cages and the area of the leaf covered by the clip cage was marked with indelible ink. The egg infested plants were confined in cages (60 cm × 60 cm × 60 cm) and immatures were allowed to develop to the third instar stage which is the most preferred stage for parasitization by Encarsia sp.( Donnell and Hunter 2002 ). When the desired third nymphal stage was reached, 4050 - nymphs were exposed to 3 mated E. bimaculata parasitoids held in a clip cage. Clip cages with parasitoids were removed after 24 hours and the plants left in cages. Developmental time, percent parasitism and percentage emergence on the three host plants were recorded as described in the age specific experiment. All experiments were repeated 6 times for each host plant.
Female adult longevity and fecundity
To determine female fecundity and longevity, a newly emerged E. bimaculata female from the same colony was mated and confined by means of a leaf cage (2.5 cm diameter) to the abaxial side of a fully expanded host plant leaf containing between 15–20 whitefly nymphs (third instar). The female was transferred to a fresh leaf every 24 hours until death to determine the fecundity (number of eggs laid by female parasitoids over her lifetime). After each day, the leaves of the test plants with B. tabaci nymphs that were exposed to the parasitoid were examined under a dissecting microscope for oviposition. Mortality was registered daily. All the host plants with nymphs that were exposed to the parasitoid were kept in cages until the parasitized nymphs contained wasp pupae. The number of eggs laid in the life time of each female was determined based on the number of wasp pupae formed and from nymphs in which the parasitoid immature died before reaching the wasp pupal stage. Leaves with wasp pupae from each host plant were enclosed in Petri dishes with the top covered with plastic for adult emergence and subsequent sex determination. The parasitoids that emerged from the pupae were counted and sexed as described by Heraty and Polaszek ( 2000 ). Survivorship and number of eggs laid each day by the F1 generation were recorded. Fecundity and longevity data were used to calculate daily and lifetime fecundity of E. bimaculata. Twenty parasitoids were tested for each host plant with each host plant replicated 6 times.
Life table and demographic parameters
Life tables were constructed using sex ratio, survivorship, age-specific (the nymphal stage parasitised) fecundity of adults and survivorship and developmental time of all immature stages to calculate intrinsic rate of increase (r m ), finite rate of increase (λ), net reproductive rate (R o ), mean generation time (T c ) and doubling time (T d ) using the formulae of Andrewartha and Birch ( 1954 ).
Statistical Analysis
Data for developmental time (egg deposition to adult emergence), percentage parasitism, and percentage emergence on different B. tabaci instars and the three host plants were analysed using one-way analysis of variance (ANOVA); lifetime fucundity (eggs laid and hatched over the female's lifetime), daily fecundity (eggs laid and hatched per day), and longevity on the three host plants were also analysed using one-way analysis of variance (ANOVA). Means were compared using Fisher's Least Significant (LSD) test at 0.05 level of significance ( SAS Institute 2001 ). | Results and Discussion
Developmental time of E. bimaulata with respect to B. tabaci host age
In a non-choice arena, E. bimaculata was capable of parasitizing all nymphal stages of B. tabaci and was able to complete development through to the adult stage.
The developmental rate of E. bimaculata varied with host age (instar parasitized) ( Table 1 ). The duration of parasitoid development from oviposition to adult emergence was significantly different (F 29,60 = 139.28, P = 0.0001) longer when the first instars were parasitized than when the second, third, fourth and pharate adults were parasitized ( Table 1 ). There was no significant difference in developmental times when third or fourth instars were parasitised. The durations of individual parasitoid stages were also influenced by host age at the time of parasitization ( Table 1 ). Egg to pre- pupa (F 29,60 = 290, df = 4 , P = 0.0001), pre-pupa to pupa (F 29,60 = 140, df = 4 , P = 0.0001) and pupa to adult (F 29,60 = 55.92, df = 4, P = 0.0001) developmental times were significantly different depending upon host age. Developmental times for these three stages were always significantly longer when first instar whiteflies were provided for oviposition ( Table 1 ). The lengthening of the egg and larvae developmental times in younger B. tabaci instars has also been reported for other heteronomous aphelenides ( Walter 1983 ).
However, these stages developed significantly faster when the third or fourth instars were parasitised than when first, second or pharate adults were parasitized ( Table 1 ). Similar findings were reported by Donnell and Hunter ( 2002 ); who observed significantly shorter developmental time and more synchronous adult emergence of E. formosa when third and fourth instars were parasitized. For egg to adult column ( Table 1 ) values do not appear to be significantly different from those reported by Hu et al. ( 2002 ) (standard error overlap) which is surprising considering the reported influence of host plant species and the fact that different wasps were used.
The general preference of E. bimaculata for older instars may be related to certain advantages these instars confer to both the ovipositing females and the would-be developing immature. Firstly, the female can assess the host resources exactly with regard to size, quality and suitability at the moment of oviposition. Secondly, eggs laid in the earlier instars will suffer the same age-related mortality risks such as predation and disease. Thirdly, shorter developmental time in the late instars reduces the period for which the offspring is susceptible to hyperparasitism. Nechols and Tauber ( 1977 ) reported that wasps that begin development in third and fourth instars nymphs exhibited higher survivorship and developmental times were shortened by approximately 38 %.
Percentage parasitism and emergence of E. bimaculata with respect to B. tabaci host age.
Percent parasitism was also influenced by host age ( Table 2 ). Percent parasitism was significantly (F 29,60 =3097 df = 4, P = 0.0001) higher when the third or fourth instars of B. tabaci were parasitized than when the pharate adults, second or first instars were parasitized ( Table 2 ). The mean percent parasitism across the various instars were not much different from the values reported by Antony et al. ( 2003 ) for E. transvena parasitizing B. tabaci instars. These authors observed that even though E. formosa oviposited in all B. tabaci instars, the third and fourth instars were mostly preferred for oviposition compared to the pharate adults or younger instars. Lopez-Avilla ( 1988 ) also reported that E. formosa, E. luteola, E. adrinae and E. cibcenses parasitized all instars of B. tabaci but the third instar had the highest percentage parasitization. Experimental evidence is lacking as to what cues wasps use to determine host size. However, Nell et al. ( 1976 ) reported that wasp may use their antennae to obtain olfactory and resonance information about hosts, and this information coupled with stimuli received while making 180° turns on the dorsum of the nymph may be used to determine host size.
When E. bimaculata parasitized third and fourth instars of B. tabaci, successful adult parasitoid emergence was significantly (F 29,60 = 124.20, df = 4, P = 0.0001) higher when third or fourth instars were parasitised as compared to when first, second or pharate adults were parasitised ( Table 2 ). Similar observations were reported by Antony et al. ( 2003 ) for E. transvena parasitizing B. tabaci instars. Nell et al. ( 1976 ) also reported higher emergence when E. formosa parasitized third and fourth instars of T. vaparariorum. The low emergence of parasitoids in the younger instars of B. tabaci could be related to the high percentage of age-related immature mortality.
Developmental time of E. bimaulata with respect to host plant species
Parasitoid development can be influenced by the nature of plant secondary compounds eaten by their herbivorous hosts (Awmack and Leather 2000). Thus, variation in secondary compounds among individual plants may cause variation in development and successful parasitism of herbivores by specialist parasitoids such as E. bimaculata. With respect to the bean species, the duration of parasitoid development on the third instar of B. tabaci from oviposition to adult emergence was significantly (F 29,60 = 45.80, df = 2, P = 0.0002) longer for parasitoids that developed on garden bean compared to cowpea or soybean ( Table 3 ).
The mean developmental time of the parasitoid on cowpea and garden bean were not much different from the range (15–15.80 d) reported by Shishehber (1996) for E. formosa at the same temperature. On the other hand, the mean value recorded on soybean was slightly different from the range (16–18d) reported by De Barro et al. ( 2000 ) for E. bimaculata on soybean under a temperature regime of between 22–30° C. Similarly, Arakawa ( 1982 ) reported that E. formosa raised on T. vaporariorum showed differences in developmental time of between 15 days on tobacco, egg plant, and tomato compared with 24.5 days on poinsettia at a mean temperature of between 22.5-25° C.
Percentage parasitism and Emergence of E. bimaculata with respect to host plant
The role of plants in the interaction between parasitoid and hosts has been known for some time ( Price et al. 1980 ; Turling et al. 1995 ). Thus, the host plant species on which the parasitoid develops plays a significant role in determining the level of parasitism in subsequent generations.
Comparatively, more nymphs were parasitized on cowpea followed by garden bean and soybean ( Table 4 ); but, the differences were not significant. Similar observation was reported by Goolsby et al. ( 1998 ) in which Encarsia sp. were found to parasitized more nymphs on rockmelon, cotton and hibiscus than tomato and soybean. The reason for the differential parasitization rates on the three bean species may be related to slight differences in leaf morphology among the bean species particularly between soybean and the other two bean species.The leaves of the soybean species used in the study were covered with hairs (trichomes) which prevent the underside of the whitefly nymphs from fitting level to the surface of the leaf thus making it difficult for E. bimaculata to oviposit on the nymphs ( Headrick et al. 1996 ). Also, these hairs may interfere with the movement of the parasitoids thereby reducing parasitism ( Headrick et al. 1996 ). This may account for the low parasitism rate on soybean compared to the other species. On the other hand, the leaves of the other two species were relatively smooth, hence the margin of the whitefly nymph's body can fit level with the leaf surface making it easier for the parasitoid to oviposit. The slight difference in percentage parasitism between cowpea and garden bean may be attributed to the comparatively larger veins on garden bean that might interfere with the foraging behaviour of the parasitoid ( Van Lenteren et al. 1976 ).
When third instars of B. tabaci were presented to E. bimaculata for oviposition, percent adult parasitoid emergence was significantly (F 29,60 = 3.96, df = 2, P = 0.0480) different ( Table 4 ). The highest emergence rate was recorded on soybean followed by cowpea and garden bean. This observation is in accordance with studies carried out by Reed et al. ( 1991 ), which showed that plant species or cultivars identity may influence the proportion of emerging parasitoids. The comparatively higher emergence rate of the parasitoid on soybean and cowpea, respectively, compared to garden bean may be attributed to the high immature mortality of the parasitoid on garden bean. The negative effect of a cultivar exhibiting antibiosis on the pre-imaginal mortality of parasitoids has also been reported by Van Emden ( 1991 ) and Farrar et al. ( 1994 ). It is well-known that secondary substances ingested by phytophagous insects may have negative effects on the biological characteristics (survival, fecundity and development) of parasitoids ( Godfray 1994 ). It has been demonstrated that parasitoids are generally more sensitive to toxic compounds than their polyphagous hosts, as they appear to be incapable of metabolizing the different concentrations of plant secondary compounds present in their host ( Turling et al. 1995 ).
Adult longevity and fecundity
Adult longevity was recorded as part of the fecundity experiment. Although most female parasitoids died by day 9, 11 and 10 on soybean, cowpea and garden bean, respectively, some females were long lived, surviving to 11 d on soybean and 12 d on cowpea and garden bean ( Figure 1 ).
There were significant (F 29,60 = 4.15, df = 2, P = 0.0439) differences in the mean longevity of E. bimaculata on the three bean species ( Table 5 ). Encarsia bimaculata lived longer on cowpea and garden bean as compared to soybean ( Table 5 ).
The mean longevity on the three bean species were lower than the value (10.4 d) reported by Lopez and Botto, ( 1995 ) for local populations of E. Formosa on tomato. These means were however, higher than the value (5.4 d) reported by Powell and Bellows ( 1992 ) at 29° C for an Eretmocerus species (likely E. eremicus ), on B. tabaci on cotton. The host plant species we used (soybean, cowpea and garden bean) were different from those used by Lopez and Botto (tomato) and Powell and Bellows (cotton). Also, Powell and Bellows used a different wasp species ( Eretmocerus sp). A combination of the above factors might have resulted in the discrepancies between our results and those of the other authors. The relatively higher survivorship of the parasitoid on cowpea and garden bean respectively compared to soybean could be attributed to differences in both surface structure and chemistry among the three bean species.
Female E. bimaculata laid an average 26.95 eggs during their lifetime on soybean, 31.10 eggs on cowpea and 30.05 eggs on garden bean. These means were within the reported range for other aphelinids ( Viggiani 1984 ). The means were significantly different (F 2,57 = 45.80, df = 2, P = 0.0002) on the three bean species ( Table 5 ). The daily mean fecundity were however not significantly different (F 4,57 = 0.3907, df = 2, P = 0.6937) on the three bean species ( Table 5 ).
Life table
Results from development and fecundity experiments were used to develop 1x-mx life tables for E. bimaculata on the three bean species. These tables were used were used to calculate the demographic parameters shown in table 6 . The sex ratio of E. bimaculata was 0.7087.
The net reproductive rates (R o ) of E. bimaculata on soybean, cowpea and garden bean are shown in Table 6 . The values recorded on soybean and cowpea were higher than that on garden bean. These values for soybean and cowpea were higher than that reported by Powell and Bellows ( 1992 ) for wasps attacking B. tabaci on cotton (11.74) but, all recorded values were lower than the value (26.50) reported by the same authors for wasps reproducing on cucumber. The low reproductive rate of the parasitod on garden bean indicates that less parasitod females were produced on it compared to soybean and cowpea, respectively.
Generation times (T o ), the finite rate of increase (λ) and doubling time (T d ) on soybean, cowpea and garden bean are shown in Table 6 . Doubling times were not much different among the bean species but, were lower on soybean followed by cowpea and garden bean. The recorded values for λ on soybean and cowpea were slightly different from garden bean
The most important parameter calculated from the life table is the intrinsic rate of increase, r m , which is the rate of increase per individual in an environment where fecundity and survivorship are maximal in the absence of external mortality factors. The larger the r m value, the greater the potential of a species in reproducing and increasing in number within a given environment. This parameter allows for the comparison of r m among species, and it also facilitates the evaluation of a parasitoid concerning its use in strategies where biological control is an option ( Jansen and Sabelis 1992 ). The r m value for soybean, cowpea and garden bean are shown in Table 6 . These values are lower than the value (0.28) reported by Lopez and Botto ( 1995 ) for E. formosa. Small differences in r m values can make remarkable differences in expected population growth over time, therefore, to compare the population growth of the parasitoid on the three bean species over time, the exponential equation for population growth N t = N o e rt was used, where N o is the initial number of parasitoid on the plant, Nt is the number of parasitoid at time t, r m the intrinsic rate of increase and t the time in days. Given a stable age distribution, the parasitoid population on soybean, cowpea and garden bean were calculated as 211.43, 236.38 and 81.11, respectively, within two generations.
Given these life table parameters, parasitoid populations should build up relatively slowly on garden bean compared to the other two bean species. Thus, the parasitoid is comparatively a poor biological control agent on garden bean compared to the other bean species. This conclusion however, cannot be applied to all populations of parasitoids on the three bean species used as the effectiveness of a parasitoid as a biological control agent is dependent, among other factors, on the morphological and chemical composition of the host plant and on the identity the parasitoids population. | Results and Discussion
Developmental time of E. bimaulata with respect to B. tabaci host age
In a non-choice arena, E. bimaculata was capable of parasitizing all nymphal stages of B. tabaci and was able to complete development through to the adult stage.
The developmental rate of E. bimaculata varied with host age (instar parasitized) ( Table 1 ). The duration of parasitoid development from oviposition to adult emergence was significantly different (F 29,60 = 139.28, P = 0.0001) longer when the first instars were parasitized than when the second, third, fourth and pharate adults were parasitized ( Table 1 ). There was no significant difference in developmental times when third or fourth instars were parasitised. The durations of individual parasitoid stages were also influenced by host age at the time of parasitization ( Table 1 ). Egg to pre- pupa (F 29,60 = 290, df = 4 , P = 0.0001), pre-pupa to pupa (F 29,60 = 140, df = 4 , P = 0.0001) and pupa to adult (F 29,60 = 55.92, df = 4, P = 0.0001) developmental times were significantly different depending upon host age. Developmental times for these three stages were always significantly longer when first instar whiteflies were provided for oviposition ( Table 1 ). The lengthening of the egg and larvae developmental times in younger B. tabaci instars has also been reported for other heteronomous aphelenides ( Walter 1983 ).
However, these stages developed significantly faster when the third or fourth instars were parasitised than when first, second or pharate adults were parasitized ( Table 1 ). Similar findings were reported by Donnell and Hunter ( 2002 ); who observed significantly shorter developmental time and more synchronous adult emergence of E. formosa when third and fourth instars were parasitized. For egg to adult column ( Table 1 ) values do not appear to be significantly different from those reported by Hu et al. ( 2002 ) (standard error overlap) which is surprising considering the reported influence of host plant species and the fact that different wasps were used.
The general preference of E. bimaculata for older instars may be related to certain advantages these instars confer to both the ovipositing females and the would-be developing immature. Firstly, the female can assess the host resources exactly with regard to size, quality and suitability at the moment of oviposition. Secondly, eggs laid in the earlier instars will suffer the same age-related mortality risks such as predation and disease. Thirdly, shorter developmental time in the late instars reduces the period for which the offspring is susceptible to hyperparasitism. Nechols and Tauber ( 1977 ) reported that wasps that begin development in third and fourth instars nymphs exhibited higher survivorship and developmental times were shortened by approximately 38 %.
Percentage parasitism and emergence of E. bimaculata with respect to B. tabaci host age.
Percent parasitism was also influenced by host age ( Table 2 ). Percent parasitism was significantly (F 29,60 =3097 df = 4, P = 0.0001) higher when the third or fourth instars of B. tabaci were parasitized than when the pharate adults, second or first instars were parasitized ( Table 2 ). The mean percent parasitism across the various instars were not much different from the values reported by Antony et al. ( 2003 ) for E. transvena parasitizing B. tabaci instars. These authors observed that even though E. formosa oviposited in all B. tabaci instars, the third and fourth instars were mostly preferred for oviposition compared to the pharate adults or younger instars. Lopez-Avilla ( 1988 ) also reported that E. formosa, E. luteola, E. adrinae and E. cibcenses parasitized all instars of B. tabaci but the third instar had the highest percentage parasitization. Experimental evidence is lacking as to what cues wasps use to determine host size. However, Nell et al. ( 1976 ) reported that wasp may use their antennae to obtain olfactory and resonance information about hosts, and this information coupled with stimuli received while making 180° turns on the dorsum of the nymph may be used to determine host size.
When E. bimaculata parasitized third and fourth instars of B. tabaci, successful adult parasitoid emergence was significantly (F 29,60 = 124.20, df = 4, P = 0.0001) higher when third or fourth instars were parasitised as compared to when first, second or pharate adults were parasitised ( Table 2 ). Similar observations were reported by Antony et al. ( 2003 ) for E. transvena parasitizing B. tabaci instars. Nell et al. ( 1976 ) also reported higher emergence when E. formosa parasitized third and fourth instars of T. vaparariorum. The low emergence of parasitoids in the younger instars of B. tabaci could be related to the high percentage of age-related immature mortality.
Developmental time of E. bimaulata with respect to host plant species
Parasitoid development can be influenced by the nature of plant secondary compounds eaten by their herbivorous hosts (Awmack and Leather 2000). Thus, variation in secondary compounds among individual plants may cause variation in development and successful parasitism of herbivores by specialist parasitoids such as E. bimaculata. With respect to the bean species, the duration of parasitoid development on the third instar of B. tabaci from oviposition to adult emergence was significantly (F 29,60 = 45.80, df = 2, P = 0.0002) longer for parasitoids that developed on garden bean compared to cowpea or soybean ( Table 3 ).
The mean developmental time of the parasitoid on cowpea and garden bean were not much different from the range (15–15.80 d) reported by Shishehber (1996) for E. formosa at the same temperature. On the other hand, the mean value recorded on soybean was slightly different from the range (16–18d) reported by De Barro et al. ( 2000 ) for E. bimaculata on soybean under a temperature regime of between 22–30° C. Similarly, Arakawa ( 1982 ) reported that E. formosa raised on T. vaporariorum showed differences in developmental time of between 15 days on tobacco, egg plant, and tomato compared with 24.5 days on poinsettia at a mean temperature of between 22.5-25° C.
Percentage parasitism and Emergence of E. bimaculata with respect to host plant
The role of plants in the interaction between parasitoid and hosts has been known for some time ( Price et al. 1980 ; Turling et al. 1995 ). Thus, the host plant species on which the parasitoid develops plays a significant role in determining the level of parasitism in subsequent generations.
Comparatively, more nymphs were parasitized on cowpea followed by garden bean and soybean ( Table 4 ); but, the differences were not significant. Similar observation was reported by Goolsby et al. ( 1998 ) in which Encarsia sp. were found to parasitized more nymphs on rockmelon, cotton and hibiscus than tomato and soybean. The reason for the differential parasitization rates on the three bean species may be related to slight differences in leaf morphology among the bean species particularly between soybean and the other two bean species.The leaves of the soybean species used in the study were covered with hairs (trichomes) which prevent the underside of the whitefly nymphs from fitting level to the surface of the leaf thus making it difficult for E. bimaculata to oviposit on the nymphs ( Headrick et al. 1996 ). Also, these hairs may interfere with the movement of the parasitoids thereby reducing parasitism ( Headrick et al. 1996 ). This may account for the low parasitism rate on soybean compared to the other species. On the other hand, the leaves of the other two species were relatively smooth, hence the margin of the whitefly nymph's body can fit level with the leaf surface making it easier for the parasitoid to oviposit. The slight difference in percentage parasitism between cowpea and garden bean may be attributed to the comparatively larger veins on garden bean that might interfere with the foraging behaviour of the parasitoid ( Van Lenteren et al. 1976 ).
When third instars of B. tabaci were presented to E. bimaculata for oviposition, percent adult parasitoid emergence was significantly (F 29,60 = 3.96, df = 2, P = 0.0480) different ( Table 4 ). The highest emergence rate was recorded on soybean followed by cowpea and garden bean. This observation is in accordance with studies carried out by Reed et al. ( 1991 ), which showed that plant species or cultivars identity may influence the proportion of emerging parasitoids. The comparatively higher emergence rate of the parasitoid on soybean and cowpea, respectively, compared to garden bean may be attributed to the high immature mortality of the parasitoid on garden bean. The negative effect of a cultivar exhibiting antibiosis on the pre-imaginal mortality of parasitoids has also been reported by Van Emden ( 1991 ) and Farrar et al. ( 1994 ). It is well-known that secondary substances ingested by phytophagous insects may have negative effects on the biological characteristics (survival, fecundity and development) of parasitoids ( Godfray 1994 ). It has been demonstrated that parasitoids are generally more sensitive to toxic compounds than their polyphagous hosts, as they appear to be incapable of metabolizing the different concentrations of plant secondary compounds present in their host ( Turling et al. 1995 ).
Adult longevity and fecundity
Adult longevity was recorded as part of the fecundity experiment. Although most female parasitoids died by day 9, 11 and 10 on soybean, cowpea and garden bean, respectively, some females were long lived, surviving to 11 d on soybean and 12 d on cowpea and garden bean ( Figure 1 ).
There were significant (F 29,60 = 4.15, df = 2, P = 0.0439) differences in the mean longevity of E. bimaculata on the three bean species ( Table 5 ). Encarsia bimaculata lived longer on cowpea and garden bean as compared to soybean ( Table 5 ).
The mean longevity on the three bean species were lower than the value (10.4 d) reported by Lopez and Botto, ( 1995 ) for local populations of E. Formosa on tomato. These means were however, higher than the value (5.4 d) reported by Powell and Bellows ( 1992 ) at 29° C for an Eretmocerus species (likely E. eremicus ), on B. tabaci on cotton. The host plant species we used (soybean, cowpea and garden bean) were different from those used by Lopez and Botto (tomato) and Powell and Bellows (cotton). Also, Powell and Bellows used a different wasp species ( Eretmocerus sp). A combination of the above factors might have resulted in the discrepancies between our results and those of the other authors. The relatively higher survivorship of the parasitoid on cowpea and garden bean respectively compared to soybean could be attributed to differences in both surface structure and chemistry among the three bean species.
Female E. bimaculata laid an average 26.95 eggs during their lifetime on soybean, 31.10 eggs on cowpea and 30.05 eggs on garden bean. These means were within the reported range for other aphelinids ( Viggiani 1984 ). The means were significantly different (F 2,57 = 45.80, df = 2, P = 0.0002) on the three bean species ( Table 5 ). The daily mean fecundity were however not significantly different (F 4,57 = 0.3907, df = 2, P = 0.6937) on the three bean species ( Table 5 ).
Life table
Results from development and fecundity experiments were used to develop 1x-mx life tables for E. bimaculata on the three bean species. These tables were used were used to calculate the demographic parameters shown in table 6 . The sex ratio of E. bimaculata was 0.7087.
The net reproductive rates (R o ) of E. bimaculata on soybean, cowpea and garden bean are shown in Table 6 . The values recorded on soybean and cowpea were higher than that on garden bean. These values for soybean and cowpea were higher than that reported by Powell and Bellows ( 1992 ) for wasps attacking B. tabaci on cotton (11.74) but, all recorded values were lower than the value (26.50) reported by the same authors for wasps reproducing on cucumber. The low reproductive rate of the parasitod on garden bean indicates that less parasitod females were produced on it compared to soybean and cowpea, respectively.
Generation times (T o ), the finite rate of increase (λ) and doubling time (T d ) on soybean, cowpea and garden bean are shown in Table 6 . Doubling times were not much different among the bean species but, were lower on soybean followed by cowpea and garden bean. The recorded values for λ on soybean and cowpea were slightly different from garden bean
The most important parameter calculated from the life table is the intrinsic rate of increase, r m , which is the rate of increase per individual in an environment where fecundity and survivorship are maximal in the absence of external mortality factors. The larger the r m value, the greater the potential of a species in reproducing and increasing in number within a given environment. This parameter allows for the comparison of r m among species, and it also facilitates the evaluation of a parasitoid concerning its use in strategies where biological control is an option ( Jansen and Sabelis 1992 ). The r m value for soybean, cowpea and garden bean are shown in Table 6 . These values are lower than the value (0.28) reported by Lopez and Botto ( 1995 ) for E. formosa. Small differences in r m values can make remarkable differences in expected population growth over time, therefore, to compare the population growth of the parasitoid on the three bean species over time, the exponential equation for population growth N t = N o e rt was used, where N o is the initial number of parasitoid on the plant, Nt is the number of parasitoid at time t, r m the intrinsic rate of increase and t the time in days. Given a stable age distribution, the parasitoid population on soybean, cowpea and garden bean were calculated as 211.43, 236.38 and 81.11, respectively, within two generations.
Given these life table parameters, parasitoid populations should build up relatively slowly on garden bean compared to the other two bean species. Thus, the parasitoid is comparatively a poor biological control agent on garden bean compared to the other bean species. This conclusion however, cannot be applied to all populations of parasitoids on the three bean species used as the effectiveness of a parasitoid as a biological control agent is dependent, among other factors, on the morphological and chemical composition of the host plant and on the identity the parasitoids population. | Developmental time, parasitism, emergence, longevity, fecundity and demographic parameters of population of Encarsia bimaculata Heraty and Polaszek (Hymenoptera: Aphelinidae), a parasitoid attacking Bemisia tabaci (biotype B) (Gennadius) (Homoptera: Aleyrodidae) infesting soybean, Glyine max L. (Merr), cowpea, Vigna unguiulata L. and garden bean, Phaseolus vulgaris L. (Fabeles: Fabaceae) were quantified and compared. Encarsia bimaculata was able to complete its life cycle independent of the B. tabaci instar parasitized. However, parasitoid development was significantly slower when first (19 d), second (15 d) instars or pharate adults (14 d) were parasitized compared to the third (13 d) or fourth (13 d) instars. Consequently, percent parasitism was higher when the third (51 %) or fourth (46 %) instars were parasitized compared to the first (22 %), second (25 %) instars or pharate adults (36 %) of B. tabaci. Similarly, percent parasitoid emergence was significantly higher when third (83 %) or fourth (76 %) instars were parasitized compared to when the first (34 %), second (64 %) or pharate adults (54 %) were parasitized. Host plant species significantly influenced egg to adult developmental time, percent parasitism and the day on which E. bimaculata nymphs hatching from eggs was first observed. More nymphs were parasitized on cowpea (40 %) followed by garden bean (36 %) and soybean (32 %), while percent hatching was significantly higher on soybean (76 %) followed by cowpea (68 %) and garden bean (42 %). Adult parasitoid females lived an average of 6.7 d on soybean, 7.6 d on cowpea and 7.2 d on garden bean and laid a lifetime average of 27 eggs on soybean, 31 eggs on cowpea and 30 eggs on garden bean. The daily mean fecundity of E. bimaculata was not significantly different on the three bean species. Life table parameters showed that the net reproductive rate (R o ) was 14.50, generation time (T c ) was 17.16, intrinsic rate of natural increase ( r m ) was 0.16, finite rate of growth (λ) was 1.17 and doubling time (T d ) was 4.44 for parasitoids on soybean. On cowpea, R o was 15.32, T c was 18.59, r m was 0.15, λ was 1.16 and T d was 4.72, while, on garden bean, R o was 8.95, T c was 19.28, r m was 0.11, λ was 1.12 and T d was 6.08. Given these life table parameters, higher population build up of the parasitoid will be expected on cowpea and soybean, respectively, compared to garden bean. Thus, for an effective augmentative release program involving E. bimaculata for the control of B. tabaci, it is important to take into consideration both the host stage of B. tabaci and the nature of the host plant on which it is developing.
Keywords | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 3; 10:28 | oa_package/fd/cf/PMC3014737.tar.gz |
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PMC3014738 | 20578881 | Introduction
In ants, solitary foundation of a new colony generally includes a nuptial flight, followed by dealation and finding a nest site. Solitary colony foundation is risky, and usually more than 99% of virgin winged queens (hereafter referred to as gynes) will die ( Hölldobler and Wilson 1990 ; Wiernasz and Cole 1995 ). Alternative reproductive strategies have also evolved in ants ( Heinze and Tsuji 1995 ; Peeters and Ito 2001 ). Fecundated gynes may return to their natal nest or another conspecific nest where they seek adoption. The consequence is a polygynous colony that may form new nests by budding ( Bourke and Franks 1995 ; Peeters and Ito 2001 ). In polygynous colonies, the gynes tend to be smaller microgynes, rather than the normal macrogynous queens.
In many ant species, queen size is dimorphic, with a large macrogynous morph (usually the normal morph) and a small microgynous morph. The significance of these morphs is rarely understood, but at least three explanations exist. First, the morphs may represent two dispersal tactics with macrogynes dispersing and microgynes filling up the area locally (Hölldobler and Wilson 1977). Some examples include Myrmica ruginodis ( Elmes 1991 ), Ectatomma ruidum ( Lachaud et al. 1999 ), Temnothorax rugatulus ( Rüppell et al. 2001 ) and Mystrium rogeri ( Molet et al. 2007 ). Unfortunately, no genetic differences have been searched between these morphs. Second, some microgynes are social parasites of the macrogynes and are genetically differentiated from their hosts. Examples include the microgynes of Myrmica rubra ( Savolainen and Vepsäläinen 2003 ) and Ectatomma tuberculatum ( Hora et al. 2005 ), now described as a distinct species ( Feitosa et al. 2008 ). Third, the selfish larvae try to develop into gynes when they should become workers with limited or no reproductive capacity ( Bourke and Ratnieks 1999 ; Beekman et al. 2003 ).
In August 1998, small alate gynes were found in a colony of Manica rubida (Latreille) (Hymenoptera: Formicidae) in the French Alps. Typically, the macrogynes of M. rubida (total length 10–13 mm) are clearly bigger than the workers (6–8 mm) ( Bernard 1968 ), but the gynes discovered were the same size as the workers. Thus these gynes were microgynes, and, apparently, the discovery was novel, as we could not find in the literature any information on microgynes of M. rubida . Later, more colonies with microgynes were found in the same area.
Because Monica is the sister genus to Myrmica (Bolton 2003), some similarities in the microgynes of M. rubida and either Myrmica ruginodis or Myrmica rubra were expected. Thus, the M. rubida microgynes were studied by investigating their size, behavior and spermatheca in relation to M. rubida macrogynes and workers. Mitochondrial DNA of the microgynes and macrogynes was also sequenced to detect possible genetic differences between them. | Materials and Methods
Manica rubida is frequent in European mountain regions from 500 to 2000 MASL ( Seifert 1996 , 2007 ) and in France in the Alps and Central Massif between 700 and 1800 MASL. It nests in open fields with low slopes where it constructs large, but superficial, subterranean nests ( Bernard 1968 ). Colonies contained several thousand workers and were either monogynous having a single queen, or polygynous with a few large, macrogynous queens ( Cammaerts and Cammaerts 1987 ). Colony foundation is semi-claustral and takes place after a nuptial flight ( Le Masne and Bonavita 1969 ).
Manica rubida was studied in the Giffre Valley (Haute-Savoie, French Alps) that runs east-west from Taninges (600 MASL, 46° 06.37 N, 6° 33.30 E) and covers about 10 km, ending in a large, touristic area (Cirque du Fer à Cheval, 1080 MASL, 46° 03.36 N, 6° 47.29 E). Manica rubida was frequent on the flat banks of the river that had little vegetation, composed mainly of alder, Alnus sp .. Other ant species in the area were Formica selysi and F. lemani . The first M. rubida colony (colony or nest A) with both macrogynes and microgynes was observed in August 1998, near Samoëns (698 MASL, 46° 04.39 N, 6° 42.43 E). This colony had been observed regularly for many years prior to 1998 without the sighting of any microgynes. Thereafter, this colony was monitored at least three times per year, particularly after the nuptial flight period at the end of May/early June and after winter, to assess the presence of alate microgynes in the nest.
In July 2002, a small nest (colony A 1 ) was observed 5 m from colony A, which suggested budding from nest A. In June 2003, another colony (H) with microgynes was observed three km east of nest A, in Morillon (46° 05.14 N, 6° 41.18 E). In June 2004, 15 colonies along an area of 6 km were found between La Rivière Enverse and Samoëns, with some colonies having both macrogynes and microgynes and some containing only macrogynes. In 2005, a large colony (P) was found in Verchaix (46° 05.33 N, 6° 39.54 E) with only microgynes. Manica rubida was searched for in a few other places: Bessans (Savoie, 1500 MASL, 45° 19.11 N, 7° 01.24 E) and Urle (Vercors, Isère, 1434 MASL, 44° 53.51 N, 5° 19.16 E), where no colonies with microgynes were found. In August 2006, one colony with both macrogynes and microgynes was discovered in the Hautes-Alpes, La Chapelle en Valgaudemar (1180 MASL, 44° 49.47–48 N, 6° 14.00–21 E), in a place where they were absent some years before.
Size of gynes and males
The maximum head width, maximum thorax width and thorax length of the macrogynes ( n = 35) and microgynes ( n = 55) of colony A were measured using a binocular microscope. The HW of males were also measured, including the eyes, from two colonies with only macrogynes ( n = 45) and two colonies with only microgynes ( n = 41).
Aggression tests
In the field, antagonistic behavior occurs between workers of different Monica nests ( Cammaerts and Cammaerts 1987 ; A. Lenoir personal observation). In fissioning species, workers of recently separated nests, however, often interact with workers of their mother nest and thus are not aggressive to each other ( Ichinose et al. 2005 ). To investigate whether nest A 1 originated (fissioned) from nest A, aggression tests were conducted between the workers of colonies A and A 1 . Several hundred foragers of colony A were collected, as was the entire colony A 1 , which contained one dealate microgyne and several hundred workers. Control experiments were set-up between workers of the same nest (nests H and M). If A 1 originated from nest A, aggression should be weak between their workers. As aggression increases with geographical distance between nests ( Cammaerts and Cammaerts 1987 ), aggression was compared between workers of nests A and A 1 and those of nests H and M, which were separated by several km from the former nests.
First three workers (residents) of one nest were placed on an arena (diameter = 85 mm, walls coated with fluon to prevent escaping); then a marked individual (intruder) from another nest was added. The intruder was placed in a tube in the middle of the arena, and after one min, the tube was removed . The bottom of the arena was covered with filter paper that had been kept for several hours in the nest of the three resident ants to impregnate their colony odor on it (Cammaerts and Cammaerts 2000). The behavior (see below) of the intruder was recorded every 5 s for 5 min. Old workers (darker color) were selected for the tests, as they are considered more aggressive than young ones (Cammaerts-Tricot 1974). A global aggression index, AI, was calculated according to a previously published formula ( Hefetz et al. 1996 ): AC is a coefficient of aggression for each behavior, f is its frequency, and n s the total number of acts, i. The aggression coefficients for each act were: 1 = threat (opening of mandibles); 2 = biting; 3 = stinging or its trial; 0 = all other behaviors. To consider the possible differences of aggression between colonies, one intruder A was tested against three B resident workers, and, reciprocally, one B intruder was tested against three residents of A. For each situation, 20 tests were carried out. The data was analyzed with the Kruskal-Wallis or Mann-Whitney test.
In founding colonies, the first workers are generally small and “nanitic” ( Porter and Tschinkel 1986 ). Therefore, to check if nest A 1 contained these nanitic workers, the size of workers from nest A 1 ( n = 50) were measured, as were workers from another nest that contained only macrogynes ( n = 51). The size index (AI) was used to weigh the head and thorax equally ( Rüppell et al. 1998 ): Where IT is size index, HW is head width, ThW is thorax width and ThL is thorax length.
Colony tasks: Egg-laying, brood retrieval, division of labor
As microgynes stayed in their natal nest and did not engage in nuptial flight, whether they exhibited any gyne or worker behaviors was tested. To compare egg-laying rate between macrogynes and microgynes, six combinations of individuals collected in the field were formed: one dealated microgyne alone ( n = 10), and with 20 workers ( n = 12); one alate microgyne alone ( n = 21), and with 20 workers ( n = 6); groups of 20 workers ( n = 10); and one macrogyne with 20 workers ( n = 7). These groups were reared in the laboratory, and all eggs laid were counted twice a week for two months.
In preliminary experiments, in groups of workers with one macrogyne, only workers retrieved brood to the nest. To further investigate whether microgynes and macrogynes differed in their behavior, two experiments were performed. (1) In broodretrieval experiments, a group of 20 workers and 10 microgynes ( n = 13), were placed one group at a time in a glass tube with some water plugged with cotton, and the tube was closed with cotton (the ants and brood were used only once). A black cover was placed over the tube to represent the conditions inside a nest, and the tube was placed in a box (10 × 15 cm). Then, 10 larvae and 10 pupae were deposited near the entrance of the tube, and the cotton plug was removed. The total time to retrieve all the brood was recorded from the first contact with the brood. (2) In the division of labor experiments, groups of 20 individuals with 20 larvae and 20 pupae were placed in a plaster nest covered with a piece of glass to allow observation of ants. Three nests were made: 10 alate microgynes and 10 workers, 10 dealate microgynes and 10 workers, and one control with 20 workers. Each individual was marked with a unique combination of color dots. The nest was linked to the arena (as above) where food was deposited. For each nest, the behavior of each individual (scan sampling) was recorded instantaneously in at least 5 min intervals on three days ( n = 100).
Foraging in the field
Dealate macrogynes were observed foraging in the field in the summer, thus verifying their non-claustral colony foundation ( Le Masne and Bonavita 1969 ). In the territories of established colonies, foraging alate microgynes were also repeatedly observed each summer. In one nest (M) in June 2004, the microgynes were offered cookie crumbs 80 cm from their nest entrance. All foraging microgynes were marked with a dot of paint, and the number of different microgynes with cookie crumbs was counted for one hour.
Spermatheca and spermatozoid count
To observe the status of their spermatheca, 50 gynes (3 alate and 11 dealate macrogynes, 26 alate and 10 dealated microgynes) were dissected and their sperm counted according to Lenoir et al. (1999). Spermathecae were isolated in a saline drop (128.3 m M NaCl, 4.7 m M KCl, 2.3 m M CaCl 2 ) and then transferred to 100 μl of the same saline solution. They were opened with forceps. The resulting suspension was gently shaken to disperse sperm and then homogenised. Three 1-μl drops of the final suspension were deposited on a clean microscope slide and air dried. The preparations were fixed by ethanol, dried again, and incubated for 10 min in a 2 μg/ml DAPI solution (4-6-diamidino-2-phenylindole) to stain nuclei. All sperm were counted in the three drops under a fluorescence microscope to obtain the total amount of sperm stored in the spermatheca.
Genetic analysis
DNA was extracted from six individuals from four nests (in total, 24 individuals): three workers (presumed macrogynes, because no macrogynes were available) and three microgynes from colony A 1 , and two macrogynes, microgynes and workers from colonies H, J5 and M. Partial cytochrome c oxidase subunit I gene was amplified using the primers C1-J-1751 (alias Ron), C1-J-2183 (alias Jerry) and TL2-N-3014T (alias Pat) (Simon et al. 1994), following the molecular methodology of Savolainen and Vepsäläinen ( 2003 ). We edited and aligned the sequences and visualized their base pair differences using Sequencher v. 4.7. (Gene Codes, www.genecodes.com ). | Results
All field observations since 1998 indicated that microgynes did not engage in nuptial flights, but, instead, overwintered in their natal nest. They kept their wings until the next spring which damaged them such that in spring some of the microgynes were dealated. It is unknown if microgynes survive more than one year.
Size of gynes and males
The head width of the macrogynes and microgynes was clearly dimorphic without overlap ( Figure 1A ). The head width of the macrogynes was 2.02 mm ± 0.085 ( n = 35), and that of the microgynes was 1.61 mm ± 0.065 ( n = 55) (Student t-test, p = 0.001). The thorax width was also bimodally distributed, though with some overlap ( Figure 1B , 1.45 mm ± 0.077, n = 41 vs. 1.08 mm ± 0.130, n = 50; Student t-test, p = 0.0009). The scutum and scutellum were more developed in the macrogynes than in the microgynes. The dotted lines indicate that the scutum increases in size in microgynes (not measured) ( Figure 2 ). The size of males was not different between the colonies producing either macrogynes or microgynes (1.04 mm ± 0.15, n = 44 vs. 1.06 ± 0.13, n = 45; Student t-test, p = 0.65, for macrogynes and microgynes, respectively). In contrast, the size of the males was dependant of the colony. For the males in the two microgyne colonies, the size was 0.94 mm ± 0.11 ( n = 14) and 1.12 mm ± 0.09 ( n = 30) (Student t-test, p < 0.0001). For the two macrogyne colonies, the size was 0.95 mm ± 0.14 ( n = 20) and 1.12 mm ± 0.11 ( n = 25) (Student t-test, p < 0.0001).
Aggression tests
Intracolonial aggression was absent in the control tests (AI within colony = 0; Figure 3 ). The workers of nest A 1 were smaller than those of nest C (Student t-test, p < 0.001). The workers of nest A 1 included two size classes (mean index IT 1.3 and 1.6), whereas the workers of nest C, a typical nest with macrogynes, were bigger (IT 1.75) ( Figure 4 ). These data are congruent with the behavioral data, indicating that the budded nest is composed of two worker types: small workers and large workers.
Colony tasks: Egg-laying, brood retrieval and division of labor
Among the 6 types of experimental colonies followed during two months, neither workers nor non-inseminated microgynes (winged or wingless) laid eggs, whereas macrogynes normally laid eggs and produced workers.
Macrogynes did not engage in brood rescue. In contrast, microgynes were efficient in brood-retrieval tests; there was no difference between microgynes (318 ± 126 s) and workers (332 ± 210 s) in mean retrieval time (Wilcoxon test, p = 0.361, n = 13).
In the laboratory, microgynes behaved like workers. They spent on average 10 to 25% of their time in caring for the brood (no significant differences between workers and microgynes in the three sets). Occasionally, a worker ant tried to cut the wings of a microgyne, but again, both workers and microgynes practiced this behavior. Behaviors that distinguished microgynes from workers were not detected.
Foraging in the field
Observations confirmed that dealate macrogynes (founding queens) foraged solitarily during their non-claustral founding period in summer. They foraged only in areas unoccupied by Manica colonies, and they never foraged in Manica territories. In mature colonies producing microgynes, some of them left the nest to forage, and they behaved like foragers, retrieving food items that were brought to the nest. In one hour, there were 53 different microgynes foraging around the same nest, which is equivalent to the foraging task of a worker.
Spermatheca and spermatozoid count
The microgynes had normal spermatheca ( Figure 5A–D ), but only 1 of 38 dissected gynes (2.8%) was inseminated. All dealated macrogynes were inseminated. The sperm counts of macrogynes were 340,388 ± 50,520 ( n = 17) (see spermatozoids in Figure 5E, F ). The single inseminated microgyne contained spermatozoids, but unfortunately the sperm could not be counted.
Mitochondrial DNA differences
For all 24 individuals, 1023 bp were obtained (Genbank accession numbers EU864121– EU864144). Only three haplotypes were found; all individuals were genetically identical, except for three workers. The two workers of nest J5 had a substitution at one third-codon position (C at position 492), and one worker of nest A 1 had a substitution at another third-codon position (C at position 594). The other individuals had T in these positions. | Discussion
The size distribution of Manica rubida gynes was clearly bimodal and not a result of random phenotypic variation. The males, however, were not dimorphic. Because these microgynes had features of both the queens (morphology, possibility to be inseminated) and workers (brood retrieval, foraging), their functional significance in the colony is not clear. There are at least four hypotheses for the existence of microgynes in ant colonies.
These hypotheses are not mutually exclusive and combine proximate (hypothesis 1) and ultimate explanations (hypothesis 2–4). (1) Numerous studies have accumulated evidence that differences in larval environment, particularly in nutrition, determine the developmental path of a larva into a reproductive worker or a gyne ( Wilson 1971 ; Hölldobler and Wilson 1990 ). Recent work on caste determination indicates, however, that it is, instead, genetically based, at least in Pogonomyrmex ( Julian et al. 2002 ; Volny and Gordon 2002 ; Helms Cahan and Keller 2003 ) and Reticulitermes ( Hayashi et al. 2007 ). Therefore, a mutation of developmental genes may have appeared, preventing larvae from developing into normal macrogynes. (2) Microgynes are a consequence of a caste conflict between larvae and the queen or workers. The selfish larvae try to escape queen or worker control over sexual production and develop into gynes rather than becoming workers with limited reproductive capacity. This is especially true for Manica workers, which are completely sterile. The brood of social Hymenoptera is generally considered to have little ability to affect the choice of their development into sexuals or workers, because they are fed by adult workers ( Bourke and Ratnieks 1999 ; Beekman et al. 2003 ). When larvae are reared in sealed cells, like in Melipona bees, they may choose to develop into workers or queens ( Ratnieks 2001 ). This may also take place in ant species with larvae that feed themselves. With a limited amount of food, miniaturization is one mechanism for diploid larvae to become queens ( Ratnieks et al. 2006 ). According these authors, it is the most likely explanation for the existence of microgynes in social Hymenoptera. (3) The microgynes are social parasites of the macrogynes and genetically differentiated from their hosts. Examples include the microgynes of Myrmica rubra ( Savolainen and Vepsäläinen 2003 ) and Ectatomma tuberculatum ( Hora et al. 2005 ), now described as a distinct species, Ectatomma parasiticum ( Feitosa et al. 2008 ). In M. rubra , the microgynous social parasite differs genetically, though only slightly, from its host ( Savolainen and Vepsäläinen 2003 ). These authors suggested that this social parasite/host pair may represent an incipient sympatric speciation process taking place through intraspecific social parasitism. Sympatric speciation has been a highly debated process, but now there are well documented cases of it, for example, in palm trees ( Savolainen et al. 2006 ). In M. rubida , mitochondrial DNA analysis showed only minor genetic differences: three workers (presumed issued from macrogynes) differed in one base pair from microgynes and macrogynes. Further genetic analyses are needed, but it seems very improbable they can be considered as two species. (4) The microgynes represent an alternative reproductive tactic and are part of a dispersal strategy. The small microgynes may spread locally after intranidal copulation from the mother nest, whereas the large macrogynes establish new colonies after a nuptial flight (Hölldobler and Wilson 1977). Some examples include Myrmica ruginodis ( Elmes 1991 ), Ectatomma ruidum ( Lachaud et al. 1999 ), Temnothorax rugatulus ( Rüppell et al. 2001 ) and Mystrium rogeri ( Molet et al. 2007 ). The presumed dispersal strategy of M. rubida is not efficient, however, as the colonies produce many microgynes that stay in the home nest as workers and forage. Foraging of Manica macrogynes is normal during the non-claustral founding stage, but it has never been observed in queens after colony foundation.
The cost of producing numerous microgynes in M. rubida is also worth discussing. If microgynes behave like workers and are of the same size as the workers, there is presumably no cost. If the microgynes work and reproduce less than the workers, however, then the colony may suffer some costs. This may be a case where an individual's interest (to become a queen instead of a worker) conflicts with the colony interest (to maximize the colony productivity). The conflict may be unstable and selected against, like semi-claustral foundation ( Peeters and Ito 2001 ). | In many ant species, queen size is dimorphic, with small microgynes and large macrogynes, which differ, for example, in size, insemination rate, ovary development, and dispersal tactics. These polymorphic queens often correspond with alternative reproductive strategies. The Palearctic ant, Manica rubida (Latreille) (Hymenoptera: Formicidae), lives mostly in mountainous regions in either monogynous colonies, containing one macrogynous queen or polygynous colonies, containing a few large macrogynous queens. In 1998, a colony of M. rubida was discovered containing macrogynes and many small alate microgynes that did not engage in a nuptial flight but, instead, stayed in the home nest the following winter. These microgynes were studied more closely by investigating their size, behavior, and spermatheca in relation to M. rubida macrogynes and workers. Mitochondrial DNA of macrogynes, microgynes and workers from four nests was sequenced to detect possible genetic differences between them. The microgynes were significantly smaller than the macrogynes, and the head width of the gynes was completely bimodal. The microgynes behaved like workers of the macrogynes in every experiment tested. Furthermore, the microgynes had a normal spermatheca and could be fecundated, but rarely (only one in several years). Finally, all the individuals were genetically identical, except three workers that differed by only one codon position. Because these microgynes have features of both queens and workers, their functional significance in the colony is not yet clear.
Keywords | Acknowledgments
We thank Nicolas Thurin and Jérémie Saulnier for fieldwork in the Giffre Valley, Christine Errard and Laurianne Leniaud for discussions, and the Academy of Finland for funding to RS. We thank two anonymous reviewers and the editors for their useful remarks.
Abbreviation
meters above sea level,
virgin winged queens | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Mar 15; 10:17 | oa_package/73/6a/PMC3014738.tar.gz |
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PMC3014739 | 20575737 | Introduction
Insects use hooks or spines to adhere to rough substrates; however, when these structures cannot engage on smoother surfaces, attachment pads are used ( Arnold 1974 ; Walker 1992 ; Fraizer 1999 ; Goodwyn et al. 2006 ). These attachment pads must adhere successfully to the substrate but also must have the capacity to be readily and easily detached ( Gorb and Scherge 2000 ; Gorb et al. 2002 ). Attachment pads vary greatly in morphology but commonly are composed of a hairy surface or a smooth flexible pad. Both types of pad are adapted to maximize contact with the diverse array of substrate with which insects potentially come into contact. This then allows the insect to rapidly locomote while still gaining a firm adhesion to the substrate ( Walker 1992 ; Gorb et al. 2000 ; Votsch et al. 2002 ; Drechsler and Federle 2006 ; Goodwyn et al. 2006 ).
The hairy pad system used by flies, beetles, and earwigs consists of many deformable seta that allow a maximal contact with the substrate giving the adhesive quality ( Votsch 2002 ; Niederegger et al. 2002 ; Langer et al. 2004 ). The smooth pad system that is used by cockroaches, grasshoppers and bugs is a soft deformable pad that mimics the contours of the substrate allowing maximal contact. ( Jiao et al. 2000 ; Beutel and Gorb 2001 ; Gorb et al. 2002 ; Federle et al. 2002 ; Drechsler and Federle 2006 ). The deformable pad is made up of mainly endocutical, with a fibrous structure. Under pressure these fibres can flatten and move close together allowing the pad to mimic the substrate, while as pressure is released, the fibers return to their original position ( Jiao et al. 2000 ; Beutel and Gorb 2001 ; Goodwyn et al. 2006 ). The construction of the pad enables it to provide stability as well as being highly flexible, meaning the pad will deform to match the substrate, therefore allowing the insect to adhere to a range of substrates found in mobile terrestrial life ( Roth and Willis 1952 ; Gorb et al. 2000 ; Jiao et al. 2000 ; Beutel and Gorb 2001 ; Gorb and Beutel 2001 ; Gorb et al. 2002 ).
In both forms, pad adhesion is facilitated by small amounts of fluid that is secreted into the area of contact. The fluid does not solely account for the adhesive ability of insects, but it is necessary for adhesion to occur ( Jiao et al. 2000 ; Votsch et al. 2002 ). The fluid secreted by the pads is a two phase hydrophobic and hydrophilic liquid ( Jiao et al. 2000 ; Votsch et al. 2002 ; Federle et al. 2002 ; Drechsler and Federle 2006 ). it is an emulsion made up of a water soluble fraction and lipid-like nano-droplets ( Votsch et al. 2002 ). The water soluble part of the secretion contains carbohydrates, consisting mainly of glucose, but xylose and mannose are also found in lower concentrations. It is also presumed that amino acids and proteins are present in the secretion, which could act to increase viscosity of the fluid; but there is no definitive evidence to support this, and it is unclear whether these elements are deliberately added to the liquid or if they occur simply as contamination of the compounds ( Votsch et al. 2002 ). The lipid-like nano-droplets of the secretion most likely are made up of fatty acids ( Votsch et al. 2002 ). The mechanism by which this fluid secretion aids adhesion is not yet fully understood, but there are a variety of explanations within the literature. The simplest explanation was that the fluid itself acted as a kind of sticky glue, but this has been largely discredited ( Jiao et al. 2000 ; Votsch et al. 2002 ). There is another theory of “wet adhesion” whereby the viscosity of the fluid aids adhesion of the pad to the substrate ( Federle et al. 2002 ; Votsch et al. 2002 ; Drechsler and Federle 2006 ). A further theory is that the fluid helps in providing greater attachment to rough substrates. The fluid fills the cavities in a rough substrate allowing the smooth deformable pad to gain maximal contact with the surface where a dry pad would only make limited contact ( Drechsler and Federle 2006 ). These types of secretions are described in many cockroach species ( Roth and Willis 1952 ; Arnold 1974 ), and therefore it can be assumed to facilitate the adhesive abilities of Gromphadorhina portentosa Schaum (Blattaria: Blaberidae).
G . portentosa, like all cockroaches, possesses smooth flexible pads found at the distal end of the leg on the tarsus. There are five pads altogether; the first four segments of the tarsus contain pads called the euplantula, while the most flexible fifth segment found at the distal end of the tarsus and is called the pretarsus, which is made up of a broadly triangular adhesive pad called the arolium and two pretarsal claws ( Roth and Willis 1952 ; Dailey and Graves 1976 ). The euplantula and arolium have been shown to have different uses during terrestrial locomotion. The euplantula are used when the legs are pushing, in walking and climbing, and have been demonstrated not to act as adhesive organs but rather in fact as friction pads used to power locomotion ( Clemente et al. 2008 ). The pretarsal claws allow cockroaches to grip and move over rough surfaces, and the arolium is the adhesive organ of the cockroach foot engaged when the legs are pulling and used in scaling and adhering to smooth surfaces ( Arnold 1974 ; Dailey and Graves 1976 ; Clemente et al. 2008 ).
There are relatively few studies on the adhesive ability of live insects, and these tend to focus on the hairy pad system of adhesion. Here, light and electron microscopy are used in combination with biomechanical testing to test the hypothesis that the adhesive ability of G . portentosa is related to substrate morphology. | Methods
Cockroach husbandry
Experiments were conducted upon five adult, female cockroaches of the species G. portentosa. Females were used as aggression by males in the colony can lead to tarsal damage, and males commonly were missing one or more tarsa. G . portentosa were kept in a tank (35 cm × 20 cm × 22 cm) at room temperature (≈ 18–22° C). The tank had approximately 2 cm of orchid bark substrate, and cardboard rolls and tree stumps were provided for housing. G . portentosa were fed a combination of vegetable matter with dried dog food for protein. Food and water were provided ad libitum.
Microscopy
Light microscope pictures were taken using a Leica MZ9s ( www.leica-microsystems.com ) stereo microscope. The images were then processed using Leica Application Suite, which allowed morphological measurements to be taken. The cockroach leg was then desiccated by placing the samples on gauze above silica gel for at least a week. Prior to slide preparation for scanning electron microscopy (SEM), the sample was washed in ethanol to remove any dirt or dust, coated with a fine layer of gold, and scanned. This process was carried out for two legs from one cockroach, a front and a rear leg, as the middle and rear legs are structurally identical ( Watson and Ritzmann 1998 ).
Mechanical Testing
G . portentosa were briefly anaesthetized by exposure to CO2 for three minutes. Then a specially constructed plastic bracket (5 × 28mm) was fixed using cyanoacrylate glue to the second thoracic section over the middle pair of legs ( Figure 1B ). The site of attachment was cleaned using ethanol to remove the waxy cuticle. The bracket was positioned so as not to interfere with the insects' movement or ability to adhere to the substrate as there was no interference with the legs or hindrance of any body flexion. Once the bracket had been attached, the cockroach was left for at least 5 h for a period of recovery prior to experimentation ( Storke 1980 ).
The force needed to remove the cockroach from the substrate was calculated using an Instron®, ( www.instron.com ) universal testing machine. This equipment is commonly used to measure tensile or compression forces via a load cell and generates highly accurate and repeatable measures ( Vincent 1992 ). The cockroaches were pulled at a constant speed and the peak force generated during the pull was recorded. A 14 cm piece of plastic twist tie was threaded through the bracket and twisted together to create a loop around the bracket and then inserted into a clamp that hung from the 100N load cell ( Figure 1A ). Each cockroach ( n = 5) was pulled twenty times from each of the ten different substrates, at a constant speed of 50 mm/second. G. portentosa was allowed a settlement period of 5 s between pulls, allowing sufficient time for pad attachment to the substrate ( Drechsler and Federle 2006 ). When each pull commenced, it continued until all legs of the cockroach were removed from the substrate. From each pull, the peak force was calculated by an interfacing computer and recorded. G. portentosa was pulled from 10 different substrates: smooth Perspex, 1 cm vertically scored Perspex, hatched Perspex (1, 0.5, 0.01 cm), roughened Perspex (fine & coarse), glass, wood, and Teflon. The experimental adhesion testing was filmed using a digital camera (Nikon Coolpix S10, www.nikon.com ), see Video .
Substrate Construction
To determine the effect of surface smoothness on adhesive ability, Perspex and glass substrates were used. These were both purchased from commercial sources. To assess the implications of increasing roughness on adhesion, the Perspex was subjected to increasing degrees of scoring. The scoring of the Perspex was conducted by hand using a razor blade 0.22 × 10-3m wide and a metal ruler. Using moderate force, the razor blade was drawn across the Perspex in one smooth action, generating the score. To enable results to be compared to substrates that were more representative of those the cockroaches may encounter in the natural habitat, testing was also conducted on wood substrate. The wood was a deciduous hardwood log (Oak, Quercus robur ) obtained from nearby woodland. The bark was removed, and the wood autoclaved to remove any foreign influences that could affect adhesion. The shape of the wood was not altered. To assess the influence of surface polarization, a Teflon-covered substrate was constructed from a non-stick baking tray.
Statistics
A one-way ANOVA was performed to determine if there was any difference between cockroaches on each substrate. The statistical test showed that there was no significant difference between each substrate. Data were analyzed using SPSS for windows (version 13.0). There was no significant difference between cockroaches; therefore, data were pooled for each substrate. As the data were not normally distributed, a non-parametric Kruskall-Wallis test was conducted. A Nemenyi post-hoc test was then run to determine which substrates were significantly different from each other. | Results
Microscopy
The light microscope pictures allowed closer examination of the different structures of the G . portentosa tarsus. It was possible, for example, to observe the euplantula pads and pretarsus in great detail. Using the microscope allowed measurements of the adhesive organs to be made so it could be assessed whether size had an influence upon the adhesive ability of G . portentosa. SEM pictures of each of the pretarsal structures clearly showed the smooth arolium between the pretarsal claws (see Figure 2 ). Under closer examination at higher magnification of the arolium of G . portentosa, it was possible to see finger-like projections near the edge of the arolium (circled area in Figure 2 ), these projections are clearly seen in Figure 3 . The “finger-like” projections were also seen on the tarsus of the front leg although it appeared some damage may have occurred to the arolium during the desiccation process and were thus not as clear as shown on the rear leg. SEM pictures of the euplantula of G . portentosa also showed the smooth structure of the pads as well as some interesting structural folds upon the pads ( Figure 2 ).
Substrate adhesion
The results from the Kruskal-Wallis test indicated that there was a significantdifference between the means (F = 16.981, p < 0.001). The Nemenyi post-hoc test revealed that a significantly greater peak force was recorded when G. portentosa was pulled from Perspex (see video in supplementary material) which had been scored along the Y axis at 1cm intervals (p < 0.05) than when G . portentosa were pulled from smooth Perspex, Perspex hatched at 0.5 cm intervals, Perspex hatched at 1 mm intervals, Perspex rubbed 40 times with fine grade sandpaper, Perspex rubbed 40 times with coarse grade sandpaper, glass, or Teflon. Perspex that had been hatched at 1 cm intervals had significantly higher peak force and Teflon. Wood showed similar significance to Perspex hatched at 1 cm; recordings (p < 0.05) than smooth Perspex, Perspex that had been hatched at 1 mm intervals, Perspex rubbed 40 times with fine grade sand paper, Perspex that had been rubbed 40 times with coarse grade sandpaper, however, wood was significantly different from Perspex hatched at 0.5 cm (p <0.05). Glass was significantly different (p < 0.05) from Perspex rubbed 40 times with coarse sand paper, Perspex rubbed 40 times with fine sand paper, and Teflon. The median peak force with Perspex hatched at 0.5 cm was significantly greater than with Perspex rubbed 40 times with fine sand paper. There were no significant differences between smooth Perspex, Perspex hatched at 1 mm intervals, Perspex rubbed 40 times with fine grade sandpaper, Perspex rubbed 40 times with coarse grade sandpaper, or Teflon. As the degree of scoring on the Perspex increased, there was a concomitant decrease in the mean peak force recorded for that substrate ( Figure 4 ). | Discussion
Previous work on the relationship between surface roughness and adhesive ability has shown that with an increase in surface roughness can lead to a decrease in adhesive ability ( Huber et al. 2007 ) and this would explain the pattern demonstrated in figure 4 . As the scoring increased the surface roughness of the Perspex the median peak force recorded showed a significant decline. The significantly higher peak force measured on the scored Perspex (1cm) could be explained due to the combined effect of adhesive pads and pretarsal claws. The morphological analysis indicated that when attempting to adhere to the scored Perspex the cockroaches would have been able to use the pretarsal claws to hook into the scores whilst also using the adhesive pads of the tarsus (euplantula and arolium) to make maximal adhesive contact with the substrate. As the distance between the scores decreased there was less chance that the adhesive pads of the tarsus have sufficient space for effective adhesive contact with the substrate. Surface roughness can influence adhesion, the increased scoring would decrease the maximal contact with the substrate that is needed for effective adhesion of the pads to occur ( Jiao et al. 2000 , Beutel and Gorb 2001 ). An increase in the degree of scoring would have also increased the number of potential attachment sites for the claws and it is only if the claws fail to engage that they are pushed out of the way by smooth surfaces which then allows arolium to come into contact with the substrate ( Arnold 1974 , Fraizer 1999 , Goodwyn et al. 2006 ). The combined effect of available attachment sites for the claws and the available space for pad attachment may have resulted in a higher peak force recordings seen for scored Perspex at 1cm intervals ( Roth and Willis 1952 , Beutle and Gorb 2001 ). However to confirm this hypothesis there would have to be clear demonstration of the role of each of the tarsal structures on the differing substrates.
There was no significant difference in median peak force between the smooth Perspex and the Perspex surfaces abraded with sandpaper. In both cases, abrasion of the Perspex surface by sandpaper likely did not provide the roughness required for the tarsal claws to engage and enable greater adhesion. Even after abrasion, the Perspex may have still been smooth enough to push the claws out of the way, and, if this is the case, adhesion would have relied solely upon the arolium, much the same effect as would be found on smooth Perspex. However, this hypothesis remains to be tested through further work from claw clipping experiments such as those seen in Voight et al. ( 2008 ). The smooth surface of Teflon did not yield a significantly different mean peak force from that of smooth Perspex or any of the Perspex abraded with sandpaper. This may mean that only the arolium was involved in adhesion. Furthermore, there were no significant differences among the smooth surfaces of the Perspex, abraded Perspex, Teflon, and the regularly scored surfaces of hatched Perspex at 1 mm. On all of these surfaces the possibility exists that only one of the pretarsal adhesive strategies could be used, and this may have been responsible for the significantly lower median peak forces recorded.
When G . portentosa were pulled off the wood substrate, there was no significantly different mean peak force from scored Perspex (1 cm). G . portentosa was tested on wood as it was believed it would represent a more relevant substrate because G . portentosa is a forest floor dwelling cockroach and therefore often encounters wood in its natural habitat ( Darmo and Ludwig 1995 ). The wood sample used in these experiments had the outer layer of bark removed and was therefore relatively smooth. It was hard to quantify the surface of wood in the same manner as for the Perspex, but it is likely that it had similar properties due to the fact that both pads and claws could have been involved in adhesion. Scored and hatched Perspex (1 cm) provided well-spaced ridges for claws to engage and ample smooth surface for the use of attachment pads. Wood did not have the regulated scores of the Perspex substrates, but wood is softer than Perspex likely allowing the claws to imbed into the wood while the attachment pads still had had ample room to facilitate the significantly greater mean peak forces generated on the wood substrate.
On Teflon, Perspex abraded with fine sand paper, and Perspex abraded with coarse sandpaper, a running action was observed as the tests began, which is most vigorously seen on the Teflon surface. However, this effect is not seen as much when G . portentosa was located on glass and smooth Perspex, probably due, in part, to van der Waals forces. These inter-molecular forces are strongly dependant upon the distance between and polarizability of the two surfaces ( Autumn 2006 ). As Teflon is a non-polarized material, van der Waals forces should not have occurred, making it harder to make adhesive contact between the attachment pads and the surface; the abrasions found on the Perspex surfaces that had been rubbed by sandpaper probably reduced the microscale contact formed by the attachment pads with the substrate, and this could have reduced the van der Waals forces between the cockroach and the substrate. The lack of van de Waals may have induced the frantic running seen at the start of these tests on smooth surfaces and could have explained, in part, the significantly higher mean peak force observed in glass as opposed to in other surfaces of an apparent similar smoothness ( Figure 4 ).
Aside from Arnold ( 1974 ), which describes and categorizes the tarsal structures of 15 species of cockroach, there is little information on tarsal structure in the cockroach. Arnold ( 1974 ) separates the tarsi into three types: type one, where the euplantula and arolium are absent or barely visible; type two, where the euplantula are prominent but the arolium is a small pad between the claws; and type three, where both euplantula and arolium are prominent. Usually cockroaches with type one or two tarsi lack the ability to climb upon smooth surfaces. It appears that G . portentosa shares the characteristics of type three tarsi, since they demonstrated the ability to climb the smooth surfaces of their tank and to adhere to smooth surfaces during mechanical testing. Furthermore, from analysis of the light microscope and scanning electron microscope pictures, it was clear that both the euplantula and arolium were prominent features of the tarsus. Another feature of the arolium that supports the idea that G . portentosa possesses a type three tarsi were the “finger-like” projections found in a ridge at the edge of the arolium on the rear leg ( Figure 3 ). Arnold ( 1974 ) describes sculpting at the leading edge of the arolium, stating that different species of cockroach possessing the ability to adhere to smooth surfaces had a sculpting of the arolium that took the form of smooth ridges, knobs, or closely adjourning rows of papillae. Arnold ( 1974 ) suggested that this sculpting aided the cockroach in the adhesion to smooth surfaces by increasing the surface area available for adhesion. These microstructures are noted in the desert locust, and these structures also are attributed to a greater climbing ability ( Kendal 1970 ). More recently these “finger-like” projections have been seen in other cockroaches that posses the ability to adhere to smooth surfaces with ease ( Clemente et al. 2008 ). However, robust mechanistic hypotheses have yet to be tested on the function of these microstructures.
This study demonstrated that foot morphology plays a key role in substrate adhesion in G. portentosa . Foot morphology is thought to relate to ecological niche ( Roth and Willis 1952 ; Arnold 1974 ), and it appears that the tarsal morphology of G. portentosa allows it to climb effectively over a wide range of substrates. | Insects are successful terrestrial organisms able to locomote over a wide range of obstacles and substrates. This study investigated how foot morphology (tarsal structure) correlates with substrate adhesion and ecological niche in the Madagascan hissing cockroach, Gromphadorhina portentosa Schaum (Blattaria: Blaberidae). Using light and scanning electron microscopy, the morphology of the different structures of the tarsus of G . portentosa was analysed. Using an Instron® universal testing machine, a series of peak force experiments were then conducted to record the force required to lift the cockroaches off different substrates. G. portentosa was pulled off 10 different substrates, which consisted of smooth Perspex; Perspex scored at 1cm intervals; Perspex hatched at 1 cm, 0.5 cm, and 1 mm intervals; Perspex abraded with fine grade sandpaper; Perspex abraded with coarse grade sandpaper; wood; glass; and Teflon. A clear relationship was seen where an increase in scoring on the Perspex caused a decrease in adhesive ability of G. portentosa . This may be due to there being adequate contact area for the attachment of the pads and to allow the claws to engage. The results obtained suggest that to achieve the greatest adhesion to substrates, G. portentosa uses a combined effect of both adhesive pads and pretarsal claws. Adhesion to a wide range of substrates appears to be an adaptation to life as a wingless forest floor dweller.
Keywords | Acknowledgements
The authors would like to thank Dr. Roland Ennos for his help in training, use of the Instron® equipment, and statistical analysis. Thanks also to Les Lockie for his help using the SEM and to Peter Tickle for his assistance during the completion of these experiments. This work was funded by the University of Manchester. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 May 8; 10:40 | oa_package/0a/43/PMC3014739.tar.gz |
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PMC3014740 | 20578882 | Introduction
In recent years, diseases caused by phytoplasmas have become increasingly important in vegetable crops of the Pacific Northwest. Since 2002, potato growers in the Columbia Basin of Washington and Oregon and Yakima Valley have experienced serious outbreaks of potato purple top disease that caused significant yield losses and a reduction in tuber processing quality ( Munyaneza 2005 ; Munyaneza et al. 2007 ). In addition, similar disease outbreaks were observed in several vegetable crops in the same area, including dry beans, carrots, and radish grown for seed ( Lee et al. 2004a , 2006 ; Munyaneza, unpublished data). It has been determined that the beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma (also known as the Columbia Basin potato purple top phytoplasma and belonging to clover proliferation phytoplasma group [16SrVI], subgroup 16SrVI-A) is the causal agent of the disease in the Columbia Basin and Yakima Valley. This pathogen is transmitted by the beet leafhopper, Circulifer tenellus Baker (Hemiptera: Cicadellidae) ( Lee et al. 2004b ; Crosslin et al. 2005 ; Munyaneza and Upton 2005 ; Munyaneza et al. 2006a , 2007 ). Despite the increasing importance of the problem, little is known about the BLTVA infectivity of beet leafhoppers invading potatoes and other vegetable crops and timing of plant infection. To provide the most effective management of phytoplasma diseases, insecticide applications targeted against insects vectoring these pathogens should be timed to correlate with both insect vector abundance and infectivity ( Weintraub and Beanland 2006 ). This pest management strategy has widely been used in the central states of the United States to manage aster yellows disease in several crops, including potatoes. The aster yellows index, which is based on numbers of the aster leafhopper, Macrosteles quadrilineatus , combined with phytoplasma infectivity, has been developed to initiate control measures before the pathogen is widely transmitted to susceptible crops ( Mahr et al. 1993 ; Foster and Flood 1995 ; Capinera 2001 ). The main objective of the present study was to determine the incidence of BLTVA phytoplasma in beet leafhoppers collected from potatoes and nearby weeds throughout the growing season in Washington. In addition, overwintering beet leafhoppers were collected throughout the Columbia Basin and Yakima Valley and tested for BLTVA to investigate if overwintering insects might constitute a source of inoculum for this phytoplasma from one year to the next. | Materials and Methods
During the 2005, 2006, and 2007 growing seasons, beet leafhoppers were collected from an 8-acre untreated potato field planted at the USDA-ARS Research Farm near Yakima, WA. The weeds in the vicinity of the potato field were also sampled for the beet leafhopper. Weeds included grasses ( Bromus spp.), mustards ( Brassica spp., Sysymbrium altissimum L. [Capparales: Brassicaceae]), kochia ( Basia scoparia (L.) Scott [Caryophyllales: Chenopodiaceae]), filaree ( Erodium cicutarium (L.) L'Her [Geraniales: Geraniaceae]), Russian thistle ( Salsola tragus L. [Caryophyllales: Chenopodiaceae]), rabbitbrush ( Chrysothamnus spp.), sagebrush ( Artemisia spp.), prickly lettuce ( Lactuca serriola L. [Asterales: Asteraceae]), hoary cress ( Cardaria draba (L.) Desv. [Capparales: Brassicaceae]), and pigweed ( Amaranthus spp.). Heavy-duty sweep nets (BioQuip Products, Inc., www.bioquip.com ), each with a 30 cm diameter net hoop, were used during the leafhopper sampling. Four 100-sweep samples were taken weekly between May and late August in both weeds and potatoes. Insects were collected in plastic bags, placed in coolers, and brought to the laboratory at the USDA-ARS in Wapato, WA.
A survey was conducted during the winter and early spring of 2005 throughout the Yakima Valley and the Columbia Basin of Washington and Oregon, to determine the incidence of BLTVA in overwintering beet leafhoppers. Sampling sites included Alderdale, Ephrata, Mattawa, Moses Lake, Moxee, Othello, Paterson, Quincy, and Warden in Washington, and Boardman and Hermiston/Umatilla area in Oregon. During this survey, beet leafhoppers were collected on overwintering weeds (mainly composed of mustards) in several locations near commercial potato fields. Because weeds were too small to be swept at this time of the year, a D-VAC suction device (Rincon-Vitova, www.rinconvitova.com ) was used to collect these overwintering beet leafhoppers. D-VAC samples were collected and processed as above.
Beet leafhoppers were sorted from the sweep net and D-VAC samples. The beet leafhoppers were then preserved in 70% alcohol until tested for BLTVA by PCR. Insect testing was performed at the USDA-ARS in Prosser, WA. A sample of 15 beet leafhoppers was tested for each sampling date at the USDA-ARS Research Farm over the three years. During the leafhopper overwintering survey, a total of 189 beet leafhoppers were collected from January to March and a sample of 98 insects was selected and tested for BLTVA by PCR. The nucleic acid extractions from individual insects were conducted as described by Crosslin et al. ( 2005 ). The first-round PCR reactions were performed using primer pair Pl and P7 and nested reactions used primer pair fU5 and BLTVA-int as described previously ( Crosslin et al. 2005 ). Insects were considered positive for phytoplasma if the expected amplification product of ≈1.2 kbp was visible after agarose gel electrophoresis.
Data on the incidence of BLTVA phytoplasma in beet leafhoppers over the three years were statistically analyzed with multiway categorical methods, testing for association between three variables: presence or absence of BLTVA in insects, collection date, and host plant (weeds or potatoes). Because BLTVA infection status in beet leafhoppers can be considered a response variable, and the remaining two variables potential explanatory variables, a logit model was developed. The resulting model is analogous to a regression model, except that response and explanatory variables are categorical. The analyses were performed using PROC CATMOD ( SAS Institute 2003 ). The level of significance was set at p = 0.05. | Results
BLTVA-infected beet leafhoppers were found in both potatoes and nearby weeds throughout the growing season during the three years of study at the USDA-ARS Research Farm ( Fig. 1 ). Phytoplasma testing results indicated that BLTVA incidence in beet leafhoppers in 2005 averaged 20.8 and 28.3% of leafhoppers collected from potatoes and weeds, respectively ( Fig. 1A ).
Results of the loglinear model assessing the combined effects of sampling date and host plant showed significant sampling date effects ( p = 0.0190) but non-significant host plants effects ( p = 0.1622). The model includes only those dates for which both weeds and potatoes were sampled ( Fig. 1 ). The likelihood ratio was marginally significant ( p = 0.055), which is evidence that the main effects model fit the data relatively poorly.
In 2006, BLTVA incidence in insects averaged 34.8 and 24.5% of leafhoppers found in potatoes and weeds, respectively ( Fig. 1B ). Loglinear model analysis of the 2006 data ( Fig. 1B ) showed that there were no significant effects of sampling date ( p = 0.3837) or host plants ( p = 0.0589), although the latter effect did approach significance. The model also suggests that the interaction between sampling date and host plant was important ( p = 0.0430), which is evidence that the magnitude or direction of host plant effects depended on the date that the sample was collected ( Fig. 1B ).
In the 2007 growing season, incidence of BLTVA in leafhoppers was considerably lower than in the two previous years, with an average of 9.2 and 5.6% infection rate in leafhoppers collected in potatoes and weeds, respectively ( Fig. 1C ). Statistical analysis indicated that there were no significant effects of sampling date ( p = 0.0699), host plant ( p = 0.1755), or the interaction between the two variables ( p = 0.5632). Results of chi-square tests performed for each sampling date separately, testing whether BLTVA infection rates in leafhoppers were different between weed-vs. potato- collected insects within a year, showed no consistent, significant differences. Year effects were explored by first collapsing data across sampling dates within each year, and comparing years using categorical analysis. Results indicated that years differed significantly in overall BLTVA infection rates in beet leafhoppers ( p < 0.0001). Using the data collapsed across sampling dates, there was no evidence of host effects in 2005 ( p = 0.1773) and 2007 ( p = 0.1767) but host effect was marginally significant for 2006 ( p = 0.0621). However, there was evidence of an interaction between year and host plant effects ( p = 0.0489), suggesting that direction or magnitude of host effects differed year-to-year.
During the leafhopper overwintering survey, beet leafhopper females were found and collected on overwintering weeds in several locations near potato fields throughout Yakima Valley and Columbia Basin, from Hermiston/Umatilla area in Oregon to north of Moses Lake in Washington. PCR results revealed that 29.6% of beet leafhoppers collected and tested for BLTVA phytoplasma carried the pathogen. | Discussion
Leafhopper-transmitted phytoplasma diseases can be very damaging if not well-managed ( Ploaie 1981 ; Lee et al. 2000 ). Pest management strategies for the control of phytoplasma diseases are currently directed at leafhoppers and other vectors of these plant pathogens. In addition to sampling for leafhopper abundance, it is also desirable to determine the proportions of leafhoppers that are infected with the phytoplasmas in order to effectively manage diseases caused by these plant pathogens. For example, in central United States, the aster yellows disease, which is caused by the aster yellows phytoplasma and mainly transmitted by Macrosteles species, causes serious damage to several vegetable crops. The aster yellows indices, based on both insect numbers and disease incidence, were developed in the Midwest and are used as action thresholds to initiate control measures before the pathogen is widely transmitted to susceptible crops ( Mahr et al. 1993 ; Foster and Flood 1995 ; Capinera 2001 ). Control of leafhoppers using insecticides is the most effective management approach available to growers to prevent the spread of aster yellows disease ( Mahr et al. 1993 ). To provide the most effective control, timing of insecticide applications is correlated with both leafhopper numbers and infectivity. In Wisconsin, recommendations concerning the development and severity of aster yellows are normally determined using a statewide average of leafhopper infectivity provided as a pest management service for growers ( Mahr et al. 1993 ). The average infectivity of leafhoppers is determined from samples collected from the initial migrating leafhopper populations. Historically, leafhoppers were collected before they entered an area and given aster plants as food. The plants were then monitored for disease symptoms to estimate the insect percent infectivity and determine the number of leafhoppers that can be tolerated on a specific crop for any infectivity level. Two percent infectivity in aster leafhoppers is commonly used as baseline for action threshold development in the Midwest ( Mahr et al. 1993 ; Foster and Flood 1995 ; Capinera 2001 ). This method can be laborious and lengthy. Recently, however, significant progress in fast detection of phytoplasmas has been made through the application of molecular biological techniques, and PCR has become the method of choice to quickly determine infectivity in leafhoppers and plants ( Goodwin et al. 1999 ; Crosslin et al. 2006 ).
The beet leafhopper is a serious pest in the western United States because it transmits plant pathogens, including beet curly top virus, Spiroplasma citri , and BLTVA phytoplasma, to several vegetable, ornamental, and horticultural crops ( Thomas and Martin 1971 ; Golino et al. 1989 ; Shaw et al. 1990 , 1993 ; Schultz and Shaw 1991 ; Capinera 2001 , Crosslin et al. 2005 ; Munyaneza and Upton 2005 ; Munyaneza et al. 2006a , 2008 ). Phytoplasma diseases have become increasingly important in vegetable crops of the Pacific Northwest. Recently, serious outbreaks of potato purple top have occurred in the Columbia Basin of Washington and Oregon and caused significant yield losses and a reduction in tuber processing quality. The beet leafhopper has been identified as the insect vectoring the BLTVA phytoplasma that causes this disease in this important potato-growing region of the United States ( Munyaneza 2005 ; Munyaneza et al. 2007 , 2008 ). This is a different vector than for the aster yellows phytoplasma associated with potato purple top in north central United States ( Banttari et al. 1993 ) and Mexico ( Leyva-López et al. 2002 ). Effective management of this potato disease in the Pacific Northwest requires knowledge of the biology and ecology of the beet leafhopper, including incidence of BLTVA phytoplasma in beet leafhoppers found in and near potato fields.
The central Columbia River area of Washington and Oregon has been reported as one of the overwintering and breeding sites of the beet leafhopper in western United States ( Hills 1937 ; Cook 1967 ). However, information on the BLTVA infectivity of local beet leafhopper populations in this potato-producing region is lacking. During the present study, it was determined that the beet leafhopper overwinters on weeds near potato fields throughout the Columbia Basin and Yakima Valley. Further BLTVA testing revealed that 29.6% of overwintered beet leafhopper females carried the phytoplasma. Contrary to previous beliefs, this plant pathogen apparently does not necessarily need overwintering weeds to survive the winter, which then suggests that leafhoppers might pass this phytoplasma from one season to the next.
The beet leafhopper is present in the Columbia Basin and Yakima Valley potatoes throughout the growing season. The seasonal occurrence and abundance of this insect pest in this region, including the sites surveyed during the present study, were recently described by Munyaneza et al. ( 2008 ). Results from the present study also indicated that BLTVA-infected beet leafhoppers were found in both potatoes and nearby weedy habitats throughout the growing season. Testing of beet leafhoppers by PCR showed that a large proportion of beet leafhoppers invading potatoes were infected with the phytoplasma, with an average of 20.8, 34.8, and 9.2% in 2005, 2006, and 2007, respectively. These phytoplasma incidence levels in leafhoppers are particularly high compared to the 2% infectivity that is used to calculate aster yellows indices to initiate control measures of potato purple top disease in the Midwest. Results further indicated that BLTVA infection rate in the leafhoppers was variable overtime and that host plant effects (weeds vs. potato) were not evident or consistent date-to-date ( Fig. 1 ). Moreover, year of study differed significantly in overall BLTVA infection rates in the insects, and study results suggest that the direction or magnitude of host plant effects differs year-to year. These observations suggest that incidence of BLTVA in leafhoppers from weedy habitats near potato fields may be used to relatively estimate the proportion of infective beet leafhoppers invading potatoes. This information may also be used in making decisions regarding whether to initiate control measures targeted against the beet leafhopper to reduce BLTVA incidence in potatoes.
The presence of BLTVA-infected beet leafhoppers in potatoes throughout the growing season threatens the crop by exposing potato plants to potential phytoplasma infection anytime during the season. However, to date, Columbia Basin potato growers have managed to keep the purple top disease under manageable levels by applying insecticides against the beet leafhopper early in the season ( Munyaneza et al. 2006b ). These observations suggest that potato plant growth stage may play an important role in susceptibility of potato to BLTVA phytoplasma. Studies to investigate this assertion are now underway, and preliminary observations indicate that, in fact, older potato plants seem less susceptible to and/or are more tolerant of the BLTVA phytoplasma ( Munyaneza et al. 2010 ).
In summary, beet leafhoppers are found in both potatoes and nearby weeds throughout the potato-growing region of the Columbia Basin and Yakima Valley. Large proportions of these leafhoppers are infected with BLTVA phytoplasma and could infect potato plants throughout the growing season. Furthermore, a large proportion of overwintering beet leafhoppers found near potato fields carry BLTVA phytoplasma and might pass on this plant pathogen from one season to the next. Effective management strategies for phytoplasma diseases in Pacific Northwest potatoes must take into consideration BLTVA infectivity of beet leafhopper local populations invading potato fields. To provide the most effective control, timing of insecticide applications should be correlated with both beet leafhopper densities and phytoplasma infectivity. Given the high phytoplasma infectivity in beet leafhoppers in the surveyed areas compared to the 2% infectivity used in the Midwest to initiate leafhopper control, further studies are needed to accurately determine treatment thresholds for the beet leafhopper in the Pacific Northwest. Information from this study will help formulate action thresholds for beet leafhopper control to reduce damages caused by BLTVA phytoplasma to potato and other vegetable crops in affected areas. Use of action thresholds also could help reduce environmental hazards and adverse impact on non-target species caused by excessive and unnecessary pesticide applications. Moreover, manipulation of weedy habitats in the vicinity of potato fields may help reduce incidence of potato purple top disease. | Phytoplasma diseases are increasingly becoming important in vegetable crops in the Pacific Northwest. Recently, growers in the Columbia Basin and Yakima Valley experienced serious outbreaks of potato purple top disease that caused significant yield loss and a reduction in tuber processing quality. It was determined that the beet leafhopper-transmitted virescence agent (BLTVA) phytoplasma was the causal agent of the disease in the area and that this pathogen was transmitted by the beet leafhopper, Circulifer tenellus Baker (Hemiptera: Cicadellidae). To provide the most effective management of phytoplasmas, timing of insecticide applications targeted against insects vectoring these pathogens should be correlated with both insect abundance and infectivity. Beet leafhoppers were collected from a potato field and nearby weeds in Washington throughout the 2005, 2006, and 2007 growing seasons and tested for BLTVA by PCR to determine the incidence of this phytoplasma in the insects. In addition, overwintering beet leafhoppers were collected throughout Columbia Basin and Yakima Valley and tested for BLTVA to investigate if these insects might constitute a source of inoculum for this phytoplasma from one season to the next. Results showed that 29.6% of overwintering leafhoppers collected near potato fields carried the phytoplasma. BLTVA-infected leafhoppers were also found in both potatoes and nearby weedy habitats throughout the growing season. PCR testing indicated that a large proportion of beet leafhoppers invading potatoes were infected with the phytoplasma, with an average of 20.8, 34.8, and 9.2% in 2005, 2006, and 2007, respectively. Similarly, BLTVA infection rate in leafhoppers collected from weeds in the vicinity of potatoes averaged 28.3, 24.5, and 5.6% in 2005, 2006, and 2007, respectively. Information from this study will help develop action thresholds for beet leafhopper control to reduce incidence of purple top disease in potatoes.
Keywords | Acknowledgments
We thank Launa Hamlin, Blaine Heilman, Aaron Lance, Megan Murray, and Nicholas Maquillan for invaluable technical assistance. We are also grateful to Dave Horton for his assistance with statistical analysis. In addition, we thank anonymous reviewers who made suggestions to an earlier draft of this manuscript. Financial support for this work was partially provided by the Washington State Potato Commission. | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Mar 15; 10:18 | oa_package/49/17/PMC3014740.tar.gz |
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PMC3014741 | 20572793 | Introduction
Mosquitoes are the vectors of pathogens that cause important human diseases including malaria, filariasis, dengue, yellow fever among others ( Paul et al. 2006 ). Aedes aegypti (Diptera: Culicidae) is the major vector of dengue viruses that cause more human mortality and morbidity than any other arthropod-transmitted viral disease ( Alphey et al. 2002 ). An estimated 50 to 100 million cases of dengue fever occur annually, and 2.5 billion people are at risk of infection ( Black et al. 2002 ; Guzman and Kouri 2002 ; Gubler 2004 ; Guha-Sapir and Schimmer 2005 ). There is no vaccine available, and there are no drugs to cure dengue fever. Dengue control is based on surveillance measures and mosquito control using insecticides and larval habitat reduction strategies ( Spiegel et al. 2005 ). However, despite these efforts, the number of cases of dengue fever and dengue hemorrhagic fever continue to rise each year, and, therefore, alternative control avenues are being investigated.
Some of these efforts have focused on the genetic manipulation of insect vectors ( Beaty 2000 ; Aksoy et al. 2001 ; Alphey et al. 2002 ) to modulate characteristics such as vector competence, the intrinsic ability of a vector to transmit a pathogen ( Woodring et al. 1996 ). Recent advances in molecular biology and the availability of genomic databases have enabled the development of new strategies for the control of vector-borne diseases. Manipulation of vector competence requires extensive knowledge on the molecular aspects of vector-parasite interactions. In this context, transgenic techniques have been used to introduce and achieve expression of foreign, antipathogenic genes in insect vectors ( Aksoy et al. 2001 ; Dotson et al. 2003 ; Abraham et al. 2005 ; Riehle and Jacobs-Lorena 2005 ). In Ae. aegypti , an engineered construct of Sindbis virus has been used to express insect immune peptides ( Cheng et al. 2001 ) or heterologous virus sequences to induce an RNA interference-like response to the target virus ( Adelman et al. 2001 ).
Population genetics studies of vector competence in Ae. aegypti have demonstrated a high variation of this characteristic among different populations ( Tabachnick 1982 ; Apostol et al. 1996 ; Paupy et al. 2000 ; Vazeille-Falcoz et al. 2001 ; Garcia-Franco et al. 2002 ; Gorrochotegui-Escalante et al. 2002 ). Susceptible and refractory strains obtained using isofemale selection demonstrated an association of vector competence with genetic components that can be affected by environmental changes ( Wallis et al. 1985 ; Miller and Mitchell 1991 ). Quantitative genetic studies have revealed that at least two genes or sets of genes control vector competence ( Bosio et al. 2000 ; Black et al. 2002 ). These studies have allowed associating a genetic component with vector competence, but the role of specific receptors or immune response-related genes that modulate arbovirus infection and replication, and the factors that determine resistance or susceptibility to arboviruses such as dengue, are still unknown.
Insects have developed precise mechanisms to protect themselves against bacterial, fungal, and parasitic infections. This immune response is innate and, depending on the type and size of the pathogen, may involve a combination of phagocytosis ( Pearson et al. 1995 ; Kocks et al. 2005 ; Watson et al. 2005 ; Lemaitre and Hoffmann 2007 ), encapsulation and melanization ( Karlsson et al. 2004 ; Bidla et al. 2005 ; Paskewitz et al. 2006 ), and production of lethal antimicrobial peptides ( Lowenberger 2001 ; Imler and Bulet 2005 ). Immune reactions begin with the recognition of cell-surface molecules of pathogens, known as pathogen-associated molecular patterns, by specific insect receptors (pattern recognition receptors) ( Michel and Kafatos 2005 ). This interaction between pathogen-associated molecular patterns and pattern recognition receptors selectively activates either of two intracellular signaling pathways. In Aedes , as in Drosophila , Gram + bacterial and fungal infections induce the Toll pathway, which results in the translocation of the NF-KB transcription factor, and Gram — bacteria triggers the Imd pathway, which results in the nuclear translocation of Relish (a NF-κB-type transcription factor) and the induction of antimicrobial peptides such as cecropin and defensin ( Lowenberger 2001 ; Bartholomay et al. 2004 ). Although these immune pathways are conserved among mosquito species, there are differences in the molecules involved ( Hoffmann and Reichhart 2002 ; Shin et al. 2003 ; Meister et al. 2005 ). Despite the knowledge of antimicrobial responses, the immune response in mosquitoes against viruses such as dengue has not been thoroughly defined ( Sanders et al. 2005 ). Other insect-virus models such as Drosophila /Drosophila virus C suggest that, in addition to Toll and Imd, a third pathway, Jak/Stat, functions as a part of an antiviral innate immune response ( Dostert et al. 2005 ; Zambon et al. 2005 ). In vertebrates, apoptosis is the first response to viral infections and later stimulates the adaptive immune system. This mechanism has also been described in some insect models infected with baculovirus as an antiviral defense strategy, but the role of apoptosis as an immune response has not been well-characterized in insects that transmit human parasites ( Clarke and Clem 2003 ; Cooper et al. 2007a ), despite the fact that apoptotic-like activity has been associated with Plasmodium infection in Anopheles gambiae ( Al-Olayan et al. 2002 ). Recent studies in Ae. aegypti have identified and characterized two initiator caspases associated with apoptosis pathways, suggesting that this immune response might function as one of the mechanisms that insect vectors use to regulate the establishment and replication of intracellular parasites such as viruses ( Cooper et al. 2007a , 2007b ).
Although Ae. aegypti is the main vector of dengue virus, there are populations or fractions of populations that do not permit virus development, presumably because they have biological barriers that impair the establishment and dissemination process ( Black et al. 2002 ). A high variability in vector competence among local populations of Ae. aegypti in Cali, Colombia ranging between 19% to 60% was identified ( Ocampo and Wesson 2004 ), indicating the presence of naturally susceptible and refractory mosquitoes to Dengue-2 virus with different infection barriers. The midgut infection barrier is one of the initial mechanisms that viruses must overcome to establish a successful infection and is one that might be genetically altered to render mosquitoes resistant to arboviruses. Therefore, it is the focus of this study.
The differential expression of midgut genes between susceptible and refractory Ae. aegypti after exposure to Dengue-2 virus is reported here. The functional annotation of pathogen-specific vector-expressed sequence tags (ESTs) that could play a role in determining or contributing to vector competence in Ae. aegypti are also described. | Materials and Methods
Study rationale
In an attempt to identify factors that determine the susceptibility of Ae. aegypti to dengue virus, midgut gene expression was evaluated in Ae. aegypti individuals that were susceptible or refractory to Dengue-2 virus 48 h post infection.
Mosquito strains
Ae. aegypti were collected in different localities from the city of Cali, Colombia and colonized at the Centro Internacional de Entrenamiento e Investigaciones Médicas insectary at 26 ± 2° C with 80% relative humidity and a 12:12 lightdark photoperiod. Ae. aegypti (Rockefeller strain) provided by the Centers for Disease Control and Prevention (Puerto Rico) were maintained in the insectary and were used as positive and negative controls in all PCR assays. The variability of susceptibility of different local populations of mosquitoes to Dengue-2 was described previously ( Ocampo and Wesson 2004 ), indicating the presence of susceptible and refractory mosquitoes in Cali. To increase the number of refractory mosquitoes, an isofemale selection was carried out. For this selection, females were allowed to feed on an infectious blood meal and were placed individually in oviposition cages. After 14 days of incubation, the phenotype of the mother, with respect to the biological barriers, was identified (midgut infection barrier, midgut escape barrier, and susceptibility) as described by Bennett et al. ( 2005 ). The eggs of each female were collected. These eggs were hatched and the emerging adults were sorted based on the phenotype of the mother. These descendents from susceptible and midgut infection barrier females were infected with a Dengue-2 infectious blood meal and midgut tissues were collected 48 h later.
Virus maintenance and mosquito infection
Dengue-2 virus New Guinea C strain, freshly grown in C6/36HT ( Aedes albopictus larvae cells) was used in oral challenges. Infected cells were incubated for 14 days at 32° C in L 15 medium supplemented with 2% heatinactivated fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine ( Higgs and Beaty 1996 ). Virus and cells were harvested and collected in a 15-ml conical centrifuge tube. Aliquots of the infected cell suspension and the mixture of blood and virus before and after the infection process were titred using the methodology described by Bennett et al. ( 2002 ). Titres in the cell suspensions ranged from 10 8 to 10 8.5 TCID50/ml in all oral challenges. Oral infections were done in a BSL2+ (biosafety laboratory) insectary with eight protection barriers. Artificial blood feeding was carried out using a membrane feeder. Infected blood was prepared by mixing defibrinated rabbit blood and Dengue-2 virus suspension (1:1 v/v) ( Higgs and Beaty 1996 ). Adult females, six to seven days after eclosion, were deprived of sucrose and water for 24 h prior to blood feeding. Mosquitoes were allowed 1 to 1.5 hours to feed ad libitum . Fully engorged mosquitoes were separated and kept in a separate cage with access to a 10% sugar solution.
Tissue dissection and RNA isolation
Forty-eight hours after infection, midguts from bloodied mosquitoes were dissected on a chilled table and thoroughly rinsed in cold DEPC-PBS to remove traces of the blood meal. Tissues were stored individually in RNA later (Qiagen, www.qiagen.com ) at -20° C for subsequent RNA isolation. The RNA later solution was removed by pipetting. Total RNA extraction from individual midguts was performed using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA was quantified using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, www.nanodrop.com ).
Detection of infection
An established nested reverse transcriptase PCR protocol ( Lanciotti et al. 1992 ) was standardized using three groups of Ae. aegypti Rockefeller strain to determine the sensitivity of reverse transcriptase PCR to detect the virus in individual midguts. These groups were: mosquitoes inoculated with Dengue-2 virus (positive controls), infected-bloodfed mosquitoes, and non-bloodfed (naïve) mosquitoes. Midguts were dissected, and RNA was extracted as described above.
In the reverse transcriptase PCR reactions, 50 ng of total RNA were reverse transcribed in a 20 μl reaction mixture containing 1X firststrand buffer (50 m M Tris-HCl (pH 8.3), 75 m M KCl, 3 m M MgCl 2 ), 5 m M DTT, 500 μ M of dNTPs mix, 50 pmol of primer D2 (5′TTGCACCAACAGTCAATGTCTTCAGGT TC-3′) and 50 units of Superscript II Reverse Transcriptase (Invitrogen, www.invitrogen.com ). Reverse transcription was conducted at 42° C for 60 min and 95° C for 5 min. The resulting cDNA was used in a 50 ìl PCR reaction containing 1X PCR buffer (50 m M KCl, 10 m M Tris-HCl (pH 9.0), 0.1% Triton® X-100), 1.5 m M MgCl 2 , 125 μ M of each dNTP, 50 pmol of primers D1 (5′TCAATATGCTGAAACGCGCGAGAAACC G-3′) and D2, and 0.05 U of Taq DNA polymerase (Invitrogen). PCR was performed with the following parameters: 95° C for 1 min; 30 cycles of 94° C for 45 s, 58° C for 45 s, and 72° C for 1 min; and a final extension at 72° C for 7 min. A second-round PCR was run with a 1:100 dilution from the first PCR reaction. PCR was performed under the same conditions used for the primary PCR with the following modifications: primer D2 was replaced with the Dengue-2 virus-specific primer TS2 (5′-CGCCACAAGGGCCATGA ACAG-3′, 50 pmol) and 35 amplification cycles were used. PCR products were resolved by 2% agarose gel electrophoresis with a 100bp DNA ladder (Invitrogen) stained with ethidium bromide and visualized under UV light.
Subtractive library construction
According to the PCR result, positive (infected) and negative (non-infected) midgut RNA samples for each phenotype were pooled separately. A total of 60 midguts were pooled for each phenotype to obtain sufficient RNA to generate the suppressive subtractive hybridization (SSH) libraries. All RNA pools were precipitated and treated with DNAse (Qiagen).
Total RNA from each pool was used to generate cDNA using the SMART PCR cDNA Synthesis Kit (Clontech, www.clontech.com ) according to the manufacturer's recommendations. This procedure generated a sufficient quantity of high-quality cDNA from small quantities of RNA for subtractive library procedures.
Libraries were built using PCR-Select cDNA Subtraction kit (Clontech) according to the manufacturer's specifications. SSH is a PCR-based technique that facilitates the detection of differentially expressed sequences in two samples by allowing exponential amplification of differentially expressed genes and suppressing the amplification of sequences common to both samples. This technique has been used previously to identify differentially expressed genes in Rhodnius prolixus in response to pathogens and parasites ( Ursic-Bedoya and Lowenberger 2007 ).
Three subtractive libraries were constructed: a library of differentially expressed genes in mosquito midguts after injection of Escherichia coli (control library), and two cDNA libraries from the midguts of Dengue-2 virus-susceptible and virus-refractory mosquitoes after the ingestion of a Dengues-infected blood meal. A predictable E. coli control library was built to confirm that the small amount of RNA available for the dengue-susceptible and dengue-refractory was sufficient to build SSH libraries.
The products of the subtracted procedure were ligated into pGemT Easy plasmid vector (Promega, www.promega.com ) and transformed by heat shock into E. coli JM109 ultra-competent cells (Promega) as previously described by Ursic-Bedoya and Lowenberger ( 2007 ). Putative transformant colonies were grown overnight in 96-well plates with 100 μl of LB medium and 0.1 μl of ampicillin (100 μg/μl). For forward and reverse libraries, a total of 384 colonies (four plates) were selected from each library for differential screening. For the bacteria-induced library, 192 colonies (2 plates) were selected.
Subtraction efficiency analysis and differential screening
The subtraction efficiency of the SSH process in all libraries was measured using PCR to amplify, before and after subtraction, a housekeeping gene that should be present in both libraries and an induced gene that should be present in only the enriched library. The Beta-actin sequence from Ae. aegypti with the primers actinF637LVP: 5′- ATTAAGGAGAAGCTGTGCTACGTC and actinR942LVP: 5′-CATACGATCAGCA TTACCTGGG was used. The PCR program was 94° C for 1 min, followed by 33 cycles of 94° C for 20 s, 60° C for 20 s, 68° C for 30 s and a final extension of 68° C for 2 min. To measure a differentially expressed gene, the Ae. aegypti lysozyme was used as described by Ursic-Bedoya and Lowenberger ( 2007 ).
Both midgut subtracted libraries were screened for differentially expressed ESTs using the PCR-select differential screening kit (Clontech) following the manufacturer's instructions. One hundred and fifty nanograms from the forward and reverse subtracted libraries were used to create a 32 P-labeled probe by random priming. Forward and reverse subtracted probes were hybridized in individual tubes with Hybond+ DNA membranes (Amersham Biosciences, www.gelifesciences.com ) containing individually spotted EST clones ( Ursic-Bedoya and Lowenberger 2007 ). 32 P-labeled probes and target EST membranes were hybridized at 65° C for 2.5 h in a rotatory oven using Rapid—Hyb buffer (Amersham Biosciences). Following hybridization, the membranes were washed with low stringency (2X SSC, 0.5% SDS; 3 times, 20 min each) and high stringency (0.2X SSC, 0.5% SDS; 3 times, 20 min each) buffers at 65° C to eliminate non-specific binding due to excess probe. Membranes were exposed to a Kodak BioMax MS film (Eastman Kodak, www.kodak.com ) overnight at room temperature. Selected colonies (strong signal with the forward and low signal with the reverse subtracted probe) were sent to BC Genome Sciences Centre (Vancouver, BC) for plasmid purification and sequencing.
Sequence analysis
Sequence homology searches were carried out using NCBI's BLAST-X ( http://www.ncbi.nlm.nih.gov/blast/ ) against nr databases with default parameters. The best annotated matches were retained. Sequences with no significant matches in NCBI's databases were translated in all possible reading frames and were analyzed using INTERPRO SCAN to identify conserved protein domains so that putative function could be assigned. Additionally, sequences with no significant match in the NCBI program were analyzed against the VECTORBASE database ( www.vectorbase.org/Tools/BLAST ). Homologies were considered statistically significant if they generated an Expect value (E) < 0.1. The EST sequences reported in this paper were submitted to the NCBI dbEST and assigned accession numbers 56768811 to 567689975 (GenBank FG107129 to FG107293). | Results
Library of ESTs found only in Dengue-2 susceptible midguts
This library was created using susceptible insects as the tester and refractory insects as the driver (forward library) in order to identify genes differentially expressed in the susceptible population. In order to increase selection of susceptibility-related genes, recombinant colonies were differentially screened by hybridization with forward (susceptible) and reverse (refractory) probes. Differentially expressed and over-expressed clones were selected. Of the 384 clones screened, only 125 were confirmed to be up-regulated by differential screening as described above.
All 125 clones were sequenced, from which 22 clones (17.6%) did not have similarities with other sequences in the databases (data not shown). A similarity search identified 57 putative genes from 103 clones that matched with annotated sequences in databases ( Tables 1 , 2 ). All identified genes were clustered in functional groups according to their putative function as cytoskeleton, nucleic acid binding, metabolism, transcription factors, immunity, ion binding and transport, receptors, mitochondrial, signaling and digestion genes. Out of 57 ESTs, 9 sequences coded for ribosomal genes (normally repressed in the suppressive subtractive hybridization), and 15 clones corresponded to hypothetical proteins. Only 11 of the putative genes had more than one copy, and 4 of them were highly repetitive (more than 3 copies) as DNA binding, Cytoskeleton, Cytochrome P450 and calcium ion binding genes that are potentially related with intracellular infections, cellular distress, and immune responses.
Library of ESTs found only in Dengue-2 refractory midguts
A total of 384 clones were spotted on membranes, but only 40 were confirmed to be up-regulated in the refractory tissues compared with the susceptible library after hybridization with the forward and reverse probes. Of those 40 clones, 5 had no significant match to other genes in the databases (data not shown). Bioinformatic analyses showed 23 (65.7%) of the EST sequences corresponded to different putative genes ( Tables 3 , 4 ). Among these transcripts, 9 sequences were hypothetical proteins. Four clones (1%) had more than one copy, but, in contrast with the susceptible library, they were not highly repetitive. Subtraction efficiency analysis by PCR showed better quality in this subtraction than in the susceptible library since only one ribosomal gene was detected.
Contrary to the susceptible subtracted library, genes related with cellular stress or immune responses were not detected ( Figure 1 ). An interesting finding was the presence of a trypsin inhibitor gene that was differentially expressed in this library ( Table 3 ). This protein could affect dengue virus infection; the inhibition or knockdown of specific trypsin molecules has been reported to reduce ( Molina-Cruz et al. 2005 ) or increase ( Brackney et al. 2008 ) the infectivity of dengue virus in Ae. aegypti . | Discussion
Insect innate immune responses have been studied against bacteria, parasites, and fungi, but antiviral responses have not been well-characterized ( Sanders et al. 2005 ). In eukaryotic organisms, antiviral innate immune mechanisms involve mechanical barriers ( Paskewitz and Christensen 1996 ; Schmid-Hempel 2001 ; Schmid-Hempel 2005 ), gene silencing (RNAi and miRNA) ( Ausubel 2005 ; Fritz et al. 2006 ; Wang et al. 2006 ), production of humoral and effector mechanisms ( Cherry and Silverman 2006 ; Seth et al. 2006 ; Zhong et al. 2006 ) and apoptosis ( Everett and McFadden 1999 ; Irusta et al. 2003 ).
It is known that the mosquito immune response mechanisms are similar to those of Drosophila melanogaster . However, D. melanogaster is not a vector of arboviruses, and therefore, certain immune mechanisms could be specific to mosquitoes such as Ae. aegypti . Although there are conserved genes among D. melanogaster, An. gambiae and Ae. aegypti , immune genes are the most divergent group even among closely related species. An estimated 285 genes related to immune response, apoptosis and oxidative stress were identified in D. melanogaster , while 338 such genes were identified in An. gambiae , and 353 in Ae. aegypti ( Nene et al. 2007 ). Phylogenetic studies indicate that genes related to pathogen recognition and signaling intracellular pathways are conserved in the three species. Genes encoding effector molecules such as antimicrobial peptides, however, may be more diverse or species specific ( Nene et al. 2007 ; Waterhouse et al. 2007 ).
Viral infection studies in D. melanogaster have revealed the role of RNA interference, Toll, Imd, Jak-Stat pathways and apoptosis mechanisms as antiviral responses. The diverse kinds of responses seen in these experiments depend on the virus life cycle and the method used in the experimental infections (oral or intrathoracic inoculation) ( Keene et al. 2004 ; Lemaitre and Hoffmann 2007 ). This study focused on identifying immune response related genes in the mosquito midgut, the primary barrier that the virus must overcome in order to infect the vector. It has been estimated that only 40–50% of the initial viral load is capable of crossing the midgut barrier ( Paskewitz and Christensen 1996 ).
In this study, differential expression analysis showed that susceptible Ae. aegypti express a higher number of metabolic and immune response-related genes than the refractory mosquitoes. Immune genes were primarily associated with both inhibition and execution mechanisms of apoptosis. The identified molecules associated with this process were: inhibitor of apoptosis, ubitiquin ligase complex, Cytochrome c, cytoskeleton genes and proteins with calcium ion binding domain. The over-expression of these molecules suggests that cell stress and apoptosis could be playing a key role during the infection process. Other studies have hypothesized that mosquitoes might use apoptosis to eliminate intracellular parasites such as viruses ( Cooper et al. 2007a , 2007b ), and that viruses might prevent it by activating the expression of inhibitors of apoptosis. The presence of anti and pro-apoptotic molecules suggests that the virus manipulates the cellular machinery to allow its replication and dissemination, as has been demonstrated in cell cultures ( O'Brien 1998 ). To this point, the differential apoptotic responses in refractory and susceptible Ae. aegypti strains after ingesting Dengue-2 were not measured, but experiments are ongoing.
In contrast, an upregulation of immune-related genes was not observed in refractory mosquitoes possibly because: a) the virus does not enter the midgut cells, b) the virus cannot establish the infection, c) there are early events of apoptosis that eliminate virus-infected cells, d) the technique used or availability of all genes in the databases did not allow the identification of other potential refractory mechanisms, or e) the mechanism is not mediated at the transcriptional level, but may be mediated by previously produced zymogens. In this context, an interesting finding in the refractory strain was the presence of a trypsin inhibitor gene. Trypsin is a digestive enzyme that has been implicated in the dengue virus infectious process ( Molina-Cruz et al. 2005 , Brackney et al. 2008 ). It is possible the trypsin inhibitor gene found in this library could affect dengue infection in the mosquito. If it is supported, this finding may suggest that refractoriness may not be due to an active immune response based on well-known and well-characterized immune processes (i.e. antimicrobial peptide expression, phagocytosis activation by TEP, phenoloxidase, melanization) but may be a function of the virus not being able to exit midgut epithelial cells, if it was able to enter initially.
The SSH technique and its ability to identify differentially expressed genes in the midguts of Dengue-susceptible and -refractory individuals were validated, but there are few similar studies with which to compare the results. The E. coli -injected control library identified some immune peptides that have been demonstrated broadly in other studies as cecropin, serine proteases, and conserved protein related to cell death (data not shown).
The limitations of the SSH technique were recognized in terms of its sensitivity, since it detects only highly over-expressed genes; however, this technique was selected for its accessibility and as a primary step in identifying potential differences between the susceptible and refractory strains. Additionally, this technique allowed us to work with the small amount of RNA that was obtained by pooling individual midguts that were previously tested.
As described above, many of the ESTs generated in this study have no known match in the databases, and they will continue to be submitted to the growing number of databases as more motifs and genomes are sequenced. The lack of match may indicate a true lack of comparable sequences in the databases, or may indicate that the ESTs map to 3′ and 5′ untranslated regions. The ESTs were examined, but none of the classic motifs found in 3′ untranslated regions were found.
Some of the more interesting proteins to which the ESTs map, and which could play a key role in the susceptibility or refractoriness to Dengue-2 virus in Ae. aegypti , are discussed below. Further studies are underway to evaluate these molecules in more detail. | Associate Editor: Jan Veenstra was editor of this paper
Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever.
Keywords | Inhibitor of apoptosis
Insect inhibitor-of-apoptosis proteins contain two baculoviral inhibitor-of-apoptosis repeat domains and a Zinc RING domain. Inhibitors of apoptosis impede activation of initiator and executioner caspases preventing either their dimerization or their binding to the active catalytic site of these enzymes ( Huh et al. 2007 ; Leu et al. 2007). Some inhibitors of apoptosis have been identified and characterized in insects, but their significance during arbovirus infection in mosquitoes has not been completely elucidated ( Blitvich et al. 2002 ; Li et al. 2007 ; Bryant et al. 2008 ). In an An. gambiae functional genomics study, 6 inhibitors of apoptosis were differentially expressed during Plasmodium berghei infection in midgut epithelial cells ( Vlachou et al. 2005 ). Likewise, there was an upregulation of apoptosis related-molecules in Ae. aegypti infected with Sindbis virus, and, among these, one inhibitor of apoptosis was over-expressed ( Sanders et al. 2005 ). What is most interesting in this study is, for the first time, the identification of apoptosis as an antiviral response in a natural Dengue/ Ae. aegypti model using wild mosquito populations.
Apoptosis as an immune response mechanism in vertebrates has been widely described. However, in invertebrates, this process has not been clearly characterized. The results of this study are consistent with other studies, suggesting this cell death process is one of the mechanisms that insect vectors use to regulate intracellular parasites such as viruses ( Cooper et al. 2007a , 2007b ).
Ubiquitin ligase complex
This enzyme complex participates in protein degradation by the proteasome in a number of key biological processes, including cell cycle progression and signal transduction ( Maniatis 1999 ). Ubiquitin-dependent proteolysis controls the abundance of many regulatory proteins and caspase activation ( Wojcik 2002 ; Arama et al. 2007 ). Several studies have shown a crosstalk between the apoptotic pathways and the ubiquitin- proteasome system ( Orlowski 1999 ; Schreader et al. 2003 ; Arama et al. 2007 ). During the cell death process, the ubiquitin ligase complex promotes caspase activation via ubiquitination and degradation of caspase inhibitors. Inhibitors of apoptosis, with ubiquitin protease ligase (E3) activity in their RING finger domain, undergo auto-ubiquitination and degradation by proteasome ( Grimm and Osborne 1999 ; Hu and Yang 2003 ). The ubiquitin-mediated pathway also regulates NF-KB factors in activation. In the D. melanogaster Toll pathway, upregulation of ubiquitin ligase levels leads to the degradation of Cactus, allowing the nuclear translocation of Dorsal ( Spencer et al. 1999 ).
Cytochromes (P450 and c )
Cytochromes are proteins involved in several cellular functions such as oxidative stress, respiration, apoptosis and xenobiotic metabolism ( Scott and Kasai 2004 ; Arama et al. 2006 ). In mammal cells, release of Cytochrome c and other proapoptotic molecules induce caspase activation and cell death via the mitochondrial apoptosis pathway ( Hengartner 2000 ; Wang 2001 ). In insects, the role of mitochondria and Cytochrome c in apoptosis has been contradictory and not completely characterized ( Abdelwahid et al. 2007 ; Goyal et al. 2007 ). Some reports have suggested that Cytochrome c release is not a necessary step to trigger apoptosis in some D. melanogaster cells ( Dorstyn et al. 2004 ; Abdelwahid et al. 2007 ). In contrast, Liu ( 2007 ) showed that Cytochrome c is required for caspase activation in Baculovirus -induced apoptosis in Spodoptera litura cells.
Trypsin inhibitor
Trypsin, a major midgut proteolytic enzyme, is essential for blood digestion in Ae. aegypti ( Noriega and Wells 1999 ) and its presence has been reported to enhance arbovirus infectivity, replication, and dissemination ( Ludwig et al. 1991 ; Xu et al. 1997 , Molina-Cruz et al. 2005 ). The addition of soybean trypsin inhibitor has been reported either to increase midgut infection rates ( Brackney et al. 2008 ) or to decrease Dengue-2 infectivity and dissemination ( Molina-Cruz et al. 2005 ).
Feeding different trypsin inhibitors blocked early trypsin activity but did not reduce late trypsin expression, and RNAi knockdown of early trypsin did not affect late trypsin expression ( Lu et al. 2006 ). The interaction between the expression of early and late trypsin is not clear. RNAi silencing of chymotrypsin, early and late trypsin had no effect on Dengue-2 infectivity whereas RNAi knockdown of a third trypsin, 5G1, reduced trypsin activity and increased dengue infectivity in the midgut ( Brackney et al. 2008 ). These studies suggest that some midgut serine proteases, acting through digestion or direct activity on viral proteins, may affect Dengue-2 infectivity of Ae. aegypti . The presence of the EST with high homology to a trypsin inhibitor ( Table 3 ) in the refractory strain suggests that inhibition of trypsin activity as a digestive enzyme or in cleaving viral proteins could contribute to the refractoriness of the wild Ae. aegypti population used in this study. However, further studies are required to determine which trypsins are affected by this inhibitor and subsequently their specific roles in limiting or enhancing Dengue-2 infectivity.
The data presented here have identified differences in gene expression between feral populations of Ae. aegypti that are naturally susceptible or refractory to Dengue-2 virus. There was an over-expression of numerous molecules and the involvement of diverse biological processes showed the complexity of viral infection and immune responses against the virus. The functional characterizations of the apoptosis-related genes have begun to be evaluated in order to elucidate their role in the susceptible or refractory phenotypes. In addition, more investigations need to be done in order to evaluate whether known immune pathways (Toll and Imd) are activated after dengue virus infection. | Acknowledgements
The authors are grateful to Paola Caicedo and Andrea Arévalo, young investigators at CIDEEVI who participated in the initial vector competence studies. We also thank Dawn Cooper and Mathias Schuetz, who contributed, respectively, to the primers design and radioactive screening of libraries in Canada. This project was supported by WHO-TDR (A30848) and COLCIENCIAS (Contract: 22290416474). Other partial support was from the CHIR grant (69558), the Canada Research Chairs program, and a Michael Smith Scholar award to CL.
Abbreviations
expressed sequence tag;
suppressive subtractive hybridization | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 May 8; 10:41 | oa_package/5c/6f/PMC3014741.tar.gz |
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PMC3014742 | 21209800 | 1. Introduction
Voriconazole, a new broad-spectrum triazole antifungal agent, was approved by the US Food and Drug Administration in May 2002. Its use has increased steadily because of its high effectiveness against a wide variety of yeasts and molds and its excellent oral bioavailability [ 1 ]. The most common side effects reported are visual disturbances, elevation in hepatic enzyme levels, and rashes [ 2 , 3 ]. Photosensitivity reactions of varying severity are frequent (1%) and well documented [ 4 – 6 ]. In contrast, only 16 cases of squamous cell carcinomas have been reported, characterized by clinical severity [ 7 – 11 ].
We report a new case of phototoxicity associated with multiple squamous cell carcinomas in a patient treated for 2 years using voriconazole. We discuss the possible mechanisms induced by voriconazole, a photosensitizing drug, in skin carcinogenesis. | 3. Discussion
Voriconazole, a broad-spectrum azole antifungal agent, inhibits the cytochrome P450-dependent 14 α -lanosterol demethylation, an essential step in fungal cell membrane ergosterol synthesis [ 1 , 3 ]. It is indicated in different fungal infections, particularly invasive aspergillosis [ 3 ]. This molecule is easily used by oral route with excellent bioavailability. It is therefore widely prescribed. However, significant side effects have been reported, with 30% visual disturbances, 10% liver abnormalities, and 8.6% skin reactions [ 1 – 3 ].
Among adverse cutaneous drug reactions, cases of generalized erythema, eczema, urticaria, bullous lichen, erythema multiform (0.05%), or Lyell's syndrome (0.1%) have been reported [ 1 – 3 ]. Photosensitivity, idiosyncratic rather than dose-dependent, is described in 1% of subjects and appears in prolonged treatment (at least 12 weeks). In most cases, skin reactions disappear on an average of 4 weeks after voriconazole discontinuation [ 4 , 5 , 12 , 13 ].
In particular, as in this case report, 15 patients presenting multiple squamous carcinomas have been recently reported in three cases and two series [ 7 – 11 ]. These 15 patients and the present case developed a phototoxic rash on sun-exposed areas on an average of 6.56 months after starting treatment (15 days to 15 months). The actinic keratoses and squamous cell carcinomas that appeared after an average of 28 months of treatment (12 to 36 months) were multiple. Skin carcinoma onset occurred at a mean age of 38 years (9 to 69 years), much earlier compared to the average age for occurrence of these lesions in the general population (76 years) [ 14 ]. Among these 16 patients, voriconazole discontinuation led to photosensitivity regression from day 14 for 7 patients and data were unavailable for 9 patients. No new carcinoma had occurred for 4 patients at 10 months after voriconazole discontinuation, and data were unavailable for 12 patients.
Immunosuppression, present in all patients treated with voriconazole, could be expected to induce these squamous cell carcinomas, as in transplant patients [ 15 ]. In the setting of immuno-suppression, EBV and HPV have also been implicated in development of nonmelanoma skin cancer [ 16 – 18 ]. In our case, EBV or HPV infection in skin lesions was not detected.
The absence of new squamous cell carcinomas concomitant with the interruption of voriconazole suggests its causal role, especially given its phototoxicity. Two mechanisms are suggested in voriconazole phototoxicity: (i) direct phototoxicity by voriconazole or its metabolites, and (ii) a retinoid-like mechanism [ 4 , 13 ]. Like fluoroquinolones, which are also phototoxic molecules, voriconazole may amplify local, systemic immuno-suppression [ 19 ] and the genotoxicity [ 20 ] induced by UV. An amplification of immuno-suppression by local enhancement of the inhibitory effect on immuno-competent cells has been shown for fluoroquinolones [ 21 ]. Concerning genotoxicity, it has been reported, in an experimental model, that fluoroquinolones, especially lomefloxacin and fleroxacin, associated with UVA radiation, enhanced tumor prevalence and drastically shortened the median latent period of tumor onset compared with UVA alone [ 22 – 24 ]. In UV-irradiated xeroderma pigmentosum group A gene-deficient mice, defective in nucleotide excision repair, the treatment with lomefloxacin induced DNA damage [ 25 ]. In our case, no mutation of POLH, a gene predisposing to xeroderma pigmentosum variant, was identified. However, two MC1R functional variants, Arg151Cys and Ser131Asn, were identified. These variants have been shown to be involved in basal and squamous cell carcinoma [ 26 ].
A plausible hypothesis is that several factors including voriconazole uptake, immuno-suppression, and the genetic background ( MC1R variants) could explain the phenotype of proliferating multiple skin carcinomas. | 4. Conclusion
Voriconazole, a new antifungal agent, is attractive by its efficiency and ease of use. However, our case and others in the literature suggest that prolonged treatments, in a context of immunosuppression complicated by phototoxicity reactions, can predispose to risk of multiple recurrent, aggressive squamous cell carcinomas. In patients at risk, an alternative therapy should be discussed whenever possible. Dermatological supervision, stringent measures of external photoprotection, and sun avoidance are recommended. | Academic Editor: Yolanda T. Becker
Voriconazole is a treatment for severe fungal infections. Prolonged voriconazole therapy may induce skin reactions, with 1% of severe photosensitivity accidents. Recently the imputability of voriconazole in skin carcinogenesis has been suggested. This report concerns a 55-year-old man suffering from pulmonary aspergillosis who presented a phototoxic reaction a few months after introduction of voriconazole, followed by multiple squamous cell carcinomas of sun-exposed skin areas. After voriconazole discontinuation, no new carcinoma was observed. The detection of EBV and HPV in skin lesions was negative. Exploration of gene mutations involved in skin carcinogenesis showed two variants of the MICR gene. The occurrence of multiple, recurrent, aggressive squamous cell carcinomas is rare with voriconazole, but its imputability is strongly suggested. A plausible hypothesis is that several factors including voriconazole uptake, immunosuppression, and genetic background could explain the phenotype of fast-developing skin carcinomas. Voriconazole therapy should be accompanied by stringent photoprotection and skin monitoring. | 2. Case Presentation
A man of 55 had been treated with corticosteroids (10 mg/day) for sarcoidosis since 1974. In February 2004, treatment comprising 200 mg voriconazole twice daily was instated for pulmonary aspergillosis in this setting of immunosuppression. His usual treatment consisted of salbutamol, budesonide, and ramipril. He had a Fitzpatrick skin phototype II and was living in a temperate area. He was an office worker and had never had intensive, prolonged sun-exposure. No previous personal or familial skin cancer was noted.
In May 2004, four months after the voriconazole instatement, a photo-exposure erythema appeared on the face, scalp, and backs of hands, with several relapses. In August 2006, while the patient was still receiving voriconazole, multiple erosive and keratosic lesions of photo-exposed areas appeared, associated with erythema of the back of hands ( Figure 1(a) ). To characterize the photoreaction, phototests were performed. UVA minimum erythema dose (MED) was normal, and UVB-MED was slightly lowered (46, 20 mJ/cm 2 ; N > 50 mJ/cm 2 ). Photopatch-tests performed in the hypothesis of a photoallergy were negative. These results argued for a phototoxic rather than a photoallergic photosensitive reaction.
The lesions worsened, becoming infiltrated and crusted. The first biopsy on the face in May 2007 showed a microinvasive squamous cell carcinoma. Subsequently, 17 squamous cell carcinomas and multiple actinic keratoses (Figures 1(b) and 1(c) ) on the face and scalp were diagnosed up to December 2008 at which time voriconazole was discontinued. Between January and December 2009, actinic keratoses were observed, but no carcinoma. Both curative and preventive treatments associating imiquimod, cryotherapy, photodynamic therapy, and surgery by excision-graft of micro-invasive carcinomas were implemented.
Using in situ hybridation we tested for Epstein-Barr-Encoded-small-RNAs- (EBERs-) positive cells with EBV-encoded RNA 1 oligonucleotide probe (Benchmark XT automat, Ventana), and for human papillomavirus using reagents and INFORM HPV III Family 16 DNA Probe (B) (P/N 800-4295) (Ventana Medical Systems Inc.), in the cutaneous lesions. No EBV or HPV infection was detected.
Given the phenotype of the multiple skin carcinomas, a search for DNA mutations in genes involved in predisposition to cutaneous carcinoma ( MC1R, POLH ) was performed. After signature of informed consent and obtaining blood samples, DNA extraction was performed, followed by amplification using PCR primers specific to the coding exons, flanking intrinsic sequences, and automated sequencing (Applied Biosystems 3130 R ). No mutation of the POLH (XPV) gene, involved in xeroderma pigmentosum variant, was detected, but two variants of the MC1R gene, Arg151Cys and Ser131Asn, were characterized. The first is a well-known loss of function in the MC1R variant that is associated with both the RHC (Red Hair Color) phenotype and an increased risk of skin cancer. The second variant, S131N, has not been previously described, to our knowledge; it occurs at a highly conserved residue and is predicted to be deleterious genomic mutation (SIFT, Polyphen). | Conflict of Interests
The authors declare that they have no conflict of interests.
Authors' Contribution
C. Morice, L. Verneuil, and D. Leroy contributed in writing the paper and reviewing of the literature as well as undertaking a comprehensive literature search; A. Acher, M. Michel, F. Comoz, and N. Soufir helped with the acquisition of data and manuscript revision. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 16; 2010:351084 | oa_package/69/ec/PMC3014742.tar.gz |
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PMC3014743 | 20569125 | Introduction
The emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), is an important pest of the North American species of Fraxinus in China ( Liu 1966 , Yu 1992 ), and after its accidental introduction to North America it is a serious pest there as well ( Haack et al. 2002 ). In China, the emerald ash borer has been reported in the northeastern provinces of Liaoning, Jilin and Heilongjiang, as well as in Shandong and Hebei Provinces and Tianjin Municipality. Damage was especially severe from 1999–2005 on velvet ash, Fraxinus velutina Torrey (Scrophulariales: Oleaceae), in tree plantations and along roadsides in Tianjin. Coastal barriers in many areas have plantings of this ash species that have been nearly eliminated by A. planipennis ( Zhang et al. 1995 , Liu et al. 1996 ). The concealed feeding and damage by A. planipennis larvae in the cambium of host trees often results in infestations going unnoticed until large ash stands are in critical condition and the trees die.
Early surveys by the senior author led to the discovery of Spathius agrili Yang (Hymenoptera: Braconidae), a previously undescribed parasitoid species and a potentially effective natural enemy of A. planipennis ( Yang et al. 2005 ). S. agrili is a gregarious idiobiont ectoparasitoid. Preliminary field observations found parasitism rates of 30–90%, indicating that S. agrili might be an obligatory natural enemy of A. planipennis larvae ( Yang et al. 2005 ). Further testing confirmed that S. agrili prefers A. planipennis as a host ( Yang et al. 2008 ). S. agrili may be a key, natural factor keeping A. planipennis under control naturally in Tianjin, and it has high potential as a biocontrol agent. Herein, the life history, basic ecology and behavior of S. agrili are described, as studied in its native range in Tianjin, China, during 2003 and 2004. | Materials and Methods
Insect collection and rearing
A. planipennis larvae were sampled from a 5 ha stand of 9- to 10-year-old F. velutina spaced 1.0 m apart in parallel rows with 1.5 m spacing in Guangang Forest Park (38°56′N, 117°29′E), Dagang District, Tianjin Municipality, China. In August 2003, a total of 12 F. velutina were debarked to locate and collect A. plenipennis larvae, which were then transported to the laboratory. Collected larvae were fed fresh ash twigs that were 1.0–1.5 cm in diameter and 5–10 cm in length. Each ash twig was split in half lengthwise with one-half having a 3 cm long groove excavated through to the bark. A field-collected A. plenipennis larva was placed in each groove, and the other half of the twig was replaced and secured with rubber bands. The two ends of each ash tree “rearing chamber” were sealed with wax to prevent desiccation of the plant tissue during feeding of A. plenipennis larva.
S. agrili cocoons were collected from their host galleries. Approximately 10 clutches were collected every 3 days from June 18 to August 28, 2003. Adult wasps that emerged from these cocoons were cultured in the laboratory and fed a 1:4 honey: water solution.
Adult emergence
S. agrili cocoons from individual host galleries were collected on March 7, April 15, and May 7 2004 and placed into single test tube (1.5 × 10 cm), one clutch per tubes. A small piece of moistened filter paper was placed in each tube to maintain humidity.
Tubes were held in the laboratory at room temperature (average 22.3°C, max 26°C, min 18°C) and checked daily for adult emergence. Surveys of forest areas were made using a sweep net and visual observation of tree trunks every 3 d beginning in late May to establish the first appearance of adults.
Adult life history
Adult longevity, sex ratios, mating behaviors, and phototaxis were determined from laboratory and field observations. Longevity of adults in the laboratory was determined using three treatments: 1) supplying 20% honey solution on pieces of cotton, 2) water only without honey, and 3) no nutrition or water provided. Mortality was recorded daily. No fewer than 14 adults of each sex were used per treatment.
Fecundity was measured by placing newly-emerged male and female pairs of adult S. agrili , along with an A. planipennis larva in an excavated ash twig, in glass test tubes (2.8 × 11.5 cm) sealed tightly with cotton plugs. A. planipennis larvae were examined every 2–3 d to determine if parasitoid eggs were laid. If S. agrili eggs were observed on an A. planipennis larva, they were counted, and an unparasitized host larva was provided in a new twig. Two treatments were used: adults supplied a 20% honey solution and adults provided water only. Pre-oviposition and oviposition periods and fecundity were recorded for each female wasp. The rate of oviposition was defined as the percentage of females in a given treatment group that laid eggs. A total of 40 pairs were observed; 17 pairs were provided the honey solution, and 23 pairs had access to water but no honey.
Determination of larval instars and stadia
S. agrili eggs were separated from the clutch of eggs and placed individually on un-paralyzed A. planipennis larvae that were in excavated ash twigs. The S. agrili eggs were separated because it would have been difficult to observe egg hatch and molting of larvae developing communally. The eggs were checked three times per day at 8:00, 15:00, and 22:00 h. First-instar larvae were placed into glass test tubes (1.5 × 7 cm) and reared individually on un-paralyzed A. planipennis larvae. Molts were monitored by marking the exuviae of the parasitoid larvae with black oil paint and examining them three times per day under the “Motic” stereo microscope (Motic, www.motic.com ). When an exuvia marked with black oil paint was shed, the molt was recorded and the fresh exuvia was marked for further observation. Body length and width were determined for each larval instar using an ocular micrometer under the “Motic” stereo microscope. Observations were made within 7 h after the insect molted and continued until pupation occurred. The duration of the pupal stage was also recorded. The development of twenty insects was recorded using the methods described above.
Parthenogenesis
Newly emerged, virgin S. agrili females were placed individually in glass test tubes (2.8 × 11.5 cm) with their host larva in an excavated ash twig. The test tubes were sealed with cotton plugs, and host larvae were examined every 2–3 d for eggs. When S. agrili eggs were found on an A. planipennis larva, they were counted, and an unparasitized A. planipennis larva in a fresh twig was provided. Two treatments were used: adults supplied with 20% honey solution and adults provided water only. The number of eggs laid, offspring development (percentage survival from egg to pupa), and offspring sex were recorded. Each treatment was replicated using 10 female S. agrili.
Life cycle and population dynamics in forests
In the research plot starting in late May, field surveys were conducted every 3 d until late October, when A. planipennis had become prepupae and entered their pupal chambers in the shallow sapwood for overwintering. During each survey, 5–10 ash trees were sampled at random for A. planipennis larvae and their parasitoids. A 10 × 30 cm bark window was removed from the trunk of each tree at 4–6 locations between 0.5–1.5 m. S. agrili eggs, larvae, and pupae associated with A. planipennis larvae or its gallery were counted and transported to the laboratory to rear. The parasitism rate (percentage of A. planipennis parasitized) and the number of S. agrili per A. planipennis larva were calculated for each survey. The life stage development and population dynamics of S. agrili in the forest were estimated from field observations combined with information on the duration of each stage observed in the laboratory.
Dissection of ovarioles of adult female
One-week-old female S. agrili that emerged from cocoons collected in field were killed with ethyl acetate vapors after mating but prior to oviposition. Specimens were fixed on a wax dish (7.0 cm diameter), immersed in 1% NaCl solution, and dissected under the “Motic” stereo microscope (80X magnification). The structure of the female reproductive system and the number of eggs contained in the ovarioles were recorded.
Data analysis
Data were analyzed with SAS version 9.1.3 ( SAS Institute Inc. 2006 ) for variance analysis by PROC GLM program. The mean values of the wasp adult longevity, preoviposition period, oviposition period, egg production, etc., under different conditions were compared using analysis of variance, and a least significant difference multiple range test was run for separation of means. Sex ratio and the rate of oviposition (percentage of females that laid eggs) between different groups were compared using the chi square method. | Results and Discussion
General life history
The life-cycle of S. agrili began in the summer with the emergence of adults. The female wasps searched for hosts by walking on the trunks and/or twigs of ash trees, tapping the bark with their antennae ( Figure 1L ). When a host was located, the female passed her ovipositor through the bark into the A. planipennis gallery ( Figure 1M ), paralyzed the A. planipennis larva by injecting venom, and then laid a clutch of 1–35 eggs on the host surface ( Yang et al. 2005 ) ( Figure 1A ). After hatching, S. agrili larvae attached themselves to the paralyzed host larva, feeding externally ( Figures 1B, 1C, 1D ). Mature wasp larvae spun silken cocoons ( Figures 1E, 1I ) within which they pupated ( Figure 1F, 1G ), and adults chewed through the bark upon emergence ( Figure 1H, 1J ). S. agrili overwintered as prepupae inside cocoons in its host gallery. A brood of cocoons usually occupied the space previously occupied by the host larva, which was reduced to a thin thread after being fed upon by the parasitoid ( Figure 1I ).
Development of life stages
S. agrili eggs were white, semitransparent, elongate, and slightly curved, with one end rounded and the other end narrowing ( Figure 1A ). Eggs were 0.8 ± 0.02 mm in length and 0.1 ± 0.002 mm in width ( Table 1 ). The parasitoid eggs were typically laid together as a clutch on the body surface of A. planipennis larvae. A fluid was exuded by the ovipositing female onto the surface of the eggs for gluing and attaching the eggs to the host larvae. Most eggs hatched in 1–3 d ( Figure 1B ) with a mean of 1.6 ± 0.16 d at room temperature (22–26°C).
Larvae were cream-colored and had 14 segments with spiracles that were not clearly visible ( Figure 1C, 1D ). There were five larval instars, and the duration of each instar is presented in Table 2 . At lower temperatures 14–21°C, the duration of the fifth instar and pupa increased ( Figure 2 ). Fifth instar larvae grew from an average of 4.4 mm (range 3.0–5.5 mm) shortly after the molt to 6.17 mm (range 5.0–7.0 mm) just prior to pupation. S. agrili larval feeding in the laboratory was impacted by the manipulation during monitoring and frequent marking. It was difficult for the 4th or 5th instar S. agrili larvae to feed on its un-paralyzed host after introduction into the test tube because the A. planipennis larvae resisted by twisting their body and crawling. Naturally occurring larvae did not encounter this problem because the hosts were paralyzed by the ovipositing female. The placement of early-instar larvae of S. agrili onto the body of A. planipennis larvae did not elicit avoidance behavior by the A. planipennis larva.
After feeding for 8–10 d, the parasitoid larvae became 5th instars, and during this stage they consumed virtually all of the host, leaving only its exuvia, which was shrunken and thread-like. The wasp larvae then aggregated, spun and formed adjacent cocoons inside the host gallery ( Figure 1I ), and pupated. The overall shape of the aggregative brood of cocoons assumed the form of the mature host larva at end of the gallery. In some cases the host larva was not completely consumed because there were only a few feeding S. agrili larvae (usually fewer than four individuals) ( Figure 1E ). Cocoons of the overwintering generation were typically brown and thicker than those of the non-overwintering generations, which were cream to light brown and thinner walled. The cocoons were 4.3–10.5 mm in length and 1.0–2.1 mm in width ( Table 1 ). At room temperature (average 20.7° C, min 13.5° C, max 29.6°C), the duration of the pupal stage was 11–29 d with a mean of 17.4 ± 0.46 d ( n = 83). Pupation was shorter at higher temperatures (11–13 d in August and 18–20 d in September).
The exarate pupae ( Figure 1F ) were cream colored. Prior to emergence, the compound eyes became red ( Figure 1G ), and then, beginning at the head, the body darkened until the abdomen and ovipositor appeared black. The ovipositor lay tightly against the abdomen until eclosion of the female S. agrili. Four to six d were required from cocoon formation to pupation in August under room conditions (average 26.8°C, min 23.1° C, max 29.8°C). When food was inadequate (i.e., host too small to support that number of parasitoids) pupation could be delayed and cocoons might not be formed prior to pupation.
Adult S. agrili were 3–5 mm in length and black-brown, with yellow legs and antennae with 25 segments ( Fig. 1L, 1M ). Females were slightly larger than males, with a 2 mm long ovipositor. Adult S. agrili seldom moved at temperatures below 15°C. They were noticeably more active at temperatures above 18° C and appeared fully active at 25–30°C (i.e., actively crawling, flying, courting, and mating). The active adults were positively phototatic, which was more strongly exhibited by females than males.
At temperatures of 21–30° C (average 24.7°C), the lifespan of female and male S. agrili adults was an average of 29.1 ± 2.5 d and 23.6 ± 5.8 d, respectively, when fed a 20% honey solution ( Table 3 ). This was significantly greater than for adults that had access to water only or that were reared without water or honey. Some individuals fed the honey solution lived as long as 90 d. Not surprisingly, adult longevity was longer when temperatures were lower. Female S. agrili usually lived longer than males under the same conditions ( Wang et al. 2007 ). Virgin S. agrili females reproduced parthenogenetically, producing only male offspring. The oviposition rate for unmated females fed a honey-water solution was 60%, while it was 20%) for unmated females provided water only.
Mating
Males of S. agrili typically eclosed prior to females, chewing an exit hole through the bark. In the same brood, males emerged prior to females 78.4% of the time. Males waited on the bark outside of the pupation chamber for mating opportunities with females exiting from the same hole. The emergence period for a single brood ranged from 5 to 44 d ( n = 56) with a mean period of 18.2 ± 1.18 d. This lengthy period suggested that not all mating occurred between siblings.
Male courtship behavior was most frequent when room temperatures reached 26–30°C, with frequent crawling, jumping, wing beating, and pursuing of females. In copulation, males clasped the female's dorsum, beat their wings, and tapped females' antennae vigorously with their own antennae, while the females were inactive. Mating time ranged from 2 to 35 s with a mean of 16 ± 3.8 s. Some males were observed to mate with multiple females during the observation period, but females usually avoided mating again after they mated once. After mating, females usually moved away from courting males. Females that did mate multiple times were paired with aggressive males that pursued them. When several males and females were introduced into a single test tube, apparent competitive mating behavior among males was exhibited with males often attacking copulating pairs. During field observations in August between 8:00–11:00 and 15:00–18:00 h, female and male groups with upwards of 10 individuals were observed on single tree trunks. Mating and oviposition were observed during this period in the same areas on trees.
Oviposition
Following mating, the pre-oviposition period averaged 9.9 ± 2.9 d, with some females laying eggs on day three and other females laying their first egg after 28 days. The oviposition period typically lasted 8–18 d with a maximum of 47 d. Individual S. agrili oviposited up to eight times on eight hosts, depositing 1–10 eggs (average 5.8 ± 0.24 eggs/host, n = 193) in each clutch, although up to 20 eggs per clutch was observed ( Figure 3 ). S. agrili has been shown to lay more eggs on larger A. planipennis larvae ( Wang et al. 2008 ), sometimes even parasitizing mature A. planipennis larvae that were partially or completely inside of the overwintering chamber, which was in the shallow sapwood ( Figure 1K ). First and second instar host larvae were avoided, probably because small A. planipennis larvae would not provide sufficient nutritional resources for the development of multiple parasitoid larvae. Single parasitoid females oviposited a total of 6–68 eggs over their lifetime. Fecundity was higher for females that received honey solution supplements, but the differences were not significant. In the honey treatment there were 17 pairs of wasps, and 12 females laid eggs; while in the water treatment there were 23 pairs of wasps but 11 females laid eggs. The χ 2 -test results comparing percentage of females that oviposited showed that there was no significant difference between treatments ( = 1.2457 < = 3.84) ( Table 4 ). The ovarioles of a 1-week-old female usually contained only a few mature eggs (mean 8.5 ± 0.87 eggs, range 6–15 eggs, n = 12). Because females could lay eggs multiple times, and because the total eggs laid on hosts was much greater than the number of mature eggs observed at the onset of oviposition, S. agrili was determined to be synovigenic.
Based on field and laboratory observations of eggs, larvae, and pupae, the clutch size of S. agrili was higher in forests than in the laboratory. In one instance, 35 eggs were found deposited on a mature field-collected host larva, although the number of ovipositional events that were involved could not be determined.
Female wasps were observed in the field tapping the bark with their antennae as they actively searched the trunks and twigs to locate host larvae for oviposition ( Figure 1L ). This activity typically peaked between 8:00–11:00 and 15:00–18:00 h, being greatest on sunny days when more than 10 individuals were regularly observed on a single trunk. After locating a host larva, the female inserted her ovipositor (length 1.4–2.5 mm, mean 1.9 ± 0.03 mm) completely through the bark ( Figure 1M ), piercing the host larva and paralyzing it with venom. The process required approximately 20 min. Eggs then were laid on the host's body surface ( Figure 1A ), during which another 40 to 90 min elapsed. During the egg-laying process, the female parasitoid was observed to contract rhythmically and shake her abdomen, which might aid in the movement of eggs through the egg channel in the ovipositor. The female remained at the site 10–20 min after removing her ovipositor from the bark. It is unlikely that the females deposited an oviposition deterrent pheromone because the paralyzed host larvae no longer feed, and S. agrili had been shown to oviposit only on larvae that are feeding (Wang et al., unpublished data).
Field observations of adult emergence, brood size, sex ratio, and parasitism rates
Adult S. agrili chewed an exit hole of approximately 1 mm in diameter in their cocoon to escape ( Figure 1H ) when they emerged. After exiting the cocoons, S. agrili then bit a round hole through the bark (mean 0.9 ± 0.03 mm diameter, range 0.6–1.8 mm diameter) ( Figure 1J ) and emerged. Typically, only one exit hole was made per brood, although two or three were observed occasionally. Adults emerged most often at the 15:00 sampling period.
Adults of S. agrili first appeared in Tianjin ash forests in late May (2004) or June (2003) and continued emerging for over 2 months ( Figure 4 ). In both years, emergence was low during the first 2–3 weeks, when A. planipennis larvae were early instars and unable to support feeding by the usually gregarious S. agrili larvae. Beginning in early July 2003 and early June 2004 there was a large increase in S. agrili emergence that lasted for approximately one month. It was during this period that the preferred older instars of A. planipennis became available. Studies have shown that S. agrili adults emerge up to several months after A. planipennis adults to allow for development of the preferred host stage, the late instar larvae ( Wang et al. 2006 ).
Field-collected single broods of S. agrili consisted of 2–18 cocoons (average of 9.1 per brood). Sex ratios were highly variable during the emergence period within and between years ( Figure 4 ). When data were pooled, females outnumbered males 2.1 to 1 ( Table 5 ). Sex ratios were also determined from a total of 316 adults of S. agrili that were reared in the laboratory from field-collected eggs and larvae. Adult emergence was grouped into three consecutive periods during the summer of 2003, and sex ratios (female: male) ranged from 3.3:1 to 3.5:1 ( Table 6 ). There was no significant difference among these sex ratios or individually from 3:1.
The parasitism rate of the first S. agrili generation was relatively low, about 10%. However, parasitism gradually increased and peaked with the overwintering generation in the late year, usually over 40% ( Figure 5 ), with parasitism in some forest stands exceeding 90%. During 2003 in Tianjin, adult S. agrili were sampled by sweep net as late as October 10, but at that time they appeared much less vigorous and typically died soon in the laboratory. These adults may have been part of the last generation of the season.
Number of generations per year
At ambient room temperature of 18–27° C (average 22.4°C), the development duration from egg to adult stages was 27–28 d, with the mean 27.9 ± 0.63 days (range 25–34, n = 26) for females and 28.2 ± 1.38 days (range 26–35, n = 6) for males. Combining estimations of developmental durations under laboratory conditions with field temperatures recorded during observation, the occurrence of all generations of S. agrili were estimated ( Figure 6 ). It was predicted that S. agrili could complete 3–4 generations per year in the forests of Tianjin, China. | Results and Discussion
General life history
The life-cycle of S. agrili began in the summer with the emergence of adults. The female wasps searched for hosts by walking on the trunks and/or twigs of ash trees, tapping the bark with their antennae ( Figure 1L ). When a host was located, the female passed her ovipositor through the bark into the A. planipennis gallery ( Figure 1M ), paralyzed the A. planipennis larva by injecting venom, and then laid a clutch of 1–35 eggs on the host surface ( Yang et al. 2005 ) ( Figure 1A ). After hatching, S. agrili larvae attached themselves to the paralyzed host larva, feeding externally ( Figures 1B, 1C, 1D ). Mature wasp larvae spun silken cocoons ( Figures 1E, 1I ) within which they pupated ( Figure 1F, 1G ), and adults chewed through the bark upon emergence ( Figure 1H, 1J ). S. agrili overwintered as prepupae inside cocoons in its host gallery. A brood of cocoons usually occupied the space previously occupied by the host larva, which was reduced to a thin thread after being fed upon by the parasitoid ( Figure 1I ).
Development of life stages
S. agrili eggs were white, semitransparent, elongate, and slightly curved, with one end rounded and the other end narrowing ( Figure 1A ). Eggs were 0.8 ± 0.02 mm in length and 0.1 ± 0.002 mm in width ( Table 1 ). The parasitoid eggs were typically laid together as a clutch on the body surface of A. planipennis larvae. A fluid was exuded by the ovipositing female onto the surface of the eggs for gluing and attaching the eggs to the host larvae. Most eggs hatched in 1–3 d ( Figure 1B ) with a mean of 1.6 ± 0.16 d at room temperature (22–26°C).
Larvae were cream-colored and had 14 segments with spiracles that were not clearly visible ( Figure 1C, 1D ). There were five larval instars, and the duration of each instar is presented in Table 2 . At lower temperatures 14–21°C, the duration of the fifth instar and pupa increased ( Figure 2 ). Fifth instar larvae grew from an average of 4.4 mm (range 3.0–5.5 mm) shortly after the molt to 6.17 mm (range 5.0–7.0 mm) just prior to pupation. S. agrili larval feeding in the laboratory was impacted by the manipulation during monitoring and frequent marking. It was difficult for the 4th or 5th instar S. agrili larvae to feed on its un-paralyzed host after introduction into the test tube because the A. planipennis larvae resisted by twisting their body and crawling. Naturally occurring larvae did not encounter this problem because the hosts were paralyzed by the ovipositing female. The placement of early-instar larvae of S. agrili onto the body of A. planipennis larvae did not elicit avoidance behavior by the A. planipennis larva.
After feeding for 8–10 d, the parasitoid larvae became 5th instars, and during this stage they consumed virtually all of the host, leaving only its exuvia, which was shrunken and thread-like. The wasp larvae then aggregated, spun and formed adjacent cocoons inside the host gallery ( Figure 1I ), and pupated. The overall shape of the aggregative brood of cocoons assumed the form of the mature host larva at end of the gallery. In some cases the host larva was not completely consumed because there were only a few feeding S. agrili larvae (usually fewer than four individuals) ( Figure 1E ). Cocoons of the overwintering generation were typically brown and thicker than those of the non-overwintering generations, which were cream to light brown and thinner walled. The cocoons were 4.3–10.5 mm in length and 1.0–2.1 mm in width ( Table 1 ). At room temperature (average 20.7° C, min 13.5° C, max 29.6°C), the duration of the pupal stage was 11–29 d with a mean of 17.4 ± 0.46 d ( n = 83). Pupation was shorter at higher temperatures (11–13 d in August and 18–20 d in September).
The exarate pupae ( Figure 1F ) were cream colored. Prior to emergence, the compound eyes became red ( Figure 1G ), and then, beginning at the head, the body darkened until the abdomen and ovipositor appeared black. The ovipositor lay tightly against the abdomen until eclosion of the female S. agrili. Four to six d were required from cocoon formation to pupation in August under room conditions (average 26.8°C, min 23.1° C, max 29.8°C). When food was inadequate (i.e., host too small to support that number of parasitoids) pupation could be delayed and cocoons might not be formed prior to pupation.
Adult S. agrili were 3–5 mm in length and black-brown, with yellow legs and antennae with 25 segments ( Fig. 1L, 1M ). Females were slightly larger than males, with a 2 mm long ovipositor. Adult S. agrili seldom moved at temperatures below 15°C. They were noticeably more active at temperatures above 18° C and appeared fully active at 25–30°C (i.e., actively crawling, flying, courting, and mating). The active adults were positively phototatic, which was more strongly exhibited by females than males.
At temperatures of 21–30° C (average 24.7°C), the lifespan of female and male S. agrili adults was an average of 29.1 ± 2.5 d and 23.6 ± 5.8 d, respectively, when fed a 20% honey solution ( Table 3 ). This was significantly greater than for adults that had access to water only or that were reared without water or honey. Some individuals fed the honey solution lived as long as 90 d. Not surprisingly, adult longevity was longer when temperatures were lower. Female S. agrili usually lived longer than males under the same conditions ( Wang et al. 2007 ). Virgin S. agrili females reproduced parthenogenetically, producing only male offspring. The oviposition rate for unmated females fed a honey-water solution was 60%, while it was 20%) for unmated females provided water only.
Mating
Males of S. agrili typically eclosed prior to females, chewing an exit hole through the bark. In the same brood, males emerged prior to females 78.4% of the time. Males waited on the bark outside of the pupation chamber for mating opportunities with females exiting from the same hole. The emergence period for a single brood ranged from 5 to 44 d ( n = 56) with a mean period of 18.2 ± 1.18 d. This lengthy period suggested that not all mating occurred between siblings.
Male courtship behavior was most frequent when room temperatures reached 26–30°C, with frequent crawling, jumping, wing beating, and pursuing of females. In copulation, males clasped the female's dorsum, beat their wings, and tapped females' antennae vigorously with their own antennae, while the females were inactive. Mating time ranged from 2 to 35 s with a mean of 16 ± 3.8 s. Some males were observed to mate with multiple females during the observation period, but females usually avoided mating again after they mated once. After mating, females usually moved away from courting males. Females that did mate multiple times were paired with aggressive males that pursued them. When several males and females were introduced into a single test tube, apparent competitive mating behavior among males was exhibited with males often attacking copulating pairs. During field observations in August between 8:00–11:00 and 15:00–18:00 h, female and male groups with upwards of 10 individuals were observed on single tree trunks. Mating and oviposition were observed during this period in the same areas on trees.
Oviposition
Following mating, the pre-oviposition period averaged 9.9 ± 2.9 d, with some females laying eggs on day three and other females laying their first egg after 28 days. The oviposition period typically lasted 8–18 d with a maximum of 47 d. Individual S. agrili oviposited up to eight times on eight hosts, depositing 1–10 eggs (average 5.8 ± 0.24 eggs/host, n = 193) in each clutch, although up to 20 eggs per clutch was observed ( Figure 3 ). S. agrili has been shown to lay more eggs on larger A. planipennis larvae ( Wang et al. 2008 ), sometimes even parasitizing mature A. planipennis larvae that were partially or completely inside of the overwintering chamber, which was in the shallow sapwood ( Figure 1K ). First and second instar host larvae were avoided, probably because small A. planipennis larvae would not provide sufficient nutritional resources for the development of multiple parasitoid larvae. Single parasitoid females oviposited a total of 6–68 eggs over their lifetime. Fecundity was higher for females that received honey solution supplements, but the differences were not significant. In the honey treatment there were 17 pairs of wasps, and 12 females laid eggs; while in the water treatment there were 23 pairs of wasps but 11 females laid eggs. The χ 2 -test results comparing percentage of females that oviposited showed that there was no significant difference between treatments ( = 1.2457 < = 3.84) ( Table 4 ). The ovarioles of a 1-week-old female usually contained only a few mature eggs (mean 8.5 ± 0.87 eggs, range 6–15 eggs, n = 12). Because females could lay eggs multiple times, and because the total eggs laid on hosts was much greater than the number of mature eggs observed at the onset of oviposition, S. agrili was determined to be synovigenic.
Based on field and laboratory observations of eggs, larvae, and pupae, the clutch size of S. agrili was higher in forests than in the laboratory. In one instance, 35 eggs were found deposited on a mature field-collected host larva, although the number of ovipositional events that were involved could not be determined.
Female wasps were observed in the field tapping the bark with their antennae as they actively searched the trunks and twigs to locate host larvae for oviposition ( Figure 1L ). This activity typically peaked between 8:00–11:00 and 15:00–18:00 h, being greatest on sunny days when more than 10 individuals were regularly observed on a single trunk. After locating a host larva, the female inserted her ovipositor (length 1.4–2.5 mm, mean 1.9 ± 0.03 mm) completely through the bark ( Figure 1M ), piercing the host larva and paralyzing it with venom. The process required approximately 20 min. Eggs then were laid on the host's body surface ( Figure 1A ), during which another 40 to 90 min elapsed. During the egg-laying process, the female parasitoid was observed to contract rhythmically and shake her abdomen, which might aid in the movement of eggs through the egg channel in the ovipositor. The female remained at the site 10–20 min after removing her ovipositor from the bark. It is unlikely that the females deposited an oviposition deterrent pheromone because the paralyzed host larvae no longer feed, and S. agrili had been shown to oviposit only on larvae that are feeding (Wang et al., unpublished data).
Field observations of adult emergence, brood size, sex ratio, and parasitism rates
Adult S. agrili chewed an exit hole of approximately 1 mm in diameter in their cocoon to escape ( Figure 1H ) when they emerged. After exiting the cocoons, S. agrili then bit a round hole through the bark (mean 0.9 ± 0.03 mm diameter, range 0.6–1.8 mm diameter) ( Figure 1J ) and emerged. Typically, only one exit hole was made per brood, although two or three were observed occasionally. Adults emerged most often at the 15:00 sampling period.
Adults of S. agrili first appeared in Tianjin ash forests in late May (2004) or June (2003) and continued emerging for over 2 months ( Figure 4 ). In both years, emergence was low during the first 2–3 weeks, when A. planipennis larvae were early instars and unable to support feeding by the usually gregarious S. agrili larvae. Beginning in early July 2003 and early June 2004 there was a large increase in S. agrili emergence that lasted for approximately one month. It was during this period that the preferred older instars of A. planipennis became available. Studies have shown that S. agrili adults emerge up to several months after A. planipennis adults to allow for development of the preferred host stage, the late instar larvae ( Wang et al. 2006 ).
Field-collected single broods of S. agrili consisted of 2–18 cocoons (average of 9.1 per brood). Sex ratios were highly variable during the emergence period within and between years ( Figure 4 ). When data were pooled, females outnumbered males 2.1 to 1 ( Table 5 ). Sex ratios were also determined from a total of 316 adults of S. agrili that were reared in the laboratory from field-collected eggs and larvae. Adult emergence was grouped into three consecutive periods during the summer of 2003, and sex ratios (female: male) ranged from 3.3:1 to 3.5:1 ( Table 6 ). There was no significant difference among these sex ratios or individually from 3:1.
The parasitism rate of the first S. agrili generation was relatively low, about 10%. However, parasitism gradually increased and peaked with the overwintering generation in the late year, usually over 40% ( Figure 5 ), with parasitism in some forest stands exceeding 90%. During 2003 in Tianjin, adult S. agrili were sampled by sweep net as late as October 10, but at that time they appeared much less vigorous and typically died soon in the laboratory. These adults may have been part of the last generation of the season.
Number of generations per year
At ambient room temperature of 18–27° C (average 22.4°C), the development duration from egg to adult stages was 27–28 d, with the mean 27.9 ± 0.63 days (range 25–34, n = 26) for females and 28.2 ± 1.38 days (range 26–35, n = 6) for males. Combining estimations of developmental durations under laboratory conditions with field temperatures recorded during observation, the occurrence of all generations of S. agrili were estimated ( Figure 6 ). It was predicted that S. agrili could complete 3–4 generations per year in the forests of Tianjin, China. | Spathius agrili Yang (Hymenoptera: Braconidae) is a gregarious larval ectoparasitoid of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) and is a recently described species. Both pest and parasitoid are native to China. In Tianjin City, China, S. agrili typically exhibited 3–4 generations per year, overwintering as a prepupa in a cocoon inside the host gallery. The multiple generations of S. agrili overlapped with its host, as did the emergence dates of the overwintering generation. From a single host, 1–18 S. agrili successfully developed to the adult stage (average 8.4), but in all cases the host was killed. The sex ratio (female: male) of the parasitoid adults emerging from field-collected cocoons was 2:1, whereas the sex ratio of parasitoids reared from field collected eggs and larvae was greater than 3:1. On average, adult females lived 29.1 d, and males lived 23.6 d when fed with 20% honey solution, significantly longer than without a nutritional supplement. Sexual reproduction is the normal mode of reproduction, but in the laboratory females did reproduce parthenogenetically, producing only males. The average fecundity was 23.3 eggs per female in the laboratory. S. agrili developed through five larval instars, and the larvae fed gregariously on the host hemolymph. The generation time from egg to adult wasp was 27–28 d at 22–26°C. Natural parasitism rates were as high as 60%, and in October they reached over 90% in some stands. This study showed that S. agrili is a promising agent for biocontrol of A. planipennis.
Keywords | Acknowledgments
This research was supported by the U.S. Department of Agriculture, Forest Service, and Animal & Plant Health Inspective Service (APHIS); International Foundation for Science (D/3689-1, 2) from Sweden, and National Natural Science Foundation of China (30671689), as well as the Key Projects in the China National Science & Technology Pillar Program (2006BAD08A12). | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 6; 10:30 | oa_package/0b/29/PMC3014743.tar.gz |
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PMC3014744 | 21209801 | 1. Introduction
Although hypercalcemia is a known metabolic complication of sarcoidosis, it is rarely a presenting manifestation. Hypercalciuria is the most common defect of calcium metabolism with a prevalence of 50–62% [ 1 – 3 ]. Although hypercalcemia in 11% and renal calculi in 10% of patients with sarcoidosis has been reported [ 4 ], clinically significant hypercalcemia is less frequent and is generally asymptomatic, occurring in less than 5% of patients [ 5 ]. Persistent untreated hypercalcemia and hypercalciuria can lead to nephrocalcinosis, renal calculi, and renal failure [ 6 ]. We report two cases of sarcoidosis, where the initial presentation was markedly elevated serum calcium levels and acute renal failure, which presented a diagnostic challenge. | 4. Discussion
Hypercalcemia is a common clinical entity encountered these days because of the routine multichannel chemistry screening. The most common cause of hypercalcemia in the outpatient clinic is primary hyperparathyroidism, whereas malignancy is the most common cause of hypercalcemia in hospitalized patients. Other causes of hypercalcemia, including sarcoidosis, are relatively uncommon. Both of our patients presented with markedly elevated serum calcium levels (13.5 and 13.3 mg/dL), and there were no other clinical clues to suggest sarcoidosis as the diagnosis. Both patients had appropriately suppressed PTH levels suggesting that the cause of hypercalcemia was not parathyroid hormone dependent. Patient 2 also had an elevated PTHrP which suggested an increased likelihood for malignancy. Both patients underwent an extensive, but futile search for malignancy.
Sarcoidosis is an idiopathic, multisystem, and granulomatous disease. It affects people of all racial and ethnic groups of all ages. Its peak incidence is between 20 and 39 years. The incidence among African Americans is approximately three times than among white Americans and tends to occur later in life [ 4 ].
The fundamental abnormality in sarcoidosis involves the formation of immune granulomas in various organs. The lung and the intrathoracic lymph nodes are the main organ systems involved, but every organ may be affected [ 7 ]. The frequent extrathoracic involvement sites include peripheral lymph nodes, eyes, skin, and liver, each being found in approximately 10–25% of cases [ 7 ]. Sarcoidosis commonly presents with the abnormalities detected on a chest X-ray (8–60%), but the presentation may vary depending on several factors such as age, sex, race, the duration of the disease, and the sites of involvement. European population is more likely to present asymptomatically or with erythema nodosum, while symptomatic and multivisceral presentations are common in African Americans. The initial symptoms of presentation include respiratory symptoms in 30% cases, constitutional symptoms such as fatigue (27%), weight loss (28%), fever (10–27%), and erythema nodosum (3–44%) [ 7 ]. Lofgren's syndrome is an acute form of disease, consisting of arthritis, erythema nodosum, and bilateral hilar adenopathy (9–34%) and presents differently in men and women [ 4 ].
As mentioned above, although hypercalcemia occurs in about 11% cases of sarcoidosis [ 4 ], clinically significant hypercalcemia is less common [ 6 ], and we believe even rarer as the presenting feature. However, we were unable to determine how often it is the presenting symptom. Similarly, although acute renal failure as the presenting feature of sarcoidosis has been described, it remains of rare occurrence [ 8 , 9 ].
The mechanism of hypercalcemia in sarcoidosis is not completely understood. Normally, the levels of vitamin D and its active metabolite 1,25-dihydroxyvitamin D play an important role in the maintenance of serum CA ++ levels.
Vitamin D is derived either from endogenous production of vitamin D in the skin or by ingestion of vitamin D. Vitamin D is hydroxylated to 25 hydroxyvitamin D in the liver, which is subsequently converted to 1,25-dihydroxy D in the proximal renal tubules by the enzyme 1, α hydroxylase. This enzyme is tightly regulated by parathyroid hormone (PTH), serum calcium, and phosphorus levels [ 10 ].
In 1939, Harrell and Fisher reported the occurrence of hypercalcemia in 6 of 11 patients with sarcoidosis [ 11 ]. On subsequent studies, in 1979, Papapoulos et al. [ 12 ] and Bell et al. [ 13 ] were among the first to recognize that levels of 1,25-dihydroxyvitamin D are elevated in patients with sarcoidosis. The high levels of 1,25-dihydroxyvitamin D are the probable cause of hypercalcemia, but overproduction of bone resorbing cytokines and PTHrP may also play a role [ 14 ]. The level of PTHrP in one of our patients was elevated, but in case 1 it was normal. Zeimer et al. described two cases of sarcoidosis with hypercalcemia and elevated PTHrP. They also demonstrated that in an archival study of 20 lymph node biopsies with the pathological diagnosis of sarcoidosis, where immunohistochemical analysis detected PTHrP in macrophages with granulomata in 17 out of 20 biopsies (85%) [ 14 ]. The renal 1, α hydroxylase is activated by PTH and suppressed by 1,25-dihydroxyvitamin D [ 10 ]. The macrophage enzyme is unaffected by any of these factors. Instead, one of its major trophic activators is cytokine interferon gamma [ 15 ]. Lack of feedback in response to increased levels of 1,25-dihydroxyvitamin D leads to an enhanced calcium absorption in the small intestine and an excess of bone resorption in patients with sarcoidosis, leading to hypercalcemia.
The diagnosis of sarcoidosis may be elusive, especially in atypical populations, given the variation of the presentation of the organs involved. Hypercalcemia may be a clue to the diagnosis, especially in cases with no pulmonary symptoms or findings on chest X-ray. Sarcoidosis should be considered in patients with hypercalcemia, low PTH levels, and elevated calcitriol levels. As mentioned above, longstanding hypercalcemia and hypercalciuria can cause nephrocalcinosis and renal failure [ 2 , 6 ]. Although severe hypercalcemia is rare in sarcoidosis [ 1 , 5 ], patients may present with severe hypercalcemia, associated with renal failure, as was the case in our patients.
Treatment of hypercalcemia due to sarcoidosis is beyond the scope of this paper. However, it has been reviewed by Conron et al. [ 1 ] and Sharma [ 16 ] and is discussed here only briefly. Therapeutic options include rehydration, a loop diuretic to promote calcium secretion, and corticosteroids [ 1 ]. Corticosteroids have been the first-line therapy in treating hypercalcemia associated with sarcoidosis because of their effectiveness in rapidly correcting hypercalcemia.
Prednisone in 20 to 40 mg/day is the drug of choice to reduce the endogenous production of 1,25 dihydroxyvitamin D. Institution of steroids causes a relatively swift decrease in circulating 1,25 dihydroxyvitamin D and serum calcium levels in 3 to 5 days [ 16 ]. Patients with asymptomatic mild hypercalcemia should be advised to avoid sun exposure and vitamin D rich diet and maintain hydration of more than 2 liters per day. The role of avoiding dairy product and low calcium diet is controversial since there is little evidence that it affects calcium balance [ 1 ].
Ketoconazole is an alternative for patients where the steroid use is contraindicated. It inhibits the cytochrome P450-linked enzyme systems involved in steroid synthesis including 25(OH) D 3 -1a-hydroxylase [ 17 ]. Hydroxychloroquine also causes similar effects and can be used in patients who cannot tolerate ketoconazole or who develop abnormal liver function tests [ 18 ]. Methotrexate and azathioprine may also help control hypercalcemia by reducing the granuloma formation [ 1 ]. | 5. Conclusion
We report two patients with sarcoidosis, where severe hypercalcemia associated with acute renal failure was the unusual presenting feature. It was only after extensive investigation that a diagnosis of sarcoidosis was confirmed. Sarcoidosis without pulmonary symptoms may pose a diagnostic challenge but should be suspected in patients with nonparathyroid-dependent hypercalcemia. Timely recognition and appropriate treatment of hypercalcemia secondary to sarcoidosis should prevent development of nephrocalcinosis and renal failure. | Academic Editor: Hermann Wasmuth
Although hypercalcemia is a known metabolic complication of sarcoidosis, it is rarely a presenting manifestation. Long-standing hypercalcemia and hypercalciuria can cause nephrocalcinosis and chronic renal failure. Acute renal failure, although described, is also a rare presentation of patients with sarcoidosis. We describe two patients with sarcoidosis, who presented with severe hypercalcemia and worsening renal function. Parathyroid hormone levels were appropriately suppressed. This led to an extensive search for the cause of hypercalcemia. Finally, after a lymph node biopsy in both cases, a diagnosis of sarcoidosis was established, hypercalcemia resolved, and renal function improved in both cases after administration of prednisone. | 2. Case 1
A 56-year-old Caucasian male was admitted three times to the hospital in 3 months for recurrent abdominal pain. He described it as an intermittent cramping pain, located in the epigastrium, with some radiation to the left lower quadrant. He also experienced intermittent nausea, vomiting, and watery diarrhea. The pain was aggravated by lying in supine position and improved with sitting upright. He denied having any fever, chills, or any other associated symptoms. He also noted excessive thirst and increased in urination but denied dysuria or hematuria. He was found to have persistent hypercalcemia and renal insufficiency (see Table 1 ). A review of his medical record revealed that he had been admitted to the hospital twice during the preceding two months with similar symptomatology. Each time he was noted to have hypercalcemia (serum calcium 13.3 and 13.5 mg/dL, resp.) and acute renal insufficiency (BUN 44 mg/dL and creatinine 2.1 mg/dL). Serum and urine protein electrophoresis were negative. 1,25-dihydroxyvitamin D level was 69 ng/mL (reference range 22–67 ng/mL), and a simultaneously obtained PTH level was 6.9 pg/mL (reference range 14–72 pg/mL). His past medical history was only significant for nephrolithiasis. He was not taking any medications regularly. Physical examination revealed blood pressure of 129/80 mmHg, heart rate 80/min regular. He was febrile and did not appear to be in acute distress. His cardiopulmonary exam was unremarkable. Abdominal exam revealed mild tenderness in the epigastrium area and left lower quadrant without any signs of guarding or rigidity. Bowel sounds were active. Acute abdominal series X-ray did not show any acute process. Laboratory investigation showed normal CBC and urine analysis. Peak serum calcium was 13.5 mg/dL (reference range 8–10 mg/dL), and ionized calcium was elevated at 1.68 mmol/L (reference range 1.10–1.30 mmol/L). Serum creatinine level was 3.4 mg/dL (0.5–1.4 mg/dL). 1,25-dihydroxyvitamin D levels were elevated and parathyroid hormone (PTH) levels were appropriately suppressed (see Table 1 ). Serum angiotensin converting enzyme level was elevated at 98 units/L (reference range 8–52 units/L). He was treated with intravenous fluid hydration and loop diuretic while the etiology of hypercalcemia was being sought, including an extensive investigation for occult malignancy and granulomatous diseases. Chest X-ray was normal. CT scans of chest, abdomen, and pelvis were performed. Bone scan was unremarkable. CT Chest revealed subtle mediastinal adenopathy ( Figure 1 ), and subsequent biopsy (Figures 2(a) and 2(b) ) confirmed the diagnosis of sarcoidosis. Corticosteroid therapy was initiated and hypercalcemia eventually resolved, and renal function also improved (BUN 32, creatinine 1.8 mg/dL).
3. Case 2
A 59-year-old Caucasian female was incidentally noted to have hypercalcemia and elevated blood pressure during routine outpatient labs screening. She stated that she had been feeling well. She denied any history of fatigue, nausea, anorexia, constipation, muscle ache, or changes in sensorium. She did not complain of polyuria or polydipsia. However, she stated that she drank 32 to 44 ounces of water during the day as a matter of habit. She had lost 25 pounds of weight within the past six months unintentionally. She had a remote history of nephrolithiasis in 1960 and occasional bilateral hip pain. She also gave a history of removal of a skin lesion on the nose, which was reported as “benign.” She denied taking any vitamin or other herbal supplements. There was no family history of endocrine disorders. She had no history of smoking tobacco, consuming alcohol, or use of illicit drugs. She had not been taking any medications. Physical examination revealed elevated blood pressure of 210/98 mmHg that substantially came down to 146/86 with treatment. Oral temperature was 99.4 F (37.4°C); pulse had a regular rhythm with a rate of 80 bpm. She was alert, oriented, and did not appear to be in acute distress. Her lungs were clear to auscultation with normal respiratory effort. A grade 2/6 systolic ejection murmur was heard over left sternal border. Abdomen was soft and not tender to palpation. Muscle strengths were normal and symmetrical in all extremities. She was found to have normochromic normocytic anemia with hemoglobin of 9.9 g/dL. Serum calcium level was noted to be persistently elevated over a period of 6 months with a peak level of 14.0 mg/dL (range from 11.4 to 14.0 mg/dL). Renal function was impaired with a peak serum creatinine of 2.4 mg/dL (range 1.7–2.4 mg/dL). BUN ranged from 26 to 32 mg/dL. 24 hours urine calcium was elevated at 448 mg and 587 mg measured on two different occasions (reference range 100–300 mg/ 24 hr). Serum phosphate and 25 hydroxyvitamin D levels were within normal limits (see Table 1 ). PTH level was appropriately suppressed and PTH related peptide (PTHrP) was elevated. A diagnosis of occult malignancy was suspected. Angiotensin-converting enzyme (ACE) was elevated (see Table 1 ). Chest and spine X-rays were negative as well as mammogram, bone marrow biopsy, and bone scan. Extensive evaluation failed to reveal malignancy. CT scan of the chest was performed as part of a malignancy workup, and it showed borderline mediastinal adenopathy in the paraaortic and pretracheal area and subcarinal region ( Figure 3 ). Biopsy of the lymph nodes confirmed the diagnosis of sarcoidosis (Figures 4(a) and 4(b) ). With steroid treatment, the hypercalcemia resolved (Ca = 9.2 mg/dL) and renal function also improved (BUN 21.3 mg/dL, creatinine 0.8 mg/dL). | Acknowledgments
The authors thank Jinie Shirey for preparing this paper and James Richard, DO for his help with the pathology material. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 16; 2010:423659 | oa_package/d6/98/PMC3014744.tar.gz |
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PMC3014745 | 20572792 | Introduction
Synanthropic mites are important pests, and their gut pH strongly affects acaricidal functions, pathogen infection success, and digestive processes. It is necessary to characterize the gut pH first to study the activity of pest digestive enzymes and gut microbial flora, ( Skibbe et al. 1996 ; Terra et al. 1996 ; Funke et al. 2008 ). Although several studies determining pH in the gut have been conducted, correctly determined pH values in the digestive tract of synanthropic mites are lacking.
In general, colorimetry, potentiometry, and conductometry are useful methods for the determination of pH ( Carsky 1981 ). Microelectrodes are the most practical and most frequently used tools to determine the pH in insects ( Brune et al. 1995 ; Harrison 2001 ; Zimmer and Brune 2005 ; Gross et al. 2008 ), but this is not possible in mites due to their small body size. Skibbe et al. ( 1996 ) showed nuclear magnetic resonance microscopy as an alternative method for determination of pH in insect guts, but this method is not commonly used.
Colorimetric determinations of pH use halochromic chemical compounds as pH indicators. The pH-dependent color changes of acid-base indicators are described by Ostwald's theory, which was later improved by Hantzsch for tautomery conception: such that the human eye is able to perceive color changes of two diverse colored forms at concentration ratios from 10:1 to 1:10, so that the transition range is about pH p K A (HIn) ± 1, where p K A (HIn) is the dissociation constant of the indicator. According to Ostwald's theory, pH indicators are weak acids or bases, and the pH closest to the equivalence point is the titration exponent pT. The color change depends on the presence of dissociated and non-dissociated forms of a given indicator, which results from the activity of hydronium ions (H 3 O + ). The indicators do not change sharply at a particular pH, but rather change over a narrow range of pH values. Indicators exhibit intermediate colors at pH values inside these transition ranges ( Brdicka and Dvorak 1977 ; Dvorak and Koryta 1983 ; Holzbecher and Churacek 1987 ).
Although the results obtained using pH indicators correspond to potentiometric determinations ( Espinoza-Fuentes and Terra 1987 ; Boudko et al. 2001 ), potentiometry is a more exact method than colorimetry. Due to their size, the only useful method for determining pH in the digestive tract of acaridid mites is colorimetry. The fact that a mite's body is thin and transparent makes it suitable for the application of colorimetry and the direct determination of the gut pH using a compound microscope. This method was previously applied to determine the physiological pH in the gut using several pH indicators such as litmus, phenol red, universal indicator, and neutral red ( Hughes 1950 ; Akimov and Barabanova 1976 , 1978 ; Akimov 1985 ). The present study is the first report of the pH in the gut of stored product and house dust mites, based on colorimetrical determinations using 18 indicators. | Materials and Methods
Experimental mites
Twelwe species of acaridid mites (Acari: Astigmata) from the following families were tested including 5 species of Acaridae ( Acorus siro Linnaeus, Aleuroglyphus ovatus Troupeau, Sancassania rodionovi Zachvatkin (syn. Caloglyphus redickorzevi Zachvatkin and Caloglyphus hughesi Samsinak), Tyrophagus putrescentiae Schrank, and Tyroborus lini Oudemans); Carpoglyphidae ( Carpoglyphus lactis Linnaeus); Chortoglyphidae ( Chortoglyphus arcuatus Troupeau); 2 species of Pyroglyphidae ( Dermatophagoides farinae Hughes and Dermatophagoides pteronyssinus Trouessart); and 3 species of Glycyphagidae ( Glycyphagus domesticus De Geer, Lepidoglyphus destructor Schrank, and Aëroglyphus robustus Banks). These species were maintained in laboratory colonies kept in the Crop Research Institute, Prague, Czechia (for a detailed description of rearing conditions, see Erban and Hubert ( 2008 )).
Application of indicators and preparation of mites for microscopic observation
The selection of indicators was based on their pT and/or p K A values and transition ranges ( Holzbecher and Churacek 1987 , Senese 2000 , Shevchenko and Kulichenko 2005 , Sabnis 2008 ). All indicators were purchased from Sigma-Aldrich. The indicators were separated into six groups: (1) Indicators determining the pH between 3 and 8 were Phenolphthalein (p K A = 9.1, 9.4, 9.53, 9.70; pT = 8.4–9.4; transition range = 8.3–10.0) and thymol blue (p K A = 1.65; 8.90; pT = 2.6 and 8.4; transition range = 1.2–2.8 and 8.0–9.2); (2) Indicators determining pH values near neutral pH were litmus (p K A = 7.00; pT = NF (not found); transition range = 5.0–8.0), phenol red (p K A = 7.9; pT = 7.0; transition range = 6.8–8.2), brilliant yellow (p K A = NF; pT = NF; transition range = 6.5–8.0) and bromothymol blue (p K A = 7.0, 7.1; pT = 6.4; transition range = 6.0–7.6); (3) Indicators determining the basic limit of gut pH were bromophenol red (p K A = 6.51; pT = 6.16; transition range = 5.2–6.8) and chlorophenol red (p K A = 6.00; pT = 6.00; transition range = 4.8–6.4); (4) Indicators determining the lower (acidic) limit of gut pH were bromophenol blue (p K A = 4.0, 3.85, 3.6; pT = 4.0; transition range = 3.0–4.6), alizarin red S (p K A = 4.5; pT = NF; transition range = 3.5–6.5), methyl orange (p K A = 3.76, 3.40; pT = 4.0; transition range = 3.0–4.4) and congo red (p K A = 4.1; pT = NF; transition range = 3.0–5.0); (5) Indicators determining pH more accurately within the acid and base limits were methyl red (p K A = 5.06, 2.3, 2.5, 4.95; pT = 4.80; transition range = 4.4–6.2), resazurin (p K A = 6.71; pT = NF; transition range = 3.8–6.5) and bromocresol green (p K A = 4.7, 4.6; pT = 4.66; transition range = 3.8–5.4); (6) Universal acidbase indicators (UABI) were UABI 3–10 (transition range = 3.0–10.0), UABI 0–5 (transition range = 3.0–10.0) and UABI 4–10 (transition range = 4.0–10.0).
The indicators were dissolved in water or a water-ethanol solution and transferred into ground wheat to obtain a 2% final concentration (wt/wt) of indicator. The moistened indicator-enriched diet was mixed using a vortex (IKA® Works, Inc., www.ika.com ) and then placed in a sealed glass tube for freeze drying (Heto PowerDry LL3000, Thermo Fisher Scientific, www.thermo.com ). The dried indicator-enriched diet was pulverized using a porcelain mortar. About 5 mg of the powder was transferred into 1.5 ml microcentrifuge tubes (Eppendorf, www.eppendorfna.com ) and at least 50 specimens were added. A piece of moistened filter paper was added to each tube. In intervals of 24, 48 and 72 hours, the mites were removed and placed in a glass Petri dish. After the mites were scattered, the specimens that had ingested indicator were collected using a camel hair brush under a Stemi 2000-C dissection microscope (Carl Zeiss, www.zeiss.com ). The mites were rinsed in a drop of physiological solution (0.9% NaCl) to remove external food particles before being examined by light microscopy.
Microscopic observations of mite gut
The color changes of the gut were observed under a compound microscope Axioskop using Axiovison software (Carl Zeiss) and a Powershot A620 digital camera (Canon Inc., www.canon.com ). The minimal design included 10 positive observations per species per indicator. Unlike in histological studies ( Sobotnik et al. 2008 ), the intercolon could not be distinguished from the gut during observation. In the present study, the color changes of the indicators in the ventriculus, caeca, colon, and postcolon were determined, and the intercolon was excluded (for detail of gut anatomy, see Figure 1 ). The color changes of the dissolved dyes in the gut lumen were taken into account, and the color changes of food or fecal boli (which contained highly concentrated indicators) were omitted. To generate color change controls, the color indicators were dissolved in Britton Robinson I buffer. This was made with a precision of 0.1 pH units, and the color changes were compared to the colors in the microscopic observations. | Results
Mites not feeding on the indicator diet had transparent bodies. The ingestion of indicator diets changed the color of the gut ( Table 1 ).
Indicators determining the pH between 3 and 8
Phenolphthalein was always colorless and corresponded to observations without any indicator. Thymol blue was always yellow and no exceptions were found.
Indicators determining pH values near neutral pH
The indicators displayed a yellow color in all cases, except red-colored litmus and a seldom-seen red coloration of the postcolon by phenol red. The color changes indicated that the pH in the gut was generally more acidic than a pH of 6.5. There were observed interspecies differences in the color of phenol red in the postcolon of all species tested. The red-colored postcolon indicated a pH of about 7 in some cases.
Indicators determining the basic limit of gut pH
This group of indicators showed that the upper limit on the pH scale was about 6.0. The indicators together showed a pH of less than 6 in the caeca and the ventriculus of all tested species of mites. In all species, the colors of both indicators were yellow in the ventriculus and caeca. In the colon and postcolon, a red color was observed, and the intensity of red coloration significantly increased from colon to postcolon. Only Dermatophagoides spp. and A. robustus showed yellow coloration of the whole gut.
Indicators determining the acidic limit of gut pH
Generally, the color changes of the indicators showed a pH higher than 4 in the mesodeal lumen. Bromophenol blue typically resulted in a blue coloration of the caeca, ventriculus and colon. However, a different situation was found in both Dermatophagoides species where they were yellow or green. In one specimen of T . lini , a green/blue coloration was observed in the ventriculus and caeca. Methyl orange and congo red ( Figure 2A ) produced similar colors in all mesodeal compartments among all species tested. Methyl red produced a yellow color and congo red ( Figure 2A ) produced a red color throughout the whole gut. Alizarin red S produced a red, or yellow, colored gut and did not correspond with other indicators.
Indicators determining pH more accurately within the acid and base limits
Methyl red indicates a pH below 5 when it is red and a pH above 5 when it is yellow. The predominantly red-colored methyl red caeca and ventriculus were therefore indicative of a pH below 5. On the other hand, a predominant yellow color was found in the colon, indicating a pH higher than 5. A yellow postcolon indicated a pH more basic than pH 6. This is in respect to the methyl red transition range (4.4–6.2) and the absence of yellow color below pH 6.2. Methyl red nicely illustrated the pH gradient across the whole gut from the middle acidic caeca and ventriculus to the rather neutral postcolon. Resazurin changed color from orange to violet within these values. Based on the titration, the fuchsia color is recognizable from a pH of about 5 and more alkaline, while violet is visible from a pH of about 5.5 and more alkaline. The prevailing color in the gut was orange. There were rare situations in which specimens had a fuchsia color in the colon and a violet color in the postcolon. Bromocresol green becomes green within the narrow pH range of 4.2 and 4.7, while the cyan blue range is between 4.8 and 5. The yellow color indicates a pH more acidic than 4.2. Between pH values of 5.1 and a neutral or alkaline pH, the indicator is blue. The ventriculus and caeca of most species were colored blue and/or green. An exception was found, however as additional yellow coloration was found in the ventriculus and caeca of four species of mites. While A. siro and C . arcuatus had, in one case, a yellow color mixed with blue, some specimens of Dermatophagoides spp. had a mostly yellow foregut. Generally, the blue coloration in the colon and postcolon did not show significant color differences between species. Additional green coloration in the colon in some cases further indicated that the colon is more acidic than the postcolon and more alkaline than the ventriculus and the caeca.
Universal acid-base indicators
This group of indicators represented cocktails that were able (with some limitations) to indicate pH with an accuracy of 1 pH unit. UABI 3–10 ( Figure 2B ) showed a light orange color in the ventriculus and caeca, corresponding to a pH between 4 and 5. The light orange color changed to yellow in both the colon and the postcolon, indicating a pH around 6 ± 0.5. The green coloration of the colon or postcolon indicated the possibility of a more alkaline environment. In addition, green coloration indicated a pH of about 7 inside the mesodeal cells and ovaria. Generally, the color produced by UABI 0–5 was blue/cyan or blue in the whole gut, indicating a pH higher than 4 for all compartments. In general, UABI 4–10 turned from orange in the ventriculus and caeca to yellow in the colon and postcolon. The orange color of UABI 4–10 indicated a pH below 5 in the ventriculus and the caeca; the yellow color indicated a pH between 5 and 6.5 in the colon and postcolon, and the green coloration indicated a pH of about 7 inside the mesodeal cells and ovaria, similar to the results from UABI 3–10 ( Figure 2B ). | Discussion
The method described here can be evaluated as a universal approach for the determination of pH by acidobasic pH indicators in the arthropod gut, where the only necessary condition is the transparency of the arthropods' bodies or dissection of the gut ( Terra and Regel 1995 ). Acidobasic indicators are suitable for the determination of pH if the size of the sample does not allow for the usage of microelectodes, which offer much more precise measurements. The disadvantages of acidobasic indicators lie mainly in the subjectivity of the determinations, since the pH indicators are susceptible to imprecise readings. For precise determination of pH using indicators, it is necessary to use many of them, since they do not change color sharply at one particular pH. Every pH indicator has a tabulated transition range, but none determine pH exactly. The color inside the transition range goes from a lower (more acidic environment) to an upper (more alkaline environment) pH limit, but the human eye starts to detect the second color at some point near the titration exponent (pT). The pT can help us in evaluation of the data, but it is necessary to note that it has some limitations. These are the main reasons why such a large palette of pH indicators is needed for the determination of pH in the gut compartment.
The use of universal acid base indicators as an initial step in the pH determinations may offer a prescreened pH in the gut, possibly reducing the number of indicators used. In this case, however, it could eliminate only the use of Phenolphthalein and thymol blue. If prescreening is used, there is the possibility of making subjective decisions that could affect the results ( Barrow and Conrad 2006 ). At least several, never one or two, pH indicators must be always used in determination of pH to obtain objective data, because each pH indicator can determine the pH only very approximately with an accuracy of pH ± 1. The physicochemical theories of Ostwald and Hantzsch must be respected in this regard also in evaluation of the data. For example, the approximation of the average value inside the transition range is not accurate.
An initial study of pH in the gut of mites showed that pH in the ventriculus and caeca of A. siro (syn. Tyroglyphus farinae ) was between 5.0 and 6.0, while the pH in the colon was determined to be above 7.0 and below 8.0 ( Hughes 1950 ). More recently, the pH in the gut of 16 mites was determined to be in the range of 5.4 to 6.3 in the caeca and ventriculus, 5.9 to 7.4 in the colon, and 6.8 to 8.0 in the postcolon ( Akimov and Barabanova 1976 , 1978 ; Akimov 1985 ). Because these results were obtained using a limited number of indicators (litmus, phenol red and neutral red), some inaccuracies would be expected. In addition, the determination of pH to an accuracy of 0.1 pH unit is impossible according to the laws of physical chemistry. For example, litmus is a pH indicator that generally determines acid or base pH since it is purple at about a pH of 7. The transition range of litmus is wide and ranges from 4.5 to 8.3. The purple color determined by Hughes in the colon of A. siro could determine pH somewhere inside the transition range, starting at a pH of about 6 to 7 (the red and blue are equal). At more acidic values, this indicator starts to become blue, which was not observed. The purple color of litmus, indicating neutral pH, was recorded in our study only for food boli in the postcolon, where we suggest that the color could be affected by a higher concentration of indicator. The neutral red and phenol red showed a pH more acidic than 7 only in the ventriculus, caeca and colon. Only the universal indicator could determine the pH more accurately, but universal indicators usually have the same color in a range of at least 1 or 2 units.
Nevertheless, based on these previous studies, the basic limit of the pH in the gut was near or below pH 8, while the acidic limit was unclear. The same results can be obtained from the determination of pH using Phenolphthalein and thymol blue. In addition, the thymol blue results exclude strong acidic pH (below pH 3). According to the results of two of the groups of indicators (indicators determining pH values near neutral pH and indicators determining the basic limit of gut pH), the basic pH limit of the postcolon and colon were 6.5–7 in the postcolon and about pH 6 in the colon. The caeca and ventriculus both had a pH lower than 6. Dermatophagoides spp. and A. robustus had different properties, with a pH of about 6 in both the postcolon and the colon. Indicators determining the acidic limit of gut pH, in general, showed a pH higher than 4 or near 4 throughout the whole gut. The results of Alizarin red S tests led to the conclusion that the presence of yellow and red in all compartments was affected by the extremely wide pH transition range (pH 3.5–6.5). Second, the coloration was affected by the presence of Ca 2+ ions ( Dahl 1952 ). Both Dermatophagoides mites displayed tendencies for buffering at pH 4 in the caeca and ventriculus. The use of indicators determining pH more accurately within the acid and base limits showed a more acidic pH in the gut of both Dermathopagoides species. This corresponds to their feeding on protein-rich food sources and the presence of cysteine proteases (formerly allergens Der p1 and Der f1) ( Tovey et al. 1981 ). Secretion and synthesis of Der p1 in the cells of the posterior ventriculus was shown using immunostaining of D. pteronyssinus sections ( Tovey and Baldo 1990 ). In insects, the acid pH optima of cysteine proteases are well documented ( Terra and Ferreira 1994 ) and could also be expected in mites.
As shown here, indicators determining pH more accurately within the acid and base limits provide for a more exact determination of pH in the morphological compartments of the mite gut, including (a) the anterior gut (the ventriculus and caeca), with a pH range of 4 to 5; (b) the midgut (intercolon/colon), with a pH between 5 and 6; and (c) the postcolon, with a pH ranging from 5.5 to 7. In addition, using the indicators red/yellow methyl red and blue/green bromocresol green, the pH in caeca and ventriculus of the mites (except Dermatophagoides ) was determined more precisely in range from 4.5 to 5 or slightly more alkaline. Dermatophagoides spp. had a more acidic anterior gut (a pH from 4 to 5), colon (a pH of 5) and postcolon (a pH of below 6).
The pH of the midgut lumen is, in most cases, actively regulated and varies with the phylogeny and feeding ecology ( Harrison 2001 ). The gut physicochemical properties are optimized with respect to utilized food source ( Zimmer and Brune 2005 ). There is a relationship between mesodeal pH and the pH optima of some digestive enzymes that hydrolyze the nutrients in the lumen contents ( Terra et al. 1996 ; Funke 2008 ). The pH in the gut is important not only for the digestive enzymes, but it also influences the solubility of food components, the dissociation or coagulation of ingested proteins, and the presence of gut microflora ( Funke et al. 2008 ). In mites, the known pH optima of enzymes correspond, in some cases, to the observed pH in the gut. The lysozyme pH optima of 4.0 to 5.0 corresponded to the lumen pH in the ventricles and caeca, where lysozyme activity was localized ( Erban and Hubert 2008 ). A similar situation was observed for α-amylases and α-glucosidases with pH optima from 5 to 6 (Akimov and Barabanova 1976a, 1976b, 1978 ; Akimov 1985 ; Sanchez-Monge et al. 1996 ; Hubert et al. 2005 ; Hubert et al. 2007 ). However, the ranges of the pH optima for cellulase (5–7.5) and chitinases (5–8) are not in accord with the pH of the gut lumen in many of the species tested (Akimov and Barabanova 1976a, 1976b, 1978 ; Akimov 1985 ). This suggests that these enzymes do not have digestive functions in the gut lumen.
Proteases were extensively studied in acaridid mites due to their allergenic importance ( Tovey et al. 1981 ). The enzymatic analyses were based usually on both specific and non-specific substrates combined with the panels of inhibitors ( Ortego et al. 2000 , Monteallegre et al. 2002 , Sanchez-Ramoz et al. 2004 ). In vitro , the enzymatic activities were measured usually in the alkaline environment. For example, mite proteases measured using azocasein as the nonspecific substrate, showed pH optima at 6.0 in body extracts and 9.5 or 10.0 in feces. The pH optima of proteolysis of haemoglobin at pH 3 or 4 indicated the presence of non-digestive acid proteases. The pH optima of the specific activities of serine and cysteine proteases were determined to be near pH 7 or 10.5 in both mite bodies and feces ( Ortego et al. 2000 , Sanchez-Ramoz et al. 2004 ). Monteallegre et al. ( 2002 ) measured activity and inhibition of serine proteases of mite bodies at pH 7.8 and 7.4.
In previous studies pH measurements were not determined in the mite gut under physiological conditions. According to the results of the present study, the alkaline gut environment does not correspond to the pH optima of the digestive enzymes. Thus, these proteases are not functional ( Terra and Ferreira 1994 ). The study of digestive enzymes needs to be focused on the physiological gut pH. | The pH of the guts of mites strongly affects their digestive processes. This study was carried out to determine the pH in the guts of 12 species of stored product and house dust mites. Eighteen pH indicators were chosen and offered to the mites in the feeding biotest. Based on the color changes of the indicators, the gut contents of acaridid mites were determined to be within a pH range of 4 to neutral. The gut contents showed a gradient in pH from the anterior to the posterior part. The anterior midgut (ventriculus and caeca) of most species had a pH ranging from 4.5 to 5, or slightly more alkaline for most of the species, while the middle midgut (intercolon/colon) had a pH of 5 to 6. Finally, the pH of the posterior midgut (postcolon) was between 5.5 and 7. Except for Dermatophagoides spp., no remarkable differences in the pH of the gut were observed among the tested species. Dermatophagoides spp. had a more acidic anterior midgut (a pH of 4 to 5) and colon (a pH of 5) with postcolon (a pH of below 6). The results characterizing in vivo conditions in the mite gut offer useful information to study the activity of mite digestive enzymes including their inhibitors and gut microflora.
Keywords | Acknowledgements
The authors are much obliged to Vladimir Sustr and Jaroslav Smrz for their comments on the manuscript and to Sarka Tuckova for technical help. We thank Pavel Klimov for review of the determination of some mite species in our rearing facilites. This study was supported by grants from the Grant Agency of the Czech Republic (Grant No: GP525/07/P253), Ministry of Agriculture of the Czech Republic (Grant No: MZE0002700604) and Ministry of Education, Youth and Sports, Czech Republic (COST FA0701 action; Grant No: OC09034). | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 May 8; 10:42 | oa_package/fe/16/PMC3014745.tar.gz |
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PMC3014746 | 20578956 | Introduction
In tropical and subtropical areas, maize Zea mays L (Poales: Poaceae) can become infected with destructive viruses including maize stripe tenuivirus, maize mosaic rhabdovirus, maize Iranian mosaic virus, and maize sterile stunt virus. These viruses are transmitted to the host plant by the planthopper Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae), which is broadly distributed, especially in tropical and subtropical regions ( Nault and Ammar 1989 ). In Argentina, P. maidis was reported as an experimental vector of maize disease caused by Mal de Río Cuarto virus (MRCV) belonging to the Fijivirus genus ( Virla et al. 2004 ). This vector was collected on maize ( Zea. mays , and Z. perennis ), sorghum ( Sorghum vulgare and S. halepense ), millet ( Panicum miliaceum ), and citrus in locations in the Formosa, Chaco, Corrientes, Entre Ríos, Jujuy, Salta, Tucumán, and Buenos Aires provinces of Argentina ( Tesón and Remes Lenicov 1989 ; Remes Lenicov and Mariani 2001 ). Several entomopathogenic fungi have been isolated from planthopper pests of rice crops in Asia, and some of these have been evaluated for control of these pest insects ( Aguda et al. 1987 ; Rombach et al. 1986a , b). Among these fungi, Beauveria bassiana (Balsamo-Crivelli) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin (Ascomycota: Clavicipitaceae) seem to be the most useful against these insects because of their ease of mass production, storage, virulence, and application ( Rice and Choo 2000 ). Although some workers have investigated the modes of infection and the histopathology of these fungi in selected insects of economic importance, there are no studies referring to the histopathology of entomopathogenic fungi in planthoppers. In the present study, adults of P. maidis were infected with B. bassiana and M. anisopliae and studied under light and scanning electron microscopy to determine the infection processes and the development of these fungi, before and after host death. | Materials and Methods
Planthoppers were reared on maize plants grown in plastic flowerpots and isolated inside 24cm × 9cm polyethylene terephthalate plastic cages in a greenhouse under 20 ± 5° C and a natural photoperiodicity. The B. bassiana strain used in this study was isolated from one adult of Oliarus dimidiatus Berg (Hemiptera: Cixiidae) associated with rice crops in Los Hornos, Buenos Aires, Argentina (34° 52′ S -57° 58′ W) and deposited in the Mycological Collections of the Centro de Estudios Parasitológicos y de Vectores (La Plata, Argentina), and at the USDA-Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (Ithaca, New York) under the accession numbers CEP 189 and ARSEF 7776, respectively. The M. anisopliae strain was isolated from unidentified species of Hemiptera: Cercopidae living on Eryngium sp. L. (Apiaceae) plants in Esteros del Iberá, Corrientes, Argentina (28° 24′ S - 57° 07′ W) and deposited in the Mycological Collections of the Centro de Estudios Parasitológicos y de Vectores, at the USDA-Agricultural Research Service Collection of Entomopathogenic Fungal Cultures (Ithaca, New York), and at the Instituto de Botánica Carlos Spegazzini (La Plata, Argentina), under the accession numbers CEP 160, ARSEF 8376, and LPSC 908, respectively. Dr. Richard Humber, insect mycologist and curator of the USDA-Agricultural Research Service Collection of Entomopathogenic Fungal Cultures, confirmed both fungal species. The fungal isolates were maintained in culture on malt extract agar for 10 d at 25° C in darkness before being used to inoculate the planthoppers. Conidial germination was calculated for each isolate according to Lane et al. ( 1988 ).
To study the infection process with a scanning electron microscope, a total of 30 brachypterous insects (15 males and 15 females) of P. maidis ( Figure 1a ) were inoculated with each fungal isolate, and 20 insects (10 males and 10 females) were used as controls. Ten-day-old insects were inoculated in groups of 10 inside glass vials of 10cm × 1cm via topical application by spreading dry conidia with a camel-hair brush on insects. After inoculation, insects were incubated at 24 ± 1° C, at high relative humidity (>90%) and with a photoperiod of 14:10 hr (L:D), in groups of 10 in plastic Petri dishes (90mm) with filter paper moistened with sterile distilled water and conditioned with maize leaves which were removed every three days. In this experiment, at 24, 48 and 72 hr after inoculation, two males and two females inoculated with each fungal strain were fixed in 2.5% glutaraldehyde for a time period of 24 hr at 4° C, then transferred to fresh 2.5% glutaraldehyde and fixed for 45 min at room temperature, washed in 0.1M cacodylate buffer for 45 min, post-fixed in 1.0% osmium tetroxide for 1 hr at room temperature, washed in distilled water for 10 min, and dehydrated in an ascending ethanol series (10, 30, 50, 70, 80, 90, 95 and 100%) for 10 min each (adapted from Becnel 1997 ). Samples were dried by critical-point, coated with a gold palladium film, and examined and photographed using a JEOL 6360 LV scanning electron microscope.
To study the entomopathogenic fungi multiplication inside the body of the insect host, sets of 20 females and 20 males of P. maidis were inoculated with B. bassiana and M. anisopliae and incubated in the way described above. Ten males and 10 females were used as controls. Two treated insects and one control insect of each sex were fixed at 24 hr intervals for 6 days, and two insects treated of each sex were fixed at 24 and 48 hr after death. Before fixation, insects were anesthetized at -20° C for 1 min, and their wings and legs were extirpated. Treated and control insects were fixed for 24 hr in formaldehyde phosphate buffer 10% (pH 6.8; Humason 1962 ). After fixation, the specimens were dehydrated in a graded series (30, 50 and 75%) of ethanol and butyl alcohol, and embedded in Paraplast®. Sagittal serial sections (5–6 μm in thickness) were prepared and stained using Masson trichromic ( Masson 1928 ) and Grocott methenamine-silver nitrate method for fungi ( Grocott 1955 ). The stained preparations were mounted in natural Canadian balsam, and then slide preparations were observed under an Olympus microscope (CH 30). | Results and Discussion
Histopathology: Conidial adhesion, germination and penetration through the insect cuticle.
Under scanning electron microscopy, B. bassiana conidia (95.5% in vitro germination) were observed at high density mainly in the sternal region of the abdomen, where these were deposited in areas near to the hairs ( Figure 1b ) and the pores of the wax glands ( Figure 1c ). Conidia were also observed between the ommatidia of the compound eye ( Figure 1d ), in the second antennal segment, between the hairs of the sensory pits ( Figure 1e–f ), and at the articulating membranes of legs. M. anisopliae conidia (100%) in vitro germination) were observed in the same regions of the host insect as B. bassiana , but in smaller concentrations ( Figure 1g ). M. anisopliae conidia were gathered near and enclosed within the spiracles ( Figure 1h–i ). The hydrophobic conidia of both fungal species were able to attach to all body regions, with a preference for surfaces containing hairs, as was reported by Boucias et al. ( 1988 ). B. bassiana conidia were found especially to be trapped by and tightly bound to these hairs ( Figure 1b ), as was also previously reported by Boucias and Pendland ( 1991 ). Few germinated conidia were observed at 24, 48, and 72 hr post-inoculation on the whole body surface. For both fungal species, germ tubes that registered at 24 and 48 hr were short, and they were observed penetrating directly through the host cuticle in regions near the hairs of the second antennal segment and on the laterosternites of abdomen. After 72 hr, long and errant germ tubes were detected on the cuticular surface. In all cases, M. anisopliae emitted only one germ tube from each conidium, as Schabel ( 1978 ) also reported. Neither the bipolar germination nor the appressoria formation observed by Mc Cauley et al. (1968) and Vestergaard et al. ( 1999 ) were detected. The most frequent method of penetration was through the cuticle (particularly for B. bassiana ) ( Figure 1c and Figure 2 a–c ), although M. anisopliae germ tubes were observed entering through the hair sockets situated on the forewing venation ( Figure 2d ). Mc Cauley (1968) reported the penetration of M. anisopliae though the solid cuticle as the most common method of entering the body cavity of Elaterid larvae, after the spiracles and pores of the sense organs. The preferential penetration sites in F. occidentalis observed by Vestergaard et al. ( 1999 ) were noted on the head, thorax, abdomen, and on the thickest part of the wings, where the conidia penetrated directly through the cuticle. B. bassiana conidia were able to penetrate directly through the integument, as well as through the respiratory system ( Pekrul and Grula 1979 ). Germination on the cuticular surface was observed at 24 hr postinoculation, but the germination percentages were low (confronted with 95.5% and 100% in vitro for B. bassiana and M. anisopliae respectively). In the study carried out by Mc Cauley et al. (1968) most of M. anisopliae conidia inoculated in Elateridae (Coleoptera) larvae germinated within 24 to 48 hr after inoculation; Vestergaard et al. ( 1999 ) obtained 100%) germination of M. anisopliae conidia at 24 hr post-inoculation of Frankliniella occidentalis (Thysanoptera: Thripidae), while the results obtained by Schabel ( 1978 ) showed that germination of the same fungal species on the cuticular surface of Hylobius pales (Coleoptera: Curculionidae) was within 35 to 132 hr post-inoculation. For some systems, the failure of fungi to invade the insect cuticle has been attributed to the presence of inhibitory compounds such as phenols, quinones, and lipids on the cuticle surface ( Smith et al. 1981 ; Kerwin 1984; St Leger 1991 ; Lord and Howard 2004 ). While Hubner (1958), Walstad et al. ( 1970 ) and Schabel ( 1978 ) also suggest the existence of antibiosis by the microbiota ( e.g . other fungi and bacteria) living on the cuticular surface of the host. Similarly, bacillus-like bacteria were present in the cuticular surface of P. maidis treated with B. bassiana and M. anisopliae ( Figure 2e–f ). In recent studies, 160 bacteria strains isolated from the cuticular surface of adults of Delphacodes kuscheli (Hemiptera: Delphacidae) and Dalbulus maidis (Hemiptera: Cicadellidae) were characterized. Almost half (45.8%) of these bacteria were Gram (+) spore-forming bacilli (Toledo et al., unpublished data). The ability of some sporeforming bacteria to inhibit different species of fungi by secreting antibiotics with antifungal properties, such as iturins, subtilins, mycosubtilins, megacins, and circulins, has been well documented ( Holland 1961 ; Katz and Demain 1977 ; Alippi et al. 2000 )
Histopathology: Entomopathogenic fungi multiplication inside the body host
In the histological sections observed under light microscopy, Masson trichromic was the most useful stain because it permitted identification of every tissue of the insect host, whereas the Grocott coloration was useful for distinguishing the hyphae and hyphal bodies, which were stained brown and black in contrast to the green coloration of the host tissues. In the colonization events observed in this study, the formation and multiplication of hyphal bodies by both fungal species inside the host body was noted. Hyphal bodies were multiplied by the budding of pre-existent cells in accordance with Madelin ( 1963 ).
Short hyphal bodies of M. anisopliae were observed inside the abdomen and thorax of males and females of P. maidis at 4 days postinoculation ( Figure 3a–b ). Within 5 days post treatment, short hyphae (2.48 μm width), were recorded at the hemocoel; in some cases hyphae branched and grew near the Malpighian tubules. At 6 days postinoculation, the entire body was invaded by hyphae. Body fat was the most affected tissue. The hyphal bodies ranged between 1 and 4 μm in diameter, according to the tissue that was invaded ( i.e . tegument 3 μm, body fat and muscle 4 μm, salivary glands 2.5 μm, lumen of the digestive tract and reproductive cavities 1–1.5 μm) ( n = 30 for each observation). Variation in length, form and vacuolation of hyphal bodies were greatest in the early stages of development. Mc Cauley (1968) reported this same variation and assumed it was due to differences in quantity and quality of the nutrients available to the fungus. A limitation of the availability of nutrients may explain why hyphae growth within the lumen of the digestive tract, Malpighian tubules, and tracheae exhibited a distinctly smaller diameter than those just outside these systems. Hyphal bodies in the muscle tissue ( Figure 3a ) and in the cephalic region near the ocular peduncle contained big vacuoles (2.4–4 μm, n = 30). At 6 days post-inoculation, hyphae were observed inside the cavities of the reproductive system and the Malpighian tubules. At 24 hr after death, hyphae invasion of the host tissues was limited to the body fat, the muscle tissue of the thorax, and the ventral nerve cord. At 48 hr after death, hyphae and hyphal bodies were observed in all the tissues, including the oocytes inside the ovarioles in females.
Hyphal bodies of B. bassiana were observed inside the abdomen and thorax of P. maidis 5 days post-inoculation ( Figure 3b ). Short septated hyphae were noted in the hemocoel of males and females (3–16.5 μm, n = 30). In both sexes, the highest concentration of hyphae was detected in the terminal region of the abdomen between the body fat cells. In females, some elongated hyphae were observed in the muscle tissue of the thorax. At 24 hr after death, hyphal bodies were observed all over the males. These hyphae and hyphal bodies were found in all the tissues, in the cavities of the digestive system, and near the Malpighian tubules and the tracheae ( Figure 3d–e ). At 24 hr after death, hyphae penetrating through the ovariole wall ( Figure 3f ) and near the tracheae were observed in the females. At 48 hr after death, the entire abdomen and the reproductive system were invaded and the muscular tissue surrounding the hyphae was under lysis. The hyphal bodies ranged between 1 and 2.8 μm in diameter ( n = 30). Neither penetration pegs nor cellular defense reactions associated with the hyphal bodies were observed in any of the saggital sections studied.
Due to the scarce number of germinated conidia observed on the insects' cuticular surface, it is likely that the bacteria over the external cuticle is interacting with fungal conidia, playing a role of antibiosis that will not allow the fungal pathogens to germinate and penetrate as suggested in the literature (Hubner 1958; Walstad et al. 1970 ; Schabel 1978 ). The in vitro antagonistic effect of the bacteria isolated from delphacids and cicadellids against the growth of B. bassiana was recently studied. This included evaluating 160 strains and recording inhibition percentages ranging between 0% and 83% (Toledo et al, unpublished data). In addition, the high quantity of wax that usually covers the cuticle of these insects could have some chemical compounds that might inhibit further germination of fungal conidia. Either or both of these scenarios could be occurring as an antagonistic and inhibitory mechanism, and further research is necessary to test this hypothesis. | Results and Discussion
Histopathology: Conidial adhesion, germination and penetration through the insect cuticle.
Under scanning electron microscopy, B. bassiana conidia (95.5% in vitro germination) were observed at high density mainly in the sternal region of the abdomen, where these were deposited in areas near to the hairs ( Figure 1b ) and the pores of the wax glands ( Figure 1c ). Conidia were also observed between the ommatidia of the compound eye ( Figure 1d ), in the second antennal segment, between the hairs of the sensory pits ( Figure 1e–f ), and at the articulating membranes of legs. M. anisopliae conidia (100%) in vitro germination) were observed in the same regions of the host insect as B. bassiana , but in smaller concentrations ( Figure 1g ). M. anisopliae conidia were gathered near and enclosed within the spiracles ( Figure 1h–i ). The hydrophobic conidia of both fungal species were able to attach to all body regions, with a preference for surfaces containing hairs, as was reported by Boucias et al. ( 1988 ). B. bassiana conidia were found especially to be trapped by and tightly bound to these hairs ( Figure 1b ), as was also previously reported by Boucias and Pendland ( 1991 ). Few germinated conidia were observed at 24, 48, and 72 hr post-inoculation on the whole body surface. For both fungal species, germ tubes that registered at 24 and 48 hr were short, and they were observed penetrating directly through the host cuticle in regions near the hairs of the second antennal segment and on the laterosternites of abdomen. After 72 hr, long and errant germ tubes were detected on the cuticular surface. In all cases, M. anisopliae emitted only one germ tube from each conidium, as Schabel ( 1978 ) also reported. Neither the bipolar germination nor the appressoria formation observed by Mc Cauley et al. (1968) and Vestergaard et al. ( 1999 ) were detected. The most frequent method of penetration was through the cuticle (particularly for B. bassiana ) ( Figure 1c and Figure 2 a–c ), although M. anisopliae germ tubes were observed entering through the hair sockets situated on the forewing venation ( Figure 2d ). Mc Cauley (1968) reported the penetration of M. anisopliae though the solid cuticle as the most common method of entering the body cavity of Elaterid larvae, after the spiracles and pores of the sense organs. The preferential penetration sites in F. occidentalis observed by Vestergaard et al. ( 1999 ) were noted on the head, thorax, abdomen, and on the thickest part of the wings, where the conidia penetrated directly through the cuticle. B. bassiana conidia were able to penetrate directly through the integument, as well as through the respiratory system ( Pekrul and Grula 1979 ). Germination on the cuticular surface was observed at 24 hr postinoculation, but the germination percentages were low (confronted with 95.5% and 100% in vitro for B. bassiana and M. anisopliae respectively). In the study carried out by Mc Cauley et al. (1968) most of M. anisopliae conidia inoculated in Elateridae (Coleoptera) larvae germinated within 24 to 48 hr after inoculation; Vestergaard et al. ( 1999 ) obtained 100%) germination of M. anisopliae conidia at 24 hr post-inoculation of Frankliniella occidentalis (Thysanoptera: Thripidae), while the results obtained by Schabel ( 1978 ) showed that germination of the same fungal species on the cuticular surface of Hylobius pales (Coleoptera: Curculionidae) was within 35 to 132 hr post-inoculation. For some systems, the failure of fungi to invade the insect cuticle has been attributed to the presence of inhibitory compounds such as phenols, quinones, and lipids on the cuticle surface ( Smith et al. 1981 ; Kerwin 1984; St Leger 1991 ; Lord and Howard 2004 ). While Hubner (1958), Walstad et al. ( 1970 ) and Schabel ( 1978 ) also suggest the existence of antibiosis by the microbiota ( e.g . other fungi and bacteria) living on the cuticular surface of the host. Similarly, bacillus-like bacteria were present in the cuticular surface of P. maidis treated with B. bassiana and M. anisopliae ( Figure 2e–f ). In recent studies, 160 bacteria strains isolated from the cuticular surface of adults of Delphacodes kuscheli (Hemiptera: Delphacidae) and Dalbulus maidis (Hemiptera: Cicadellidae) were characterized. Almost half (45.8%) of these bacteria were Gram (+) spore-forming bacilli (Toledo et al., unpublished data). The ability of some sporeforming bacteria to inhibit different species of fungi by secreting antibiotics with antifungal properties, such as iturins, subtilins, mycosubtilins, megacins, and circulins, has been well documented ( Holland 1961 ; Katz and Demain 1977 ; Alippi et al. 2000 )
Histopathology: Entomopathogenic fungi multiplication inside the body host
In the histological sections observed under light microscopy, Masson trichromic was the most useful stain because it permitted identification of every tissue of the insect host, whereas the Grocott coloration was useful for distinguishing the hyphae and hyphal bodies, which were stained brown and black in contrast to the green coloration of the host tissues. In the colonization events observed in this study, the formation and multiplication of hyphal bodies by both fungal species inside the host body was noted. Hyphal bodies were multiplied by the budding of pre-existent cells in accordance with Madelin ( 1963 ).
Short hyphal bodies of M. anisopliae were observed inside the abdomen and thorax of males and females of P. maidis at 4 days postinoculation ( Figure 3a–b ). Within 5 days post treatment, short hyphae (2.48 μm width), were recorded at the hemocoel; in some cases hyphae branched and grew near the Malpighian tubules. At 6 days postinoculation, the entire body was invaded by hyphae. Body fat was the most affected tissue. The hyphal bodies ranged between 1 and 4 μm in diameter, according to the tissue that was invaded ( i.e . tegument 3 μm, body fat and muscle 4 μm, salivary glands 2.5 μm, lumen of the digestive tract and reproductive cavities 1–1.5 μm) ( n = 30 for each observation). Variation in length, form and vacuolation of hyphal bodies were greatest in the early stages of development. Mc Cauley (1968) reported this same variation and assumed it was due to differences in quantity and quality of the nutrients available to the fungus. A limitation of the availability of nutrients may explain why hyphae growth within the lumen of the digestive tract, Malpighian tubules, and tracheae exhibited a distinctly smaller diameter than those just outside these systems. Hyphal bodies in the muscle tissue ( Figure 3a ) and in the cephalic region near the ocular peduncle contained big vacuoles (2.4–4 μm, n = 30). At 6 days post-inoculation, hyphae were observed inside the cavities of the reproductive system and the Malpighian tubules. At 24 hr after death, hyphae invasion of the host tissues was limited to the body fat, the muscle tissue of the thorax, and the ventral nerve cord. At 48 hr after death, hyphae and hyphal bodies were observed in all the tissues, including the oocytes inside the ovarioles in females.
Hyphal bodies of B. bassiana were observed inside the abdomen and thorax of P. maidis 5 days post-inoculation ( Figure 3b ). Short septated hyphae were noted in the hemocoel of males and females (3–16.5 μm, n = 30). In both sexes, the highest concentration of hyphae was detected in the terminal region of the abdomen between the body fat cells. In females, some elongated hyphae were observed in the muscle tissue of the thorax. At 24 hr after death, hyphal bodies were observed all over the males. These hyphae and hyphal bodies were found in all the tissues, in the cavities of the digestive system, and near the Malpighian tubules and the tracheae ( Figure 3d–e ). At 24 hr after death, hyphae penetrating through the ovariole wall ( Figure 3f ) and near the tracheae were observed in the females. At 48 hr after death, the entire abdomen and the reproductive system were invaded and the muscular tissue surrounding the hyphae was under lysis. The hyphal bodies ranged between 1 and 2.8 μm in diameter ( n = 30). Neither penetration pegs nor cellular defense reactions associated with the hyphal bodies were observed in any of the saggital sections studied.
Due to the scarce number of germinated conidia observed on the insects' cuticular surface, it is likely that the bacteria over the external cuticle is interacting with fungal conidia, playing a role of antibiosis that will not allow the fungal pathogens to germinate and penetrate as suggested in the literature (Hubner 1958; Walstad et al. 1970 ; Schabel 1978 ). The in vitro antagonistic effect of the bacteria isolated from delphacids and cicadellids against the growth of B. bassiana was recently studied. This included evaluating 160 strains and recording inhibition percentages ranging between 0% and 83% (Toledo et al, unpublished data). In addition, the high quantity of wax that usually covers the cuticle of these insects could have some chemical compounds that might inhibit further germination of fungal conidia. Either or both of these scenarios could be occurring as an antagonistic and inhibitory mechanism, and further research is necessary to test this hypothesis. | Editor: Fernando Vega was editor of this paper
The planthopper Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae) is an important vector of maize viruses in tropical and subtropical areas. Planthoppers are biologically controlled with several species of entomopathogenic fungi that have been isolated from these insect pests of rice in Asia. Beauveria bassiana (Balsamo-Crivelli) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae) appear to be the most useful against planthoppers because of their ease of mass production, storage, virulence, and application. In the present study, adults of P. maidis infected with B. bassiana and M. anisopliae were observed under light and scanning electron microscopy to characterize morphologically the process of infection and the development of these fungi, prior to and after the death of the host. The hydrophobic conidia of both fungal species were able to attach to all body regions, with a preference for surfaces containing hairs. Few germinated conidia were observed on the insect's body surface at 24, 48, and 72 hr post-inoculation. On the cuticular surface of P. maidis treated with B. bassiana and M. anisopliae , bacillus-like bacteria were observed. These microorganisms could be interacting with fungal conidia, playing a role of antibiosis that will not allow the fungal pathogens to germinate and penetrate. In the colonization events observed in this study, the formation and multiplication of hyphal bodies by both fungal species inside the host's body was noted. The host's whole body was invaded by hyphae between five and six days post-inoculation, and body fat was the most affected tissue.
Keywords | Acknowledgements
The authors wish to thank Mr. Topa for his collaboration with histological preparations, Dr. Micieli for assistance developing the protocol for scanning electron microscopy, Ms. Sarmiento and Mr. Urrejola for technical support with scanning electron microscopy, Dr. Richard Humber for confirmation of fungal identification, Ms. Karen Hansen for the fungal cultures preservation in the USDA-ARS Collection of Entomopathogenic Fungal Cultures Lic. Pan for the manuscript review. This study was partially supported by the National Research Council of Argentina. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 17; 10:35 | oa_package/a4/d9/PMC3014746.tar.gz |
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PMC3014747 | 20578952 | Introduction
Lady beetles (Coleoptera: Coccinellidae) are known to be voracious predators of plant pests such as aphids (Hemiptera: Aphididae) ( Hodek 1973 ; Gordon 1985 ). It is often assumed that aphidophagous lady beetles are highly polyphagous, consuming most (if not all) aphid species that they encounter ( Pedigo and Rice 2006 ). However, there is evidence that not every aphid species is equally suitable for every lady beetle species ( Obrycki and Orr 1990 ; Phoofolo and Obrycki 1997 ; Kalushkov 1998 ; Michaud 2000 ; Kalushkov and Hodek 2004 ; Mignault et al. 2006 ). For example, Michaud ( 2000 ) conducted choice tests with seven lady beetle species and two aphid species, Toxoptera citricida and Aphis spiraecola . Although all lady beetles tested consumed both aphid species, four species Coccinella septempunctata , Coleomegilla maculata fuscilabris , Coelophora inaequalis , and Olla v-nigrum , were not able to complete their developmental cycle with either aphid species. Depending on the aphid species consumed and the addition of supplements (pollen) to the diet, the other three species, Hippodamia convergens , Cycloneda sanguinea , and Harmonia axyridis , varied considerably in the number of eggs laid, egg viability, larval development time, and adult weight.
Lady beetles are commonly released to combat a diverse range of pests ( Gordon 1985 ; Koch 2003 ), despite the fact that little is known about specific prey preferences of different species. The success of such pest control measures depends, in part, upon the willingness of the lady beetles to consume the pest in question. Releases of non-native species may supplement pest control by native species when their prey species do not overlap or when prey is plentiful. Native lady beetle abundance, however, may be reduced through competition with non-native species with overlapping prey preferences. Additionally, non-native lady beetles may alter aphid community structure. Determining differences in prey consumption by different lady beetle species may provide insight into changes that occur in systems where non-native species become established. In the laboratory, one native and three non-native lady beetle species were provided four different species of aphid prey and their consumption was recorded. To determine if any differences documented in the laboratory were reflected in the field, lady beetle species were observed for their association with these aphids under field conditions. | Materials and Methods
Study species
The four lady beetle species chosen for this study are aphidophagous ( Gordon 1985 ) and abundant in Maine in the same habitats ( Finlayson et al. 2008 ). The native lady beetle species used was Coccinella trifasciata perplexa Mulsant, which is found from Labrador south to New Jersey and west to California and Alaska ( Gordon 1985 ). The non-native lady beetle species used were Coccinella septempunctata L., Harmonia axyridis Pallas, and Propylea quatuordecimpunctata L. These three species are Palearctic in origin and were intentionally and inadvertently introduced in North America. C. septempunctata has been established in North America since 1973 ( Angalet and Jacques 1975 ), H. axyridis since 1988 (Chapin and Brou 1991; Tedders and Schaefer 1994 ), and P. quatuordecimpunctata since 1968 ( Wheeler 1990 ).
Four aphid species that are abundant and readily available in the region were chosen to serve as the prey for the selected lady beetle species. The potato aphid, Macrosiphum euphorbiae (Thomas), feeds on over 200 plant species ( Blackman and Eastop 1984 ). The green peach aphid, Myzus persicae (Sulzer), feeds on over 40 different plant families ( Blackman and Eastop 1984 ). The hosts of the rose aphid, Macrosiphum pseudorosae (Patch), include the genus Rosa and a variety of herbaceous plants ( Foottit and Maw 1997 ). The lupine aphid, Macrosiphum albifrons Essig, is a specialist, feeding exclusively on plants in the genus Lupinus ( Blackman and Eastop 1984 ). While M. persicae is believed to be Palearctic in origin ( Blackman and Eastop 1984 ), the other three aphid species are Nearctic ( Stroyan 1981 ; Blackman and Eastop 1984 ).
Laboratory trials
Lady beetles were collected from the field 48–72 hours before test initiation, maintained on a 50/50 diet of honey/egg yolk, then provided with water, but no food, for 48 hours before test initiation. Lady beetles were collected from a variety of locations and plants in Orono, Maine (44.8835° N, 68.6721° W), that included mixed shrub ( Solidago sp., Rubus sp., Prunus sp., Rosa sp., Cornus sericea , Alnus sp.), apple ( Malus sp.), grain ( Hordeum sp., Avena sp.), mixed organic crops ( Solanum lycopersicon , Allium sp., Brassica sp., Pisum sp., Phaseolus sp.) and fallow fields ( Phleum pratense , Trifolium sp., Cirsium sp., Vicia sp., Fragaria sp.).
Potato aphids and green peach aphids were obtained from colonies maintained in the laboratory. The colonies were originally founded by aphids collected from potato, Solanum tuberosum (Solanales: Solanaceae), in Presque Isle, Maine, and then maintained for at least 20 generations on excised potato foliage in the laboratory. Rose and lupine aphids were collected in the field from host vegetation including multi-flora rose, Rosa multiflora Thunberg (Rosales: Rosaceae), and lupine, Lupinus polyphyllus Lindley (Fabales: Fabaceae), respectively, and then maintained in the laboratory on excised host vegetation for up to 3 days before use in trials.
For each experiment, 10 aphids of the same species were placed, using a paintbrush, on an excised leaflet held within a 100 × 15 mm polystyrene Petri dish. Leaves used in trials were from the host plants from which aphids were collected in the field, as previously stated. Each trial was initiated when a single lady beetle previously housed in a separate Petri dish was added to the Petri dish containing the aphids by quickly exchanging lids between the two Petri dishes when the lady beetle was on the lid. After 24 hours, the beetle was removed and the number of aphids remaining in the dish was recorded. When a partial aphid remained, it was estimated to the 0.25 aphid. The experiment was conducted separately with adult apterae and with first to second instars. Sixty trials were conducted with each lady beetle species/aphid species pairing: 30 replicates with adult aphids and 30 replicates with the nymphs.
Lady beetles, aphid colonies, and test dishes were housed in Percival I-33VL Intellus environmental chambers at a 16:8 L:D photoperiod and 20° C. Trials with M. euphorbiae and M. albifrons were conducted in 2005, from June 16 to August 12 and from June 2 to August 12, respectively. Trials with M. persicae and M. pseudorosae were conducted in 2006, from May 24 to August 16 and from August 10 to August 24, respectively. Trials were conducted continuously throughout the range of dates and in random order with respect to beetle species, aphid species, and choice of aphid nymph or adult.
Field observations
Plots of L. polyphyllus , S. tuberosum , and R. multiflora were observed for 30 minutes each in Orono, ME (44.8974°N, 68.6873°W). Observations were made between 10:00 am and 2:00 pm in plots at least 0.1 ha in size where the vegetation of interest was dominant (≥ 50%). The number of adult lady beetles on host vegetation where aphids were absent (designated “absent”) or where M. albifrons , M. euphorbiae , or M. pseudorosae were present (designated “present”) was recorded. Because aphid populations were fairly contiguous where present, with no break in distribution greater than approximately one meter, each of the plots observed was designated as either “absent” or “present.” Forty observation trials were conducted for each of the three species. M. persicae were not found in the field in numbers sufficient to conduct observations. M. albifrons colonies were observed from June 2 to July 12, 2005, M. euphorbiae colonies were observed from June 17 to July 30, 2005, and M. pseudorosae colonies were observed from June 20 to August 24, 2006.
Statistical analyses
Normality of laboratory-generated data was tested using the Wilk-Shapiro test (PROC UNIVARIATE; SAS Institute Inc. 2002 ). The data were transformed using rank transformations (Conover and Iman 1989). Means and standard errors reported in this paper were calculated from the untransformed data. Differences between lady beetle species were analyzed separately for each aphid species using one-way ANOVA followed by Tukey's multiple comparison test (PROC GLM; SAS Institute Inc. 2002 ). Analyses were conducted separately for aphid nymphs and adults.
Poisson regression (PROC GENMOD; SAS Institute Inc. 2002 ; SAS Institute Inc. 2005 ) was used to analyze lady beetle count data generated during field observations. Each plant species observed was analyzed separately, with the number of lady beetles as the response variable and lady beetle species and aphid presence/absence as the predictor variables. Overdispersion for M. albifrons and M. pseudorosae was corrected using a multiplicative overdispersion factor (Pearson chi-square divided by degrees of freedom) ( Cox 1983 ; Allison 1999 ; SAS Institute Inc. 2005 ). | Results
Laboratory trials
There were always significant differences in the numbers of aphids consumed by different lady beetle species ( Figure 1 ). H. axyridis consumed the most nymphs and adults of M. persicae (nymphs: F 3,116 = 6.27, p < 0.0006; adults: F 3,116 = 37.37, p < 0.0001), M. euphorbiae (nymphs: F 3,116 = 11.98, p < 0.0001; adults: F 3,116 = 20.67, p < 0.0001), and M. pseudorosae (nymphs: F 3,116 = 32.59, p < 0.0001; adults: F 3,116 = 48.47, p < 0.0001) compared with the other three lady beetle species, while P. quatuordecimpunctata consumed the fewest adults of these three aphid species and the fewest nymphs of M. persicae and M. euphorbiae. C. septempunctata consumed the lowest numbers of M. pseudorosae nymphs compared with the other three beetle species.
Lady beetles generally consumed fewer M. albifrons ( Figure 1 ) compared with the other three aphid species. C. trifasciata and P. quatuordecimpunctata consumed a greater number of M. albifrons nymphs compared with the other two beetle species (F3,116 = 11.86, p < 0.0004); C. trifasciata also consumed the greatest number of lupine aphid adults (F3,116 = 6.46, p < 0.0006).
Field observations
All four lady beetle species were found on S. tuberosum , while only H. axyridis and P. quatuordecimpunctata were found on R. multiflora and only C. trifasciata and P. quatuordecimpunctata were found on L. polyphyllus ( Table 1 ). There were significant differences in mean numbers of lady beetle species documented in two of the three vegetation types observed. The most abundant species in S. tuberosum was P. quatuordecimpunctata , followed by C. septempunctata (X 2 = 18.17, p < 0.0001), H. axyridis (X 2 = 22.02, p < 0.0001), and C. trifasciata (X 2 = 18.84, p < 0.0001). On L. polyphyllus, P. quatuordecimpunctata was more abundant than C. trifasciata (X 2 = 5.52, p = 0.0188). However, there was no difference in the relative abundance of P. quatuordecimpunctata and H. axyridis on R. multiflora . Although mean lady beetle numbers were higher in six out of the eight occasions where aphids were present compared to absent ( Table 1 ), those differences were not significant. | Discussion
Consumption rates of the four aphid species differed among the four lady beetle species. With the exception of M. albifrons, H. axyridis was the most voracious predator, while P. quatuordecimpunctata removed the least prey. There may be a number of reasons for these differences. First, consumption rates may have been affected by the size of the beetles and/or the size of the prey. C. septempunctata is the largest of the lady beetles studied, followed by H. axyridis, C. trifasciata , and P. quatuordecimpunctata (Finlayson unpublished data). Being the smallest in size, P. quatuordecimpunctata may be satiated with fewer aphids compared with the other species. M. albifrons is larger than the other aphid species, thus fewer M. albifrons may satiate a beetle compared with the other species offered. Consumption rate may also be affected by differences in handling ( Pervez and Omkar 2005 ), nutritional suitability of prey ( Houck 1991 ; Roger et al. 2001 ; Gagné et al. 2002 ), or chemical deterrence ( Pasteels et al. 1983 ; Nishida and Fukami 1989 ).
Observations of adult beetles on field plots were generally consistent with expectations based on their consumption of aphids in the laboratory. H. axyridis consumed the most M. pseudorosae in laboratory trials and was one of two species found in the field with M. pseudorosae . C. trifasciata consumed the most M. albifrons in laboratory trials and was one of two species found in the field with M. albifrons . The other beetle species found with M. pseudorosae and M. albifrons was P. quatuordecimpunctata , the species that consumed the second largest number of M. pseudorosae and M. albifrons , although this difference was only statistically significant for M. albifrons nymphs. It is also not surprising to find P. quatuordecimpunctata in all observations because this species is probably the most abundant lady beetle in Maine ( Finlayson et al. 2008 ).
Three of the species tested in this study, H. axyridis , C. septempunctata , and P. quatuordecimpunctata , are not native to Maine ( Gordon 1985 ). Because lady beetle species differ in their prey consumption, decreases in the relative abundance of native species following the establishment of non-native lady beetle species that has been reported in a number of studies ( Elliot et al. 1996 ; Brown and Miller 1998 ; Colunga-Garcia and Gage 1998 ; Michaud 2002 ; Brown 2003 ; Turnock et al. 2003 ; Alyokhin and Sewell 2004 ) may favor some aphid species over others. For example, Alyokhin et al. ( 2005 ) observed a significant reduction in both density and the amplitude of annual oscillations of populations of M. persicae and Aphis nasturtii following the establishment of H. axyridis and P. quatuordecimpunctata .
When compared with other aphidophagous coccinellid species, H. axyridis has been shown to have superior competitive abilities regarding its feeding rate ( Michaud 2002 ), intraguild predation ( Hironori and Katsuhiro 1997 ; Yasuda et al. 2001 ; Yasuda et al. 2004 ), and interactions with natural enemies ( Dutcher et al. 1999 ; Saito and Bjørnson 2006 ; Finlayson et al. 2009 ). Similarly, in this study, H. axyridis exhibited greater prey consumption of three of the four aphid species tested compared with the other lady beetle species tested. The true voracity of H. axyridis , however, may have been underestimated because it consumed close to the upper limit of what was made available in trials. Providing more than ten aphids may have improved the resolution of species differences.
M. albifrons is native to the study area ( Stroyan 1981 ) and is known to sequester toxic compounds from its host plant that have been shown to cause a “narcotizing effect” on C. septempunctata ( Gruppe and Roemer 1988 ). It is thus notable that H. axyridis and C. septempunctata , both introduced species without historical exposure to M. albifrons , consumed the lowest numbers of this species. In contrast, C. trifasciata , which is native to the area, consumed the most M. albifrons adults. It would seem that C. trifasciata may have evolved the ability to tolerate these compounds, whereas the recently introduced non-native species have yet to do so. By virtue of being able to exploit lupine aphids, C. trifasciata may enjoy a refuge from prey competition with the non-native species. These differences in prey consumption suggest that different lady beetle species should not be considered equal consumers of aphids. | Associate Editor: J.P. Michaud was editor of this paper.
The acceptability of four different aphid species Macrosiphum albifrons (Essig), Macrosiphum euphorbiae (Thomas), Macrosiphum pseudorosae Patch, and Myzus persicae (Sulzer) (Hemiptera: Aphididae), as prey for four lady beetle species, one native species Coccinella trifasciata L, and three non-native Coccinella septempunctata L, Harmonia axyridis Pallas, Propylea quatuordecimpunctata L (Coleoptera: Coccinellidae) were tested in the laboratory. The relative field abundance of adults of the same lady beetle species on host vegetation, Lupinus polyphyllus Lindley (Fabales: Fabaceae), Solanum tuberosum L (Solanales: Solanaceae), and Rosa multiflora Thunberg (Rosales: Rosaceae), both with and without aphids present was also observed. In the laboratory, H. axyridis generally consumed the most aphids, while P. quatuordecimpunctata consumed the fewest. The exception was P. quatuordecimpunctata , which consumed a greater number of M. albifrons nymphs, and C. trifasciata , which consumed a greater number of M. albifrons nymphs and adults, compared with the other two beetle species. Lady beetles consumed fewer M. albifrons compared with the other three aphid species, likely because of deterrent compounds sequestered by this species from its host plant. In the field, P. quatuordecimpunctata was the most abundant species found on L. polyphyllus and S. tuberosum .
Keywords | Acknowledgments
The authors would like to thank the following for their assistance in the field and laboratory: Lauren Little, David Ginsberg, Allison Fleming, and Todd Finlayson. We would also like to thank Joseph Cannon, John Jemison, Black Bear Food Guild, and Orono Land Trust for providing access and guidance on their land, and Frank Drummond and Malcolm Hunter, Jr., J.P. Michaud, and two anonymous reviewers for providing comments on the manuscript. This research was supported by the Maine Agricultural and Forest Experiment Station (Hatch ME08466-01) and the National Science Foundation's GK-12 Teaching Fellows Program (Grant # DGE — 0231642 to S. Brawley et al.). This is Publication No. 3034 of the Maine Agricultural and Forest Experiment Station. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 6; 10:31 | oa_package/20/25/PMC3014747.tar.gz |
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PMC3014748 | 20578883 | Introduction
Imported fire ants are major pests of urban, agricultural and wildlife areas ( Adams 1986 ; Allen et al. 1995 ; Boman et al. 1995 ; Taber 2000 ; Wojcik et al. 2001 ). The black imported fire ant, Solenopsis richteri Forel (Hymenoptera: Formicidae), was introduced first in Mobile, Alabama about 1918 and the red imported fire ant, Solenopsis invicta Buren, was introduced between 1933–1945 in the same port. Imported fire ants can now be found throughout the Southeast and in scattered localities in the extreme Southwest United States ( Callcott and Collins 1996 ; Taber 2000 ; Tschinkel 2006 ). An important way for imported fire ants to disperse is the mating flight ( Markin et al. 1971 ; Morrill 1974 ). The most comprehensive studies of fire ant mating flights were by Markin et al. ( 1971 ). They found an average yearly expansion of the S. richteri range of ∼3–5 miles. Using airplanes with retractable nets and passive black light traps at different heights on a TV transmission tower, they found females up to 800 feet high and males to 1000 feet. They believed males did not swarm over a swarm marker like most hymenoptera, but rather flew in a layer 200– 500 feet high. Males flew from late morning or early afternoon to near dusk, while females remained aloft 30 minutes or less, and 95% returned inseminated. No direct observations of swarming or mating were made and apparently none have been made by others. Vogt et al. ( 2000 ) investigated flight energetics and dispersal capability of S. invicta alates by recording flight distance and speed in a flight mill and recorded respiration in closed-system and flow-through flight respirometers. In the absence of wind, S. invicta female alates flew less than 5 km. They also recorded wingbeat frequencies but did not study the detailed motion of the wings. In mosquitoes and Chironomidae it is known that frequencies/tones generated by wingbeat are important for attraction between males and females in swarms ( Roth 1948 ; Wishart and Riordan 1959 ; Belton and Costello 1979 ; Ogawa and Sato 1993 ; Fyodorova and Azovsky 2003 ; Gibson and Russell 2006 ). The present study using high-speed video is the first of a detailed investigation of fire ant flight that attempts to understand wingbeat induced sound waves and associated air flows that may be important in communication and mate localization.
High-speed video techniques have been widely used to capture wing motion of bird and insect flights ( Norris 2001 ; Sudo et al. 2001 , 2005 ; Dalton 2002 ; Gorman 2003 ). Recent advances in high-speed imaging technique make it possible to capture insect wing motion not only at high phase resolution but also at high digital resolution, e.g. the Photron Ultima APX high-speed camera used in our lab is capable of capturing partial frames of 1024×256 pixels at 8,000 frames per second (fps). High digital resolution and high phase resolution together enable a precise analysis of the wing motion within a wingbeat period. However, at least two images from different view angles, e.g. provided by a stereo imaging system, are necessary to determine the three dimensional shape and orientation of the wing surface. A stereo imaging system requires at least two cameras that view an object from two different directions, and in an ideal situation, these two view directions should be perpendicular to each other, ( Sudo et al. 2001 , 2005 ). Since one advanced high-speed camera is already very expensive for most research laboratories, building a stereo imaging system with two high-speed cameras is usually not possible. Therefore, a stereo high-speed imaging system was constructed using a single high-speed camera for the fire ant alate tests. One important reason for conducting the stereo high-speed imaging tests is to collect the necessary data for computational fluid dynamics (CFD) simulations of the fire ant alate flight. CFD has been widely used to solve fluid flow problems and the accompanied acoustic problems in engineering areas since the invention of computers, and it was recently used to solve problems of insect flight ( Kazuhiro 2000 ; Sane 2003 ; Mao and Gang 2003 ; Cheng et al. 2006 )
In the present work, stereo high-speed image frames were acquired for wing motions of many black and red imported hybrid fire ant males and females that were tethered with fine metal wires and stimulated to fly with a mini blower in the lab. Two candidates from the males and females, respectively, were selected for further detailed analyses to determine the three-dimensional wingtip positions and the wing surface orientation angles. The resulting fire ant wing motion data were verified with a numerical reconstruction of the stereo image frames. This allowed the development of a numerical model for CFD simulation. Since some of the near field sound waves were simultaneously measured with the wingbeat motion test, the wingbeat motion data was be used to explain some features of the sound waveforms. | Materials and Methods
Specimen collection and rearing in the laboratory
Colonies of S. richteri were sampled for alates in Lafayette County, Mississippi including the University of Mississippi Oxford campus, Holly Springs National Forest. University of Mississippi field station and other localities. A limited number of ants were dug up if alates were present, placed in trays with sides coated with Fluon to prevent escape, and maintained in the laboratory at room temperature. In each tray, tubes of water and 10% sucrose were placed as well as moth pupae and dry dog food.
Measurement system and stereo image frame
A photograph of the stereo high-speed imaging system for the fire ant alate flight tests is shown in Figure la . The system included a Photron Ultima ( www.photron.com ) APX camera with a 60-mm Nikon ( www.nikon.com ) Micro Nikkor lens, a pair of mirrors, a pair of ground glass plates as back lighting screens, and a stereo adaptor between the lens and the high-speed camera. The tested fire ant alates were glued (initially using super glue and later contact cement) on the tip of a fine metal wire (0.3 mm) so that the ant body could be put in a fixed position and the relative motion of the wings could be investigated. As schematically illustrated in Figure lb , in the test section, the tethered fire ant alate was placed between the two mirrors and in front of the backlighting screens, which were illuminated with an Intralux ( www.intralux.com ) 5100 High Intensity Cold Halogen Light Source through a pair of fiber optic light guides. The stereo adaptor included a cuneiform block and two mirrors. Because of the cuneiform block the fire ant alate could not be directly imaged, whereas the two images of the mirrors in the test section were imaged through the apertures between the inside mirrors and the cuneiform block to the two halves of the image sensor, respectively. In the test the tethered fire ant alate was oriented so as to acquire images of both the side and front views. Once the distances and angles among the image sensor, the lens, the mirrors and the tested fire ant alate were properly setup, the angle between the view directions of the two images was around 90°, and a desired imaging magnification was achieved.
The Photron Ultima APX camera was operated at 8000 fps with a resolution of 1024×256 pixels, so that two 512×256 imaging regions could be used for each of the two views. This was sufficient to clearly capture images of flying fire ant alate wings for quantitative analyses. The shadow imaging technique based on back lighting is generally used for high-speed imaging, and it worked extremely well for fire ant studies because the alate wing is transparent, except for the wing veins, so that the shape and structure of the wings can easily be recorded in shadow images. Tests showed that most fire ant alates may fly steadily for a few minutes in the laboratory room with temperature around 27° C when stimulated by a mini blower, and in some cases, the alate flew continuously for hours. The image acquisition took a quarter second for 2,000 frames at 8,000 fps. Microphones were used to record the sound wave to monitor the variation of the wingbeat frequency. The working distance of the stereo imaging system was adjusted according to the size of the tested fire ant alate so that the image-to-object ratio was 22.5 pixel/mm for the males and 20 pixel/mm for the females. Figure 2 shows one of the stereo image frames that was improved with digital image processing tools. As shown in Figure 2 , the XY plane information of the fire ant wings is projected to the front view shadow image on the left, and the YZ plane information is projected to side view shadow image on the right. If a wing surface can be assumed as a planar surface, it can be determined with three points indicated in the figure, i.e. the root of the wing ( O ), the tip of the wing ( T ), and the third point that can be chosen at the edge of the wing ( 3 ). The origin of the image coordinate ( X,Y,Z ) is defined here at the lower left corner of the image frame. The line that connects the root and tip of the wing is defined as wing axis, i.e. OT .
As shown in Figure la , two free-field 1⁄4-inch microphones were used to simultaneously record the wingbeat induced sound waveforms from bottom and rear, respectively.
Wing surface parameters and reconstruction method
Figure 3 and 4 show a few important parameters that are traditionally used for aerodynamic analysis of insect flight ( Ellington 1984 , Dudley 2002). The longitudinal axis of the fire ant body is in the yz -plane, and it forms an angle χ with respect to horizontal axis oz . In Figure 3 the blue image indicates the highest position of the forewing tip; the yellow image shows the lowest position of the forewing tip; and the horizontal position of the wings is given by the pink image. The line that is determined from a linear regression of wing tip positions in the side view defines the angle of the stroke plane ( β ). Note that in Figure 3 the origin of the physical coordinate ( x,y,z ) is defined at the center of the neck of the ant body. The root position of a fire ant wing marked with red dot in Figure 3 can be estimated with the joint point of wing axis lines of deferent image frames. In order to determine parameters that may be used for an aerodynamic analysis, the coordinate system (x,y,z) is rotated and shifted to (x* ,y* ,z*), so that x*y* -plane overlaps with the stroke plane and the root of the forewing is on z*-axis. As shown in Figure 4 , the angular position in the stroke plane and the wing angle in the revolution surface φ* are determined, and the wingbeat angular amplitude is obtained as Φ =( max - min ). Other parameters shown in Figure 4 include air flow velocity V , revolving velocity U , relative velocity U r and attack angle α τ . An aerodynamic analysis cannot be completed until the magnitude of V is determined with flow measurement or CFD simulation.
Figure 5 left shows the coordinates and parameters for determining a single wing surface marked with pink color. The wing surface passes through the coordinate origin that is set at the root of the wing, and it has angle γ when cut with XY-planes and α when cut with XZ-planes, i.e. γ is the wing surface angle in the front view and α in the side view. With these two angles, the wing surface can be represented in local coordinate system ( X',Y',Z' ) as: γ X ′ - Y ′ + tan α · Z ′ = 0. Another way to represent the wing surface is to use wing axis OT and the wing rotational angle φ that is the angle between the wing surface and the reference surface (marked with yellow in Figure 5 ), which is determined with wing axis OT and the coordinate axis OZ . For a wingbeat with a constant frequency, the wingtip position and the above mentioned angles are functions of the phase that is represented here as t/T , wherein T is the wingbeat period and t is the time in the wingbeat period. The start of the wingbeat period (i.e. t=0 ) is defined when the wingtip is around the maximal y-position and rotational angle φ equals zero. As shown in Figure 5 right, a high resolution digital image of the tested fire ant wing is used to provide detailed information of the wing structure with wing surface coordinate system ( L , H ). The shape of a fire ant wing is determined with trailing edge width a , leading edge width b at position l on the wing axis, the length of the wing is recorded as C .
Assume that the wing surface parameters in Figure 5 left are determined and the high-resolution wing image in Figure 5 right has gray value distribution G(L,H) . For an arbitrary point on the wing surface P , which is determined with coordinates ( x' , y' , z' ), the distance from P to the wing axis OT can be determined as H , and the projected length of OP on axis OT can be determined as L . Then the gray value of the three dimensional image at point P is obtained as g(x' , y' , z' ) = G(L,H) . Since the coordinates x' , y' , z') have only two independent variables, the wing images projected to XY-plane (front view), YZ-plane (side view) and XZ-plane (top view) can be determined as g(x' ,y'), g(y' ,z') and g(x' ,y') . When the stereo images of both the forewing and hindwing are reconstructed, they can be assembled according to the wing root positions as shown in Video 3 and 4 . | Results
While tests were conducted with many male and female fire ant alates, only data sets for those alates that appeared normal in their flight behavior have been kept in the data base. The high-speed video of each test case was inspected to exclude data sets that indicated defective wings, defective or missing body parts, interfered wing motions, unbalanced wing motions, non-periodical wing motions, and other behaviors that were considered abnormal. Image recordings of 9 female and 9 male black imported fire ant alates were considered as valid. Each of these image recordings consisted of more than 2000 frames, and the time interval between frames was 1/8000 second. In the tests the slowest wing motion had a frequency of 81 Hz, so that at least 20 wingbeat periods were recorded in the one quarter second imaging acquisition time. Test data demonstrated a constant wingbeat period for each test case.
A representative male and a representative female fire ant alate ( S. richteri ) were selected for detailed data analyses. The male live weight was 5.5 mg and the female live weight was 11.4 mg. Their respective body lengths were 6.2 mm and 8.3 mm, while their wingbeat frequencies were 108 Hz and 96 Hz. Some wingbeat parameters were obtained for these two alates by evaluating the high-speed image frames ( Figure 6 ). Figure 6c and 6f show that the wingbeat angular amplitude ( Φ ) of the male was about 10% larger than that of the female for both the forewing and the hindwing, and that the angular amplitude of the hindwing was around 15% larger than that of the forewing for both the male and the female. Since the fire ant alates were tethered during the tests, the measured stroke plane angle β and the body angle χ may be different from those during natural flight, however, the sum of these two angles (i.e. β + χ ) may not be affected by the tethering. The tests show that the angle between the wingbeat stroke plane and the longitudinal axis of the fire ant body was almost the same (i.e. 67.0° & 66.7°) for both the male and the female. The wing root position ( x 0 , y 0 , z 0 ) was determined in the physical coordinate system as shown in Figure 3 , which is necessary for reconstructing the stereo image frames. The wingtip coordinate and wing surface angle distributions in the wingbeat period are given in Table 1 and 2 for the male and female fire ant alate, respectively. The angular position distributions in the stroke plane and the wing angles in the revolving surface are shown in Figure 7 for the selected fire ant alates.
Since the stereo high-speed image frames were obtained with relatively low digital resolutions, i.e. 22.5 pixel/mm for the male and 20 pixels/mm for the female, shadow images of both the forewings and hindwings were taken with a ratio of 128 pixel/mm after the stereo imaging tests, and they are shown in Figure 8 . The high resolution wing images in Figure 8 provide not only the dimension and shape of the wings but also the structural details of the wing veins. Detailed data of the wings are given in Table 3 .
With the wingbeat parameters, wingtip position distributions, wing surface angle distributions, and wing structure images given above, stereo image frames were numerically reconstructed. In Video 1 (male alate) and Video 2 (female alate) the reconstructed stereo wing images (below) were compared with those recorded in the stereo high-speed imaging tests (above) in 74 (male) and 83 frames (female). Since the view angles of the two cameras were perpendicular, there was no problem determining the three dimensional position of a visible marker (e.g. wingtip and wing root) on the wing surface. It would be an ideal test case when the body axis of the tested fire ant alate is in the YOZ-plane. In most cases, however, there was a small deviation angle between the fire ant body axis and YOZ-plane, so that the side view images of the two forewings and two hindwings overlapped incompletely. The angular deviation could be calculated according to the difference between the left and right forewing data. In the cases of Video 1 and Video 2 , the angular deviations were determined to be 4° and 5°, respectively. The angular deviations were considered in the reconstructed wing images. The animated frames in the videos demonstrate that the reconstructed stereo wing images match the captured stereo high-speed video images very well, however, slight deviations could be observed at t/T=0.08∼0.16 when the two hind wings touched together, and t/T=0.56∼0.68 when the forewings rotated very fast. The three view reconstructions of the selected male and female fire alate are illustrated with Video 3 and Video 4 , respectively.
The data presented in Figure 6 , Table 1 , 2 and 3 provide inputs for a mathematical description that can easily be used to build a numerical model for CFD simulation. Further deductions of the data can be used to explain some aerodynamic or aeroacoustic phenomena. For example, the periodical distributions of the wingtip speed and wing surface rotational speed were deduced from the data and are shown in Figure 9 , which shows that, for the forewing and the hindwing of both the male and the female, the maximal wingtip speed of the downwards wing motion was around t/T=0.35, whereas the maximal upward wingtip speed was around t/T=0.75.
The wing surface rotational speed had negative high amplitude peaks between t/T=0.5 and 0.6. The sound waveforms measured about 10-mm away from the bottom of a male and a female fire ant alate are shown in Figure 10 , in which the sound pressure distributions of five periods overlapped. The sound pressure had a minimum at t/T≈0.36 for both the male and female case, which should result from the maximal downward wingtip speed. In the male fire ant case, the sound pressure went up to maximum at t/T≈0.75 that may be related to the maximal upward wingtip speed. It seems that the high-speed rotation of the fire ant wings between t/T=0.5 and 0.7 generated a negative pressure pulse to inhibit the sound pressure increase and formed a local minimum at t/T≈0.65. In the female case, since the negative rotational speed of the hindwing was much higher than the forewing of the female and both the forewing and hindwing of the male, the sound pressure increase was greatly inhibited, so that no maximum could be seen around the maximal upward wingtip speed. | Discussion
The stereo high-speed imaging system applied a simple optical configuration to avoid a much more expensive two-camera system for taking stereo videos of fire ant alate wingbeat motion. Unlike the usual stereo imaging adaptors, in our optical configuration the focus planes of the two views are perpendicular to each other, so that the three dimensional positions of the fire ant alate wings can be directly read from the stereo image pairs. With post-processing of the image the background noise is removed and clear images are obtained. The basic requirement of the camera lens is that the undistorted imaging area width should not be less than double that of the image sensor width. In addition, there should be enough room between the camera lens and the image sensor to install a pair of mirrors. With the 60 mm lens, the field of view of the presented stereo image system is around 24×12 mm 2 that is sufficient to image the largest fire ant alate. When the working distance and the outside mirror angles are adjusted, this system can be used for larger or smaller winged insects.
In this work the flight parameters and wing motion details of a male and a female fire ant alate were completely described, so that the three-dimensional wing motions could be numerically reconstructed. Based on the twoview image frame from the stereo high-speed imaging test, wing images of arbitrary view direction were obtained with numerical reconstruction, e.g. the top-view images in Video 3 and Video 4 . When the thickness and proper profile of fire ant wings are considered, the obtained fire ant alate flight data can be used for a further computational fluid dynamic (CFD) analysis.
Further deductions of the fire ant alate flight data can be used to explain some aerodynamic or aeroacoustic phenomena. For example, the wingtip speed and wing surface rotational speed were used to explain some waveform features of the wingbeat-induced near field sound.
In this work a fire ant wing was assumed to have a planar surface, so that it could easily be determined with the coordinates of three points that could be read from the stereo image frame. This assumption works very well in 80% of the wingbeat period. The animated frames in Video 1 and Video 2 show that the two hindwings were obviously bent when they touched together within t/T =0.08∼0.16, and that the forewings were slightly deformed when they rotated rapidly during t/T =0.56∼0.68. A higher order surface fit may be necessary to include the wing surface bending and deformation in the data, however, this would require a much more complicated algorithm and a higher digital resolution of the imaging system. It should also be mentioned that the flight of tethered fire ant alates may differ from the flight of free flying alates. | Editor: John Ewer was editor of this paper
The wing motions of a male and a female fire ant alate, which beat their wings at 108 and 96 Hz, respectively, were captured with a stereo imaging system at a high frame rate of 8,000 frames per second. By processing the high-speed image frames, the three-dimensional wingtip positions and the wing surface orientation angles were determined with a high phase resolution, i.e. 74 and 83 phases per period for the male and the female, respectively. A numerical reconstruction of the stereo wingbeat images demonstrated that the data collected described almost all the details of the wing surface motion, so that further computational fluid dynamic simulations are possible for fire ant alate flight.
Keywords | Acknowledgments
This study was supported by grants from the United States Department of Agriculture to Drs. Henry E. Bass and John M. Seiner (Grants: USDA 58-6402-1-102, USDA 58-6402-6 NCPA).
Abbreviations
trailing edge width;
leading edge width;
wing length;
computational fluid dynamics;
frames per second;
forewing;
hindwing;
position at wing axis;
wing surface coordinates;
wing axis;
revolutions per minute;
time in wingbeat period;
wingbeat period;
revolving velocity;
relative velocity;
air flow velocity;
stereo image coordinates;
physical coordinates;
local coordinate;
side view wing surface angle;
attack angle;
wingbeat stroke plane angle;
front view wing surface angle;
angular wing position;
wing rotational angle;
wingbeat angular amplitude;
wing angle in revolution surface;
maximal angular position;
minimal angular position;
body orientation angle | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Mar 15; 10:19 | oa_package/5c/31/PMC3014748.tar.gz |
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PMC3014749 | 21209802 | 1. Introduction
Chediak-Higashi syndrome (CHS; MIM 214500) is an autosomal recessive disorder characterized by oculocutaneous albinism, increased susceptibility to pyogenic infections, defective natural killer (NK) activity, delayed bactericidal activity of neutrophils, and the presence of giant lysosomes in many cell types [ 1 – 3 ]. We previously reported that abnormally downregulated protein kinase C activity is responsible for the impaired cellular functions of polymorphonuclear leukocytes, fibroblasts and NK cells of CHS mice and patients [ 4 – 9 ]. The manifestation of CHS may result from defective trafficking of proteins into late multivesicular endosomes [ 10 ]. Most CHS patients die young due to a lymphoproliferative histiocytosis called the accelerated phase unless they undergo bone marrow transplantation.
The genetic defect resulting in CHS was identified in 1996 [ 11 , 12 ]. The human gene, CHS1 , was also called LYST (Lysosomal Trafficking Regulator). A similar disorder has been identified in beige mice and many other mammalian species. Human CHS patients and beige mice have homologous disorders associated with the CHS1 mutation [ 11 – 13 ]. CHS1 consists of 51 coding exons with an open reading frame of 11,406 bp [ 12 ]. The CHS1 protein is cytosolic and is composed of 3801 amino acids with a molecular weight of 429 kDa. It is known that CHS1 has a pleckstrin homology domain, a BEACH domain, and WD-40 repeats in the C-terminal region [ 14 ]. While the exact function of the CHS1 protein has not been elucidated, the protein suggested to regulate lysosomal size or lysosomal fission and affect cellular events, such as those of nuclear phosphatidylinositol-4, 5-bisphosphate [ 15 , 16 ].
Thus far, 31 mutations in the CHS1 gene have been reported, including frameshift, nonsense, and missense mutations [ 17 – 21 ]. Only five Japanese CHS patients have been examined to date. Two patients had a one-base substitution, and one patient had a deletion, whereas no mutation of the CHS1 gene was detected in the other two patients [ 17 ]. Thus, we attempted to examine the mutations in other Japanese CHS patients. Here, we report novel heterogenous mutations of the CHS1 gene identified in Japanese patients with CHS. | 2. Patients and Methods
Informed consent for this study was obtained from the patients or their parents. The study protocol was approved by the Ethics Committee of the University of Yamanashi.
Patients 1, 2, and 3 (siblings) were 23-year-old male, 20-year-old female, and 17-year-old female, respectively. Giant granules were observed in polymorphonuclear cells from these three patients ( Figure 1 ). They all had albinism of the skin and hair ( Figure 2 ). However, there was no history of severe infection in any patient. Their parents were normal and healthy.
Patient 4 was a 6-year-old female with visual disturbance and hypopigmentation of the skin and hair ( Figure 3 ). The diagnosis of CHS was determined by the presence of myeloperoxidase-positive giant granules in leukocytes ( Figure 4(a) ). At the age of 4, she suffered from hemophagocytic lymphohistiocytosis ( Figure 4(b) ), which is known as accelerated phase. At the time of the study, she had high fever, bleeding tendency, hepatosplenomegaly, and pancytopeny. She had low NK activity and decreased bactericidal activity of neutrophils. In addition, she had been treated with cyclosporine and steroids and then underwent bone marrow transplantation.
Patient 5 was a 21-year-old female who suffered from recurrent infections in childhood. At the time of the study, she had visual disturbance and neurological dysfunction including gait disturbance. Detailed information on this patient was not available.
Peripheral blood samples were obtained from five CHS patients who had not undergone bone marrow transplantation. Blood samples from parents of patients 1, 2, and 3 were also obtained. Genomic DNA was obtained from blood samples. Extraction of DNA was performed using the QIAamp DNA Blood kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer's protocols. Polymerase chain reaction (PCR) primer pairs (25–35 bp) designed from intron sequences flanking each exon were used to amplify genomic DNA segments spanning each exon. For example, exons 9-10 were amplified using the F primer 5′-ATTTTTGCCACTAGATCTTCTAAATG-3′, and R primer was 5′-AGAAGCCATTATTATCAACTTTTCAC-3′. The F primer of exon 18 was 5′-TGCTACTGGCCACTAAGGTTGTGTGTC-3′, and R primer was 5′- GACTTTGATGACGAGATGAGTATCACTGC-3′. The F primer of exon 30 was 5′-CATTGTATCTATTACATCTAATACACCTGATACAC-3′, and R primer was 5′-ACGTATAATACAGTCAACATAAAACCTCTATTTCC-3′. PCR was performed using KOD-Plus-Ver. 2 according to the manufacturer's protocols (TOYOBO, Tokyo, Japan). The PCR products (220–3600 bp) were separated by electrophoresis on agarose gels. DNA was isolated from each band using the QIAquick Gel Extraction kit (QIAGEN Inc.). Direct sequencing using dye termination cycle sequencing was performed at FASMAC Co., Ltd. (Atsugi City, Kanagawa, Japan). The mutations were analyzed using Sequence Scanner Ver. 1 (Applied Biosystems, Tokyo, Japan) and were also checked at the cDNA level. For this purpose, total RNA was extracted from blood samples using Isogen-LS (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer's protocols. cDNA was synthesized using PrimeScript reverse transcriptase (Takara Bio. Inc., Shiga, Japan), and PCR was performed as described above. | 4. Discussion
The sequence pattern of CHS1 mutations described here has not been reported previously. All mutations were predicted to halt production of the complete CHS1 protein. Karim et al. [ 17 ] demonstrated that missense mutant alleles that likely encode CHS1 polypeptide with partial function were found in adolescent and adult forms of CHS, whereas functionally null mutant CHS1 alleles were found in case with severe childhood CHS. Westbroek et al. [ 19 ] also demonstrated that cellular defects in CHS correlate with the molecular genotype and clinical phenotype. In the present study, patient 4 and 5 exhibited early-onset CHS and were predicted to have a truncated CHS1 protein. Although the other three patients (patients 1, 2, and 3) were also predicted to have the truncated protein, they had clinically milder forms of CHS. In these patients, only single mutation in the CHS1 coding exons was detected. Since these patients were relatively healthy, it is possible that the mutant protein is acting as a dominant negative, resulting in a mild phenotype. In addition, we cannot exclude a possibility that the second mutation lies in the intron sequence or splice mutation site. However, the real reasons remain unknown, as we could not examine protein levels in these patients. In the previous report [ 17 ], no mutation in the CHS1 gene was found in 10 CHS patients, and only single mutations were found in 4 CHS patients. These findings suggest a possibility that the mutation lies in a gene other than CHS1 , which affects the generation of lysosome-related organelles. In patient 5, two heterogenous nonsense mutations were identified. Recently, two heterogenous nonesense mutations in the CHS1 gene were reported in an African-American patient [ 20 ]. In that report, the patient exhibited severe childhood CHS. These findings support that the functionally null CHS1 mutant alleles are detected in severe childhood CHS.
In Japanese CHS patients, only three mutations have been identified so far, whereas no mutation in the CHS1 gene was found in two other patients [ 17 ]. We have described four patterns of novel mutations that are expected to result in a truncated CHS1 protein. However, the relationship between these mutations and the phenotype remains to be resolved. Examination of a large number of CHS patients will be required to clarify the genotype-phenotype correlation. | Academic Editor: Marie-Cécile Nassogne
Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by oculocutaneous albinism, recurrent bacterial infections and progressive neurological dysfunction. We demonstrate novel heterogenous mutations of CHS1 , the responsive gene of CHS, identified in five Japanese patients with CHS. Patients 1, 2, and 3 were siblings, and they had albinism of the skin and hair. They all had a heterogenous two-base deletion (c.5541-5542 del AA, p.Q1847fsX1850) in exon 18. Patient 4 had a heterogenous single-base insertion (c.3944-3945 ins C, p.T1315fsX1331) in exon 10. The patient exhibited severe early-onset phenotype and suffered from hemophagocytic lymphohistiocytosis. Patient 5 had two heterogenous nonsense mutations; c.7982C>G, p.S2661X in exon 30 and c.8281A>T, p.R2761X in exon 31. The patient suffered from infections in childhood and had visual disturbance and albinism of the skin and hair. The CHS1 mutations described here have not been reported previously. | 3. Molecular Analysis
All 51 exons of the CHS1 gene of the five patients from three families were sequenced, and four patterns of novel heterogenous mutations were identified. In patients 1, 2, and 3, a two-base deletion (c.5541-5542 del AA) in exon 18 resulted in a frameshift mutation that eventually led to the formation of a stop codon (p.Q1847fsX1850) ( Figure 5(a) ). The second mutation was not found in the coding exons. The same heterogenous mutation was detected in their father. In their mother, no mutation was found in the CHS1 exons. Since their parents had no symptoms, it was possible that the second mutation lies in the intron sequence or splice mutation site. It was also possible that this mutation shows a mild phenotype associated with heterozygosity. Another possibility is the mutation in another gene, which affects the generation of lysosome-related organelles.
In patient 4, we detected a heterogenous one-base (C) insertion (c.3944-3945 ins C) in exon 10, resulting in a frameshift mutation that led to the formation of a stop codon (p.T1315fsX1331) ( Figure 5(b) ). The second mutation was not found in any coding exon. The blood samples of her parents were not available.
In patient 5, two heterogenous mutations were identified; C-G substitution (c.7982 C > G) in exon 30 resulted in a nonsense mutation (p.S2661X) ( Figure 5(c) ), and A-T substitution (c.8281A > T) in exon 31 resulted in a nonsense mutation (p.R2761X) ( Figure 5(d) ). Blood samples of her parents were not available. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 15; 2010:464671 | oa_package/d5/04/PMC3014749.tar.gz |
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PMC3014750 | 20578884 | Introduction
Neonicotinoid insecticides were introduced into the market in the early 1990s and, today, are one of the most important chemical groups used to control sucking insects as the major insecticide replacing organophosphate and carbamate insecticides. Imidacloprid, the first neonicotinoid insecticide, is particularly effective against sucking insects such as aphids and whiteflies, as well as several beetles, flies, and moth species, with its systemic and broad-spectrum activities; imidacloprid, however, is not toxic to phytophagous mites at normal field rates ( Elbert et al. 1991 , Nauen et al. 2001 , Nauen and Bretschneider 2002 ).
Imidacloprid has a mixed reputation regarding its safety to natural enemies of pests ( James and Price 2002 ). It was reported that using imidacloprid against sucking insects is safe for natural enemies of other pests such as spiders and some predatory beetles and bugs ( Hough-Goldstein and Whalen 1993 , James 1997 , Kunkel et al. 1999 ; James and Vogele 2001 , Elzen 2001 ), but other studies showed that imidacloprid was highly toxic to certain species of spiders, predatory beetles, and bugs ( Mizell and Sconyers 1992 , Stark et al. 1995 , Delbeke et al. 1997 , Sclar et al. 1998 , James and Coyle 2001 ). In addition, the application of systemic insecticides, as well as some non-systemic contact poisons like DDT and several synthetic pyrethroids, in fields often causes resurgence of non-target pest insects and mites. Recently, field outbreaks of the two-spotted spider mite, Tetranychus urticae , following acetamiprid (Assail) treatments have been reported ( Beers et al. 2005 ). Moreover, in laboratory experiments, James and Price ( 2002 ) reported a significant increase in egg production in T. urticae after spray and systemic applications of imidacloprid at field-relevant rates for hop yards. In contrast, significantly reduced oviposition in T. urticae following drench or foliar applications of imidacloprid and acetamiprid at field-relevant rates was reported in another study ( Ako et al. 2004 ). Ako et al. ( 2006 ) suggested that the ovipositional response of T. urticae to field-recommended doses of imidacloprid is strain-dependent. It is generally understood that imidacloprid and, most likely, neonicotinoids in general, used at their field-recommended rates, are not the sole factors contributing to the propagation of mite pests by oviposition stimulation, and one possible explanation currently under investigation is interspecies competition.
The carmine spider mite, Tetranychus cinnabarinus Boisduval (Acari: Tetranychidae), is a polyphagous spider mite pest of vegetable crops and ornamental plants in warm zones throughout the world ( He 1990 ). T. cinnabarinus is an important mite pest of horticultural and field crops in Southwestern China and is exposed to imidacloprid in many crop systems, particularly those that have aphids and whiteflies as principal pests. T. cinnabarinus shares the same ecological niche with several other important pests, such as aphids and whiteflies, in greenhouses and the open field. When these insects are chemically controlled, T. cinnabarinus is a non-target pest insect that is also exposed to imidacloprid. Currently, research on the impact of imidacloprid on T. cinnabarinus is lacking. Therefore, the present study was undertaken to assess the potential effects of imidacloprid on the survivorship, reproduction, and vitellin content of the carmine spider mite through a laboratory experiment. | Materials and Methods
Mites
The stock culture of the carmine spider mite, T. cinnabarinus , was collected from the cowpea bean, Vigna unguiculata Endlicher (Fabales: Fabaceae), in 2003 in Chongqing, China. This culture was maintained on potted V. unguiculata plants in a walk-in insect rearing room at 28 ± 1°C, 75–80% relative humidity, and a photoperiod of 14:10 hours (L:D). This colony was maintained for more than two years without the use of any pesticide. In order to obtain homogeneous individuals for the experiments, a synchronized mite culture was established in 2006 on V. unguiculata in the greenhouse with the same conditions as insect rearing room. Thirty mated female mites were placed on the third leaf from the top of V. unguiculata plants (three females each) on the 12 h. Thereafter, the adults were removed, and the offspring were kept until the progeny had developed into preovipositional females. Cotton strips were used to keep the mites from escaping. In order to increase the number of male mites and thereby increase the mating chance for young pre-ovipositional females, 15 additional males collected from the stock culture were transferred to the V. unguiculata mite rearing plants before the deuteronymphal stage of the synchronized population.
Bioassays
Imidacloprid (10% WP, Jiangsu Wujiang Pesticide Ltd. Co., China) was tested at sublethal doses (0.5778, 1.4247, and 2.7308 mg/L, corresponding to LD 10 , LD 20 , and LD 30 respectively and the field-relevant dose (23.4730 mg/L) previously determined for Myzus persicae ( Zeng and Wang 2007 ). Approximately 100–150 young mated female mites were transferred to 10 V. unguiculata leaf discs (diameter 1 cm) that were placed on a moistened sponge in the Petri dish (diameter 15cm) for 24 h. The adults were then removed, and each leaf disc with 30 eggs was immersed in treatment concentrations of imidacloprid for 10 s. Tap water immersion was used as the control. The hatch rate of the carmine spider mite was observed daily for 10 d. A total of 250 hatched nymphs for each treatment was kept under aforementioned conditions until they developed into pre-ovipositional females. Pre-imaginal survivorship was determined by recording the number of offspring that survived until adulthood in each treatment. Then, 100 female mites from each treatment with the cotton strip were transferred individually to V. unguiculata leaf discs as previously described. Males also were introduced to each female for mating. The number of eggs oviposited was counted under a stereomicroscope using a manual counter every 2 d for 16 d. Female mites that escaped or died were excluded from the analysis. After recording the number of eggs, the female mite was transferred carefully with a soft brush from the previous leaf disc to a new leaf disc. Each treatment was replicated three times temporally.
Determination of vitellin content
Three hundred eggs from the above treatment were homogenized manually in 200 μ l NaCl solution (0.4 M) and centrifuged at 10,000 g for 15 min at 4°C. The resulting supernatants were used to determine the content of vitellin of the carmine spider mite using bovine serum albumin as a standard ( Abdel-Aal et al. 1990 ). Absorbance was read in the spectrophotometer with the wavelength of 595 nm. The determination also was replicated three times as a bioassay.
Statistical analyses
Difference in hatch rate of eggs, preimaginal survivorship, fecundity, and vitellin content of the carmine spider mite were subjected to analysis of variances (ANOVA) by using the SPSS 10.0 for Windows ( SPSS 1999 ). General linear model procedure was used and means were separated by Fisher protected least significant difference (LSD) test when significant F -values were obtained (p < 0.05). The percentage of the egg hatch rate was transformed to the arcsin squareroot before analysis to stabilize error variance. | Results
Hatch rate of eggs and pre-imaginal survivorship
Compared with the control, exposure to the field-relevant dose of imidacloprid (23.47 mg/L) did not significantly affect the hatch rate of T. cinnabarinus eggs. However, exposure to the sublethal dose rates (0.5778, 1.42, and 2.73 mg/L) significantly increased the age-specific egg hatch rate ( Table 1 ). The highest total hatch rate (95.23%) was observed with exposure to 1.42 mg/L imidacloprid. In general, the pre-imaginal survivorship was relatively high in all treatments. Compared with the control, the pre-imaginal survivorship was significantly higher with the exposure to the three sublethal doses of imidacloprid; however, no differences in pre-imaginal survivorship were observed between the control and the field-relevant dose of imidacloprid ( F =24.64; df = 4, 10; p < 0.001; Figure 1 ).
Adult fecundity and longevity
The age-specific adult fecundity and longevity of T. cinnabarinus under the impact of imidacloprid is presented in Table 2 and Figure 1 . In general, the observed ovipositional pattern showed an increase in egg number from the second day after adulthood (day 2) onward, with a maximum number of eggs laid at day 8 and followed by a constant decrease until the death of the female ( Table 2 ). Compared with the control, the imidacloprid-treated populations led to a small increase in mite fecundity from day 2 to day 16, but the difference was not significant ( Table 2 ). In most cases, the highest fecundity was observed for the 0.5778 mg/L imidacloprid-treated population. The adult longevities did not differ significantly among the treatments ( F = 0.398; df = 4, 391; ns; Figure 1 ).
Vitellin content
Compared with the control population, the vitellin contents in the imidacloprid-treated eggs were all significantly increased ( F = 40.70; df = 4, 10; p < 0.001; Figure 2 ). Among the tested concentrations of imidacloprid, the highest vitellin content of T. cinnabarinus (6.07 μ g/300 eggs) was recorded for the 1.42 mg/L imidaclopridtreated population, and the lowest vitellin content (3.72 μg/300 eggs) was recorded for the 23.47 mg/L treated population ( Figure 2 ). | Discussion
Previous investigation showed that there is no acute, lethal effect of imidacloprid against T. cinnabarinus . The mite is particularly serious in vegetable crops with aphids and whiteflies as principal pests that are controlled routinely by imidacloprid (Wang et al. unpublished data). In this study, the hatch rate of eggs, pre-imaginal survivorship, age-specific fecundity, longevity, and the vitellin content of T. cinnabarinus on V. unguiculata leaves were compared under laboratory controlled conditions to determine the impact of imidacloprid. The results showed that sublethal imidacloprid doses led to significantly earlier hatch time, greater total hatch rate, and increased pre-imaginal survivorship to adult. However, when treated with the field-relevant rate of imidacloprid, these parameters did not differ significantly from the control. Results of this study also showed that exposure to imidacloprid significantly increased the vitellin content of T. cinnabarinus , which, in turn, led to an increase in the speed of egg hatch and total hatch rates. The highest vitellin content was recorded at the sublethal imidacloprid dose 1.42 mg/L. The speed of hatching was also the fastest, and the total hatch rate was the highest for this population. It is known that the sublethal doses of many insecticides stimulate pest resurgence, and the cause of pest resurgence is usually due to the suppression of a natural enemy or the reproductive stimulation of pests ( Morse and Zareh 1991 , Nandihalli et al. 1992 , Nemoto 1993 ). In southwestern China, imidacloprid is typically applied on commercial vegetable crop fields as foliar spraying, and the active ingredient of this chemical is systemic. The concentrations on the plants would decrease gradually to sublethal doses as the plant aged. Thus, T. cinnabarinus often is exposed to field-relevant or sublethal doses of imidacloprid. The present study suggests that through the stimulation of egg hatching and enhancement of the pre-imaginal survivorship, the application of imidacloprid may be one of the reasons for field outbreaks of T. cinnabarinus in southwestern China. However, other factors such as climate, agronomic practices, and pest resistance could also interact with imidacloprid, yielding less predictable results than those obtained under controlled conditions. Therefore, further investigation is needed, particularly under field conditions, in order to shed light on the factors which may interact with imidacloprid to lead to mite population increase.
Ako et al. ( 2006 ) reported that the fecundity of two strains of T. urticae (namely GSS, an acaricide-susceptible strain, and WI, an organophosphate-selected strain) treated with the field-relevant doses of imidacloprid decreased, while two other strains (namely USA, a largely uncharacterized strain, and Akita, a mitochondrial electron transport inhibitor, acaricide-resistant and cross-resistant to dicofol strain) did not differ from the untreated control. The same phenomena also were observed for sex ratio, hatch rate of eggs, and pre-imaginal survivorship of T. urticae . However, James and Price ( 2002 ) reported a significant increase in oviposition of T. urticae after drench or foliar applications of imidacloprid at concentrations of 0.011 and 0.013% A.I. in laboratory. According to Luckey ( 1968 ), the phenomenon of reproductive stimulation of pests or beneficials after exposure to sublethal doses of systemic insecticides was the basic hypothesis of hormoligosis. However, Cohen ( 2006 ) suggested that hormoligosis cannot be claimed for cases in which the observed stimulatory effects were due to exposure of non-target pests (i.e., mites) to pesticides (DDT, carbaryl, insecticidal pyrethroids or imidacloprid). Instead, pesticide-induced homeostatic modulation is suggested as a broader term to include both hormesis and stimulatory effects of pesticides on non-target pests. A possible reason for these contradicting results are differences in terms of methodology and the mites used in studies. Only one population of T. cinnabarinus was used in the present study, and the different reactions of the mites may be due to differences in laboratory and field studies. The present study, nonetheless, provided some basic information on the impact of imidacloprid on the survivorship, reproduction, and vitellin content of T. cinnabarinus . The widespread stimulation of fecundity in T. cinnabarinus and T. urticae by imidacloprid and other chloronicotinyls, if confirmed, would have great significance and importance to many crop protection and integrated pest management programs throughout the world. Since imidacloprid has a mixed reputation regarding its safety for natural enemies of pests and non-target pest species. Further, detailed studies of the impact of imidacloprid on T. cinnabarinus and its natural enemies will be necessary for the development of integrated pest management programs for mite control. | Associate Editor: Eileen Cullen was editor of this paper
Occasional reports linking neonicotinoid insecticide applications to field population outbreaks of the spider mite have been a topic of concern for integrated pest management programs. To elucidate the impacts of a neonicotinoid insecticide on the carmine spider mite, Tetranychus cinnabarinus Boisduval (Acari: Tetranychidae), the survivorship, reproduction, and vitellin contents of the mite were investigated after exposure to various concentrations of imidacloprid on the V. unguiculata leaf discs at 25°C, 80% RH and a photoperiod of 14:10 (L:D) in the laboratory. The results showed that the field-relevant dose of imidacloprid did not significantly affect the hatch rate of eggs or pre-imaginal survivorship of the mite, while sublethal doses of imidacloprid, previously determined for Myzus persicae , led to a significant increase in the hatch rate of eggs and pre-imaginal survivorship of the mite compared to the untreated control. Adult longevity and fecundity of T. cinnabarinus for imidacloprid-treated populations were slightly prolonged and increased, respectively, but the difference from the untreated control was not significant. The vitellin content in eggs increased significantly after exposure to imidacloprid. Imidacloprid may be one of the major reasons for the outbreak of T. cinnabarinus in the field.
Keywords | Acknowledgements
This research was funded in part by a grant from the Ministry of Agriculture (nyhyzx07-057), the Program for New Century Excellent Talents in University (NCET-04-0854) of China to Jin-Jun Wang. | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Mar 16; 10:20 | oa_package/80/d4/PMC3014750.tar.gz |
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PMC3014751 | 20578953 | Introduction
Knowledge of the blood-feeding preferences of a mosquito species provides important insight into the dynamics of virus transmission and allows vector control authorities to design and implement efficient strategies for vector control ( Tempelis 1975 ; Vinogradova 2000 ). The significance of Culex quinquefasciatus Say (Diptera: Culicidae) in the transmission of West Nile virus ( Flaviviridae : Flavivirus ) (WNV) in Mexico is poorly understood. However, it is well documented that this species is a principal vector of WNV in the United States ( Turell et al. 2005 ). Cx. quinquefasciatus has accounted for more than half of the WNV-infected mosquito pools reported during some transmission seasons in the United States ( Hayes et al. 2005 ).
Cx. quinquefasciatus populations from the United States feed readily on both birds and mammals ( Reisen and Reeves 1990 ; Reisen et al 1990 ; Zinser et al. 2004 ). For instance, in Tucson, Arizona, 50% of engorged Cx. quinquefasciatus had fed on humans, 32% had fed on birds, and 12% had fed on dogs ( Zinser et al. 2004 ). Cx. quinquefasciatus populations in East Baton Rouge Parish, Louisiana fed most frequently on dogs (69%), followed by birds (16%) and humans (11%) ( Niebylski and Meek 1992 ). In Harris County, Texas, 52%) of engorged Cx. quinquefasciatus had fed exclusively on mammals, and 39% had fed exclusively on birds; the remainder contained mixed avian and mammalian blood meals ( Molaei et al. 2007 ). Dogs were the most common (41%) vertebrate host followed by mourning doves (18%) and cats (9%). Cx. quinquefasciatus populations from the United States are relatively efficient laboratory vectors of WNV under laboratory conditions ( Goddard et al. 2002 ; Turell et al. 2005 ). Taken together, these findings suggest that Cx. quinquefasciatus serves both as an important amplification and as a bridging vector of WNV in the United States.
Serologic evidence has demonstrated widespread WNV activity in Mexico ( Blitvich et al. 2003 ; Estrada-Franco et al. 2003 ; Farfan-Ale et al. 2004 , 2006 ; Loroño et al 2003 ). The seroprevalence for WNV in horses sampled in Yucatan State in 2002 was 1.2% ( Loroño et al. 2003 ). In a longitudinal study of WNV infection in birds conducted in Yucatan State from 2000 to 2003, the seroprevalence for WNV was 0.06% ( Farfan-Ale et al. 2004 ). In a more recent study, 52% of horses sampled in the neighboring state of Quintana Roo in 2003 were seropositive for WNV ( Farfan-Ale et al. 2006 ). Several WNV isolates have also been collected in Mexico. One isolate was from a pool of Cx. quinquefasciatus collected in northern Mexico in 2003 ( Elizondo-Quiroga et al. 2005 ); all other WNV isolates were from sick or dead vertebrates ( Beasley et al 2004 ; Blitvich et al. 2004 ; Deardorff et al 2006 ; Elizondo-Quiroga et al. 2005 ; Estrada-Franco et al. 2003 ). This study was conducted to determine the host-feeding preferences of Cx. quinquefasciatus in an urban area of Yucatan State, Mexico because WNV has been isolated from Cx. quinquefasciatus in Mexico and serves as an important ampiflying host and a bridging vector of WNV in the United States. | Materials and Methods
Description of study sites
Mosquito collections were made in the city of Merida (20° 58′ 12′′ N, 89° 37′ 12′′ W) in Yucatan State, Mexico. Merida has a warm and humid climate throughout the year with an average annual temperature of 26°C (79°F) and distinct seasons, rainy from May to October and dry from November to April. The average annual precipitation is 929 mm (766 mm in the rainy season, 163 mm in the dry season). The average elevation in Merida is 10 meters. The population is approximately 750,000.
Mosquito collections
Diurnal outdoor resting mosquitoes were collected in the backyards of 40 houses. The houses were located in a 36 square-mile area of Merida. The houses were spaced evenly apart; each was approximately 0.5 to 1 mile from the closest study site. Mosquitoes were collected from each backyard five times per week from January 8, 2005 to December 22, 2005. Collections were made using resting wooden boxes (0.5 m 3 ) painted red on both the inside and outside ( Reisen and Pfunter 1987 ). One resting box was placed in each backyard. Resting boxes were placed on the ground in a sheltered position facing west to prevent morning sunlight from entering. Mosquitoes were removed from resting boxes between 0600 and 0900 hours using handheld, battery-operated aspirators. Mosquitoes were transported alive to the laboratory, euthanized in a -70°C freezer, and then identified on chill tables according to species, sex, and blood feeding status using morphological characteristics ( Carpenter and LaCasse 1955 ; Darsie and Ward 1981 ).
Census of vertebrate hosts
A census was taken of humans and domestic animals (birds and mammals) at each site. This was done by interviewing the occupants of each residence. If the number of vertebrate animals at a site changed (i.e. due to death), the site was no longer used for this study. Sites were only used if the number and species composition of vertebrates remained constant during the study period. Due to practical constraints, the numbers of freeranging birds temporally and spatially associated with each study site were not estimated. The numbers of humans and domestic vertebrates in neighboring houses were not counted.
Blood meal identification
Abdomens were removed from engorged females and individually placed into 1.5 ml eppendorf tubes. Abdomens were manually homogenized in 600 ul of phosphate-buffered saline (pH 7.4) using a sterile micro-pestle. Homogenates were applied to QIAshredder spin columns (Qiagen, www.qiagen.com ) and centrifuged (14,000 g for 3 minutes at 4° C). DNA was extracted from supernatants using the QIAamp DNA extraction kit following the manufacturer's instructions. Extracted DNA was analyzed by PCR using universal avian-and mammalian-specific primers that amplify 508 and 772-nt fragments, respectively, of the mitochondrial cytochrome b gene ( Ngo and Kramer 2003 ; Cicero and Johnson 2001 ). DNA from each avian-derived blood meal was further examined in a single PCR reaction using 3 pairs of order-specific primers that differentiate among Passeriformes, Galliformes and Columbiformes; these primers amplify 165, 210 and 333-nt fragments, respectively, of the mitochondrial cytochrome b gene. PCR products derived from mammalian blood meals were subjected to restriction enzyme analysis using Alu I to differentiate between human-, dog-, cat-, pig-and horse-derived blood meals ( Zehner et al 1998 ). The performance of the restriction enzyme assay was validated using DNA extracted from the blood of known vertebrate species. Species used for the validation experiments were: chicken (Galliformes), rock pigeon (Columbiformes), Yucatan jay (Passeriformes), human, dog, cat, horse and pig. Prior to the analysis of field-caught mosquitoes, the specificity of each primer pair was validated by PCR using DNA from the above mentioned vertebrate species. Each primer pair produced a PCR product of the expected size when tested with DNA from its intended target(s). Nucleotide sequencing confirmed that the amplified products were indeed mitochondrial cytochrome b DNA and that the expected Alu I restriction enzyme sites were present (Genbank Accession numbers FJ160756, FJ160757, FJ160758, FJ160759, FJ160760, FJ160761, FJ160762 and FJ160763) None of the primer pairs generated a detectable PCR product when tested with DNA from non-target species (data not shown).
Data analysis
The human blood index (HBI), which is defined as the proportion of freshly engorged mosquitoes containing human blood, was calculated as described by Garrett-Jones ( 1964 ). The forage ratio (FR), which quantifies vector selection of a particular vertebrate host rather than other available hosts, was also measured ( Boreham and Garrett-Jones 1973 ). FRs were calculated by determining the percent of Cx. quinquefasciatus females containing blood of a particular host, divided by the percent of the total available host population represented by that particular host ( Hess et al. 1968 ). An FR of 1.0 indicates neither a selective bias nor avoidance of a particular host animal; FRs significantly > 1.0 indicate a selective bias, and values < 1.0 indicate avoidance of a host in favor of other available hosts.
The host feeding index (HFI), which is defined as the observed proportion of feeds on one host with respect to another divided by the expected comparative proportion of feeds on these two hosts, was calculated using the formula described by Richards et al. ( 2006 ). The formula is as follows: where N x and N y are the mean numbers of blood meals taken from hosts x and y per study site, respectively, and A x and A y are the mean numbers of hosts x and y per study site, respectively. An index of 1.0 indicates equal feeding on the two hosts. Results < 1.0 and > 1.0 indicate a decrease or increase, respectively, in feeding on the first host relative to the second. HFIs were calculated for each pair of hosts. One advantage of the HFI versus the FR is that it does not require a full animal census ( Kay et al. 1979 ) | Results and Discussion
Mosquito collections
A total of 4,644 Cx. quinquefasciatus were captured in this study ( Table 1 ). Of these, 1600 were female, and 3,044 were male. A total of 658 females were classified as engorged, 233 as gravid, and 709 did not contain blood. Cx. quinquefasciatus were present year-round, but they were most abundant in August (17.4% of the total collection was made at this time) and November (15.7%).
Hood feeding preferences
For the blood meal identification experiments, 240 engorged females were used (20 per month). Overall, 197 (82.1%) mosquitoes contained avian-derived blood meals and 43 (17.9%)) contained mammalian-derived blood meals ( Figure 1 ). The proportion of mosquitoes containing avian-derived blood each month ranged from 60% (May) to 100% (December). No mosquitoes contained mixed blood meals. Forty percent of the DNA samples failed to yield a detectable PCR product. By comparison, 27% of field-collected engorged Culiseta and Anopheles spp. mosquitoes from New York did not yield a detectable PCR product ( Ngo and Kramer 2003 ). Additional mosquitoes were analyzed when PCR negative samples were encountered, until the monthly total of PCR positive samples reached 20.
The most frequent vertebrate hosts for Cx. quinquefasciatus were Galliformes (47.1%), Passeriformes (23.8%) and Columbiformes (11.2%) ( Table 2 ). These findings suggest that Cx. quinquefasciatus populations in Merida are strongly ornithophilic. The proportion of mosquitoes that had acquired blood meals from galliformes each month ranged from 20% (September) to 85% (March) ( Figure 1 ). The proportion of mosquitoes that had acquired blood meals from passeriformes also exhibited considerable seasonal variation, as this avian order was fed upon more frequently in the latter half of the year. From January to July, the proportion of mosquitoes that had acquired blood meals from passeriformes each month ranged from 10% to 25% (mean: 13.6%). From August to December, the proportion of mosquitoes that had acquired blood meals from passeriformes each month ranged from 30% to 45% (mean: 38.0%). August is the beginning of the long-distance migration season for many species of passerine birds that migrate from the United States to the Yucatan Peninsula of Mexico. Thus, the increase in the number of mosquitoes feeding on passerines in the latter half of the year could have been due to an increase in the relative abundance of passerines as a consequence of long-distance migration.
The most frequent mammalian hosts for Cx. quinquefasciatus were dogs (8.8%) and humans (6.7%) ( Table 2 ). Cats, horses, and pigs were not a common source of blood. The proportion of mosquitoes that had acquired blood meals from dogs was highest in September (25%) and November (20%) ( Table 3 ). No blood meals were obtained from mammals in December.
One limitation of this study is that a subset of mosquitoes could have acquired blood meals from vertebrate hosts that were located in nearby houses and therefore were not included in the census. Mark-release-recapture studies have shown that Cx. quinquefasciatus can travel up to 1.0 km/day to acquire blood meals ( Reisen et al. 1991 ). However, the census was restricted to the 40 houses used for the mosquito collections because mosquitoes are more likely to feed on vertebrate hosts in their immediate vicinity and because of the logistical restraints associated with censusing all vertebrate hosts within the Cx. quinquefasciatus flight path.
Human blood index and forage ratio values
The overall HBI was 6.7% ( Table 2 ). On five occasions, the monthly HBI values were 10%. The highest monthly HBI values occurred in May (15%), and three months were 0% (February, March, and December) ( Table 3 ). The numbers of humans and domestic animals (mammals and birds), residing at each house sampled in this study were counted. Overall, 88 (41%)) of the vertebrates were human, 45 (21%) were chickens and turkeys (galliformes), 32 (15%) were dogs, 14 (7%) were cats, 5 (3%) were passeriformes and 4 (2%) were horses. There were no pigs, cows or columbiformes Humans were the most common vertebrate species in the study area. However, the FR for humans was < 1.0, indicating that Cx. quinquefasciatus had a preference for other vertebrate hosts ( Table 4 ). Forage ratios were calculated for other vertebrate hosts; and those with FRs > 1.0 were Passeriformes (9.1) and Galliformes (2.0) birds.
Host feeding indices
Calculation of the HFIs for each pair of vertebrate hosts revealed that humans were the least preferred source of blood relative to species of Passeriformes, and Galliformes birds, and dogs, horses and cats ( Table 5 ). HFI values could not be calculated for Columbiformes or pigs because none were present at any study sites. In congruence with the FR values, the HFI data suggested that Passeriformes were the preferred source of blood for Cx. quinquefasciatus, followed by Galliformes.
Summary
This study suggests that Cx. quinquefasciatus populations in Merida, Yucatan State feed most frequently on birds year-round. These findings suggest that Cx. quinquefasciatus could be an important amplification vector of WNV in this region. In addition, the data refute the hypothesis that the low incidence of WNV illness in Mexico is due to the lack of interaction between Culex spp. mosquitoes and avian reservoir hosts in this region. Ornithophilic feeding behavior also was reported recently for Cx. quinquefasciatus populations in the city of Monterrey, northern Mexico; 44% to 73% of engorged mosquitoes collected outdoors had acquired their blood meals from chickens ( Elizondo-Quiroga et al. 2006 ). The overall HBI in the present study was low (0.1) indicating that humans were not a preferred host for Cx. quinquefasciatus in Merida. Nevertheless, a small proportion (6.7%)) of engorged Cx. quinquefasciatus contained human blood, indicating that this mosquito species could also transmit WNV to humans in this region. Vector competence studies are needed to determine the role that Cx. quinquefasciatus plays in the transmission of WNV in Mexico. | Results and Discussion
Mosquito collections
A total of 4,644 Cx. quinquefasciatus were captured in this study ( Table 1 ). Of these, 1600 were female, and 3,044 were male. A total of 658 females were classified as engorged, 233 as gravid, and 709 did not contain blood. Cx. quinquefasciatus were present year-round, but they were most abundant in August (17.4% of the total collection was made at this time) and November (15.7%).
Hood feeding preferences
For the blood meal identification experiments, 240 engorged females were used (20 per month). Overall, 197 (82.1%) mosquitoes contained avian-derived blood meals and 43 (17.9%)) contained mammalian-derived blood meals ( Figure 1 ). The proportion of mosquitoes containing avian-derived blood each month ranged from 60% (May) to 100% (December). No mosquitoes contained mixed blood meals. Forty percent of the DNA samples failed to yield a detectable PCR product. By comparison, 27% of field-collected engorged Culiseta and Anopheles spp. mosquitoes from New York did not yield a detectable PCR product ( Ngo and Kramer 2003 ). Additional mosquitoes were analyzed when PCR negative samples were encountered, until the monthly total of PCR positive samples reached 20.
The most frequent vertebrate hosts for Cx. quinquefasciatus were Galliformes (47.1%), Passeriformes (23.8%) and Columbiformes (11.2%) ( Table 2 ). These findings suggest that Cx. quinquefasciatus populations in Merida are strongly ornithophilic. The proportion of mosquitoes that had acquired blood meals from galliformes each month ranged from 20% (September) to 85% (March) ( Figure 1 ). The proportion of mosquitoes that had acquired blood meals from passeriformes also exhibited considerable seasonal variation, as this avian order was fed upon more frequently in the latter half of the year. From January to July, the proportion of mosquitoes that had acquired blood meals from passeriformes each month ranged from 10% to 25% (mean: 13.6%). From August to December, the proportion of mosquitoes that had acquired blood meals from passeriformes each month ranged from 30% to 45% (mean: 38.0%). August is the beginning of the long-distance migration season for many species of passerine birds that migrate from the United States to the Yucatan Peninsula of Mexico. Thus, the increase in the number of mosquitoes feeding on passerines in the latter half of the year could have been due to an increase in the relative abundance of passerines as a consequence of long-distance migration.
The most frequent mammalian hosts for Cx. quinquefasciatus were dogs (8.8%) and humans (6.7%) ( Table 2 ). Cats, horses, and pigs were not a common source of blood. The proportion of mosquitoes that had acquired blood meals from dogs was highest in September (25%) and November (20%) ( Table 3 ). No blood meals were obtained from mammals in December.
One limitation of this study is that a subset of mosquitoes could have acquired blood meals from vertebrate hosts that were located in nearby houses and therefore were not included in the census. Mark-release-recapture studies have shown that Cx. quinquefasciatus can travel up to 1.0 km/day to acquire blood meals ( Reisen et al. 1991 ). However, the census was restricted to the 40 houses used for the mosquito collections because mosquitoes are more likely to feed on vertebrate hosts in their immediate vicinity and because of the logistical restraints associated with censusing all vertebrate hosts within the Cx. quinquefasciatus flight path.
Human blood index and forage ratio values
The overall HBI was 6.7% ( Table 2 ). On five occasions, the monthly HBI values were 10%. The highest monthly HBI values occurred in May (15%), and three months were 0% (February, March, and December) ( Table 3 ). The numbers of humans and domestic animals (mammals and birds), residing at each house sampled in this study were counted. Overall, 88 (41%)) of the vertebrates were human, 45 (21%) were chickens and turkeys (galliformes), 32 (15%) were dogs, 14 (7%) were cats, 5 (3%) were passeriformes and 4 (2%) were horses. There were no pigs, cows or columbiformes Humans were the most common vertebrate species in the study area. However, the FR for humans was < 1.0, indicating that Cx. quinquefasciatus had a preference for other vertebrate hosts ( Table 4 ). Forage ratios were calculated for other vertebrate hosts; and those with FRs > 1.0 were Passeriformes (9.1) and Galliformes (2.0) birds.
Host feeding indices
Calculation of the HFIs for each pair of vertebrate hosts revealed that humans were the least preferred source of blood relative to species of Passeriformes, and Galliformes birds, and dogs, horses and cats ( Table 5 ). HFI values could not be calculated for Columbiformes or pigs because none were present at any study sites. In congruence with the FR values, the HFI data suggested that Passeriformes were the preferred source of blood for Cx. quinquefasciatus, followed by Galliformes.
Summary
This study suggests that Cx. quinquefasciatus populations in Merida, Yucatan State feed most frequently on birds year-round. These findings suggest that Cx. quinquefasciatus could be an important amplification vector of WNV in this region. In addition, the data refute the hypothesis that the low incidence of WNV illness in Mexico is due to the lack of interaction between Culex spp. mosquitoes and avian reservoir hosts in this region. Ornithophilic feeding behavior also was reported recently for Cx. quinquefasciatus populations in the city of Monterrey, northern Mexico; 44% to 73% of engorged mosquitoes collected outdoors had acquired their blood meals from chickens ( Elizondo-Quiroga et al. 2006 ). The overall HBI in the present study was low (0.1) indicating that humans were not a preferred host for Cx. quinquefasciatus in Merida. Nevertheless, a small proportion (6.7%)) of engorged Cx. quinquefasciatus contained human blood, indicating that this mosquito species could also transmit WNV to humans in this region. Vector competence studies are needed to determine the role that Cx. quinquefasciatus plays in the transmission of WNV in Mexico. | Studies were conducted to determine the host-feeding preference of Culex quinquefasciatus Say (Diptera: Culicidae) in relation to the availability of human and domestic animals in the city of Merida, Yucatan State, Mexico. Mosquitoes were collected in the backyards of houses using resting wooden boxes. Collections were made five times per week from January to December 2005. DNA was extracted from engorged females and tested by PCR using universal avian- and mammalian-specific primers. DNA extracted from avian-derived blood was further analyzed by PCR using primers that differentiate among the birds of three avian orders: Passeriformes, Columbiformes and Galliformes. PCR products obtained from mammalian-derived blood were subjected to restriction enzyme digestion to differentiate between human-, dog-, cat-, pig-, and horse-derived blood meals. Overall, 82% of engorged mosquitoes had fed on birds, and 18% had fed on mammals. The most frequent vertebrate hosts were Galliformes (47.1%), Passeriformes (23.8%), Columbiformes (11.2%) birds, and dogs (8.8%). The overall human blood index was 6.7%. The overall forage ratio for humans was 0.1, indicating that humans were not a preferred host for Cx. quinquefasciatus in Merida.
Keywords | Acknowledgments
The authors thank Francisco Solis-Heredia and Fernando Puerto-Manzano for providing blood samples for the validation of the restriction enzyme assays and Hugo Valenzuela-Nahuatl for support in the field. This study was supported in part by grant 2004-01-131 from the Fondo Sectorial Conacyt-Salud Mexico, by The National Institutes of Health grant # 53-4569-2, and by the Centers for Disease Control and Prevention grant # G-4457-2. Sequencing experiments were supported by The National Institutes of Health, grant 5R21AI067281.
Abbreviations
West Nile virus,
human blood index,
forage ratio,
host feeding index | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 6; 10:32 | oa_package/da/89/PMC3014751.tar.gz |
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PMC3014752 | 20578885 | Introduction
The bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae) is one of the most damaging pests of the kidney bean, Phaseolus vulgaris L. (Fabales: Fabaceae) in the Mediterranean area. It causes losses of up to 30% of stored beans ( Pemonge et al. 1997 ). Its oviposition and growth are continuous, and the larvae feed on the seeds. After emergence from the seeds, the adults reproduce either in the field or in the stored seeds in a continuous cycle ( Labeyrie 1962 ).
The Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), is one of the major pests in industrial flour mills in temperate climates ( Jacob and Cox 1977 ). Larvae reduce product quality by their presence and by the production of frass and webbing, and they also cause direct damage by feeding ( Johnson et al. 1997 ). The Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), is a cosmopolitan pest that infests a wide range of stored products including nuts, beans, processed foods and dried fruits ( Simmons and Nelson 1975 ). The control of these pests in storage systems mainly depends on fumigants such as methyl bromide or phosphine. However, methyl bromide was banned in many countries starting in 2004 because of its ozone depleting properties ( Hansen and Jensen 2002 ). Many alternatives have been tested to replace methyl bromide fumigation for stored-product and quarantine uses. There is an urgent need to develop safe alternatives that have the potential to replace the toxic fumigants, yet are effective, economical and convenient to use ( Ayvaz et al. 2008 ). Many spices and herbs, and their extracts, are known to possess insecticidal properties that are frequently present in the essential oil fraction ( Brattsten 1983 ; Schmidt et al. 1991 ; Shaaya et al. 1991 ). Most of the essential oil constituents are monoterpenoids, which are secondary plant chemicals and considered to be of little metabolic importance. The toxicity of a large number of essential oils and their constituents have been evaluated against a number of stored-product insects.
Over the past 15 years, interest in botanical insecticides has increased as a result of environmental concerns and insect populations becoming resistant to conventional chemicals. Botanical insecticides are naturally occurring insecticides that are derived from plants ( Isman 2000 ). The insecticidal activity of essential oils and plant extracts against different stored-product pests has been evaluated ( Shaaya et al. 1991 ; Sarac and Tunc 1995 ; Tunc et al. 2000 ; Kim et al. 2003 ; Lee et al. 2003 ; Asian et al. 2005 ; Cetin and Yanikoglu 2006 ; Negahban et al. 2007 , Ayvaz et al. 2009 ). In spite of the wide-spread recognition that many plants possess insecticidal properties, only a handful of pest control products directly obtained from plants are in use because the commercialization of new botanicals can be hindered by a number of issues ( Isman 1997 ). Botanicals used as insecticides presently constitute 1% of the world insecticide market ( Rozman et al. 2007 ). Essential oils from different plant species possess ovicidal, larvicidal, and repellent properties against various insect species and are regarded as environmentally compatible pesticides ( Isman 2000 ; Cetin et al. 2004 ).
In the present study, the chemical constituents of essential oils from Satureja thymbra L. (Lamiales: Lamiaceae), Origanum onites L. (Lamiales: Lamiaceae), and Myrtus communis L. (Rosales: Myrtaceae) were determined, and the insecticidal activity of these essential oils was tested against the adult stages of the stored-products pests: E. kuehniella, P. interpunctella and A. obtectus. No study has been reported previously concerning the activity of these compounds as fumigants against these stored product insects. The essential oils were applied primarily on adults to prevent mass egg production and further damages from larvae. | Materials and Methods
Insect cultures
The founding insect culture of A. obtectus was collected in infested kidney beans ( P. vulgaris ) that were stored in 5-liter plastic containers at a storehouse from Kayseri Province of Turkey. Adults oviposited on P. vulgaris beans, and the larvae developed inside the beans until adult emergence in 1-liter glass jars. To allow air passage, a hole 2 cm in diameter was opened in the center of each jar lid, and a sterile cloth was glued to the underside of each lid.
The Mediterranean flour moth, E. kuehniella, culture was obtained from the Department of Plant Protection, Faculty of Agriculture of Ankara University. E. kuehniella larvae were reared using a mixture consisting of one kg wheat flour, 55 g yeast and 30 g germs of wheat ( Marec et al. 1999 ).
The Indian meal moths, P. interpunctella, used in this experiment were taken as larvae from naturally infested dried apricot collected from Kayseri province. The larvae of P. interpunctella were maintained continuously on a diet containing 10% glycerol, 50% dried apricot and 40% wheat flour with wheat bran mixture. Throughout the experiments insect cultures were maintained at constant temperature (27 ± 1°C), photoperiod (14L:10D) and relative humidity (60% ± 5) ( Ayvez et al. 2007 , 2009 ).
Plant materials
The plants savory ( S. thymbra ) and myrtle ( M. communis ) were collected in the middle of July 2007 from the fields of Mersin (Southern Turkey), and oregano ( O. onites ) was collected in the middle of August 2007 from the fields of Canakkale (Western Turkey). Leaves were randomly collected from plant parts and shade dried.
Extraction of essential oil
The plant leaves were air dried at room temperature and chopped into small pieces using a mill with rotary knives. The essential oil was extracted from the plants using a Clevenger-type water steam distillation apparatus ( Papachristos and Stamopoulos 2004 ). The distilled essential oils were stored in a refrigerator at 4°C until being used in the treatments.
Analysis of volatile compounds
The composition of the volatile constituents was established by gas chromatography-mass spectrometry/Quadropole detector analyses using a Shimadzu QP 5050 system fitted with a Free Fatty Acid Phase (50 m × 0.32 mm (i.d.) film thickness: 0.25 μm) capillary column. Detector and injector temperature were set at 230°C. The temperature program for the column was from 120°C (1 min) to 230°C at a rate of 6°C/min and than held at 200°C for 35 min. Helium was used as a carrier gas at a flow of 14 psi (Split 1:10), and the injection volume of each sample was one μl. The identification of the components was based on comparison of their mass spectra with those of Wiley and Nist Tutore Libraries ( Adams 2001 ). The ionization energy was set at 70 eV.
Insecticidal activity
In order to test the toxicity of essential oils on the adults (< 24 h age) of E. kuehniella and P. interpunctella, ten adults were put into the 1000 ml glass jars. Essential oils were applied on a filter-paper strip measuring 3 × 3 cm that was attached to the lower side of the jar's lid. Doses were calculated based on nominal concentrations and assumed 100% volatilization of the oils in the exposure vessels ( Papachristos and Stamopoulos 2004 ). The same procedure was applied for the adults (< 48 h age) of A. obtectus, but this species was provided with kidney beans as food. All the insecticidal activity experiments were conducted at constant temperature (27 ± 1°C), photoperiod (14L: 10D) and relative humidity (60% ± 5). The adults of E. kuehniella and P. interpunctella were exposed to essential oil vapors (1.5; 3; 6; 9; and 25 μl/l air) for 24 h. However, because of high tolerance of A. obtectus adults were treated with higher doses (65; 130; and 195 μl/l air) and longer exposure time (24, 72 and 144 h) than that of E. kuehniella and P. interpunctella. A dose-mortality line depending on the exposure time(s) was developed, and the lethal concentration of essential oil needed to kill 50 or 99% of the pest population (LC 50 and LC 99 , respectively) was determined. Three replicates were set up for each dose and exposure time. A complete set of controls was maintained and replicated three times for each treatment. All replicates ran simultaneously during the experiments.
Statistical analysis
The data were corrected using Abbott's formula ( Abbot 1925 ) for the mortalities in the controls, and the data were subjected to probit analyses using SPSS ( 2001 ) for Windows to estimate LC 50 and LC 99 values of the essential oils against each stored-product insect species. Percentage mortality values for different exposure times were subjected to analysis of variance (one-way ANOVA) using the same statistical program ( SPSS 2001 ) for probit analysis. Data were transformed using arcsine √x transformation to meet normality, which is recommended for analysis of variance (ANOVA) ( Steel and Torrie 1980 ). Means were separated at the 5% significance level by the least significant difference (LSD) test. | Results
Chemical composition of essential oils
The chemical constituents of three different essential oils are given in Table 1 . The results of analysis showed that the chemical contents of oregano and savory were similar, but myrtle had a different chemical composition. The main compounds found in the savory were characterized as carvacrol (53.7%), γ -terpinen (17.6%), thymol (13%) and p- cymene (10.1%). Of all the compounds found in the oregano, the percentage of the carvacrol was the highest (70.3%) as in the savory. The main other compounds were linalool (11.9%) and thymol (9.3%). The chemical constituents of the myrtle were different from other oils tested, and the main components were linalool (31.3%), linalyl acetate (17.8%), and 1.8-cineole (14.7%). The other constituents of the savory, oregano and myrtle are given in Table 1 .
Insecticidal activity of essential oils against the E. kuehniella adults
The increasing doses of essential oils caused a significant increase in the mortality when the E. kuehniella adults were exposed to these oils for 24 h (for myrtle: F = 55.98; d.f. = 5, 12; p < 0.0001; for savory: F = 23.90; d.f. = 5, 12; p < 0.0001; and for oregano: F = 80.08; d.f. = 5, 12; p < 0.0001). Percent mortality of E. kuehniella was 90% at the 25 μl/l air dose when exposed to myrtle, and the same dose caused complete mortality (100%) when exposed to savory and to oregano ( Figure 1 ).
Probit analysis showed that LC 50 values of these essential oils against E. kuehniella were 7.52, 10.34 and 12.74 μ l/l air for oregano, savory and myrtle, respectively. LC 99 values revealed that E. kuehniella was more susceptible to oregano (12.72 μl/l air) than savory and myrtle (21.27 and 29.43 μl/l air for savory and myrtle, respectively) ( Table 2 ).
Insecticidal activity of essential oils against the P. interpunctella adults
The mortality values significantly increased depending on the increasing essential oil concentration when the P. interpunctella adults were exposed to myrtle, savory and oregano (for myrtle: F = 135.32; d.f. = 5, 12; p < 0.0001; for savory: F = 067.73; d.f. = 5, 12; p ;< 0.0001; and for oregano: F = 100.48; d.f. = 5, 12; p < 0.0001). The mortality values reached 96.7 and 100% when the adults were exposed to 6 μ l/l air concentrations of savory and oregano, respectively, and all the adults were killed by a 9 μ l/l air or higher concentration ( Figure 2 ). The mortality effect of myrtle was lower than those of the other oils, and the rate of adult mortality was 66.7% at the 25 μ l/l air myrtle concentration. LC 50 and LC 99 values of the essential oils tested are summarized in Table 2 , and these values revealed that the most effective essential oil against P. interpunctella adults was oregano, the same as for E. kuehniella. LC 50 and LC 99 values of the oregano against P. interpunctella were 4.06 and 5.77 μ l/l air, respectively ( Table 2 ).
Insecticidal activity of essential oils against the A. obtectus adults
Among the tested insects, A. obtectus was the most tolerant species against the essential oils. To kill the adults of this insect by using these three essential oils, higher concentrations and exposure times are required than are required for the other two species. The concentration (25 μl/l air) that caused complete mortality for E. kuehniella and P. interpunctella did not affect A. obtectus adults. The myrtle was more effective than the other essential oils against A. obtectus adults. Increasing mortality was observed for the A. obtectus adults when the concentrations and exposure times increased. Although 100% mortality was obtained after 144 h at 195 μl/l air when A. obtectus was exposed to oregano and savory, the myrtle showed the same effect at 65 μl/l air and 72 h of exposure ( Figure 3 ).
LC 50 and LC 99 values of the essential oils tested for the length of the time are summarized in Table 3 , and the LC 50 and LC 99 values of the myrtle against A. obtectus were 33.56 and 50.97 μl/l air, respectively, for the highest exposure time. The doses required for 99% mortality (LC 99 ) for using oregano and savory against A. obtectus were 127.36 and 76.05 μ l/l for the 144 h exposure, respectively ( Table 3 ). | Discussion
The insecticidal constituents of many plant extracts and essential oils are monoterpenoids. Due to their high volatility they have fumigant activity that might be of importance for controlling stored-product insects ( Konstantopoulou et al. 1992 ; Regnault-Roger and Hamraoui 1995 ; Ahn et al. 1998 ). In the current study, the essential oils obtained from savory, oregano and myrtle showed insecticidal activity against the adults of E. kuehniella, P. interpunctella and A. obtectus. A. obtectus was the most tolerant species against these oils as the doses required to kill the adults of this species were much higher than those required for the other two pest species for all treatments. The adults of A. obtectus were more susceptible to myrtle oil than oregano or savory. The toxic effects of the myrtle could be attributed to major constituents such as linalool (31.3%), linalyl acetate (17.8%) and 1.8-cineole (14.7%). The high toxicity of linalool, linalyl acetate and 1.8-cineole was reported against the rice weevil Sitophilus oryzae and Rhyzopertha dominica ( Rozman et al. 2007 ). Due to the linalool, linalyl acetate and 1.8-cineole constituents, the myrtle could also be used effectively against S. oryzae and R. dominica. These results, and those reported earlier, indicate that the insecticidal activity of the essential oils varies depending on the stage of the insect, the species and the plant origin of the essential oil ( Tunc et al. 2000 ; Chiasson et al. 2001 ; Choi et al. 2003 ; Sedy and Koschier 2003 ; Negahban et al. 2007 ).
The essential oils of oregano and savory were highly effective against P. interpunctella and E. kuehniella, and 100%) mortality was obtained after 24 h at 9 and 25 μl/l air for these species, respectively. The major components of these two essential oils are monoterpenes, primarily carvacrol and thymol. Reported biological activities of plant terpenoids include repellency and deterrence, reduced palatability, growth inhibition through altered protein availability, enzyme inhibition, and direct toxicity ( Harborne 1993 ). The plants oregano and savory belong to the family Lamiaceae. Jacobson ( 1989 ) pointed out that the most promising botanical insect-control agents are in the families of Annonaceae, Asteraceae, Canellaceae, Lamiaceae, Meliaceae and Rutaceae.
The insecticidal activity against A. obtectus of the essential oils obtained from oregano and savory was higher at the lowest dose and longest exposure period than at the highest dose and the lowest exposure period ( Figure 3 ). El-Nahal et al. ( 1989 ) stated that the period of exposure appears to be more important than dosage in affecting the efficiency of the vapors of Acorus calamus essential oil to adults of five stored-product insect species. Similar results have been reported for the toxicity of methanol extract of the rhizome from Acorus gramineus to adults of S. oryzae and Lasioderma serricorne ( Park 2000 ).
Similarly, the essential oils of oregano and savory have been found to be lethal to P. interpunctella and E. kuehniella but less toxic to A. obtectus. Carvacrol and thymol are usually extracted from Satureja, Coridothymus, Thymbra, and Origanum species and have been found to be lethal to turnip aphids ( Chiasson et al. 2001 ). There are numerous reports on the insecticidal activity of the essential oils from Origanum species, and the major components of this species, such as carvacrol, thymol, γ-terpinen and terpinen-4-ol, are based on fumigant and repellent activity rather than contact toxicity ( Tunc et al. 2000 ; Erler and Tunc 2005 ; Erler 2005 ).
Carvacrol has broad insecticidal and acaricidal activity against agricultural, stored-product and medical pests, and it acts as a fumigant. It is highly toxic to nymphs of the termite Reticulitermes speratus, adults of the rice weevil S. oryzae, the pulse beetle Callosobruchus chinensis, the cigarette beetle, L. serricorne ( Ahn et al. 1998 ), and the mite, Tetranychus urticae ( Isman 2000 ).
The insecticidal activity of the myrtle was lower than that of the oregano and savory against E. kuehniella and P. interpunctella. The percent mortality of both moth adults was 100% at the 25 μl/l air and 24 h exposure when treated with oregano and savory. However, percent mortality of E. kuehniella and P. interpunctella was 90% and 67%, respectively, when exposed to myrtle.
If the cost-effective commercial problems can be solved, the essential oils obtained from these plants can be effectively used as part of integrated pest management strategies ( Rozman et al. 2007 ). The essential oil content of aromatic plants is about 1–3% ( Cakir 1992 ). Therefore large quantities of plant material have to be processed in order to obtain the essential oils in quantities sufficient for commercial-scale tests ( Tunc et al. 2000 ). It would be useful to breed the plants containing desired essential oils in elevated quantity.
The high activity of these compounds could make it a potential substitute for methyl bromide in various uses in stored-product control programs and can be used in coordination with microbial insecticides, attractants and traps, and beneficial insects and mites (natural enemies of pests) as a component of the integrated pest management. The observed fumigant activity shows that essential oils are sources of biologically active vapors that are potentially efficient insecticides. Consequently, the possibility of employing these natural fumigants to control insects in stored products may be worthy of further investigation. | Associate Editor: Eileen Cullen was editor of this paper
This study was conducted to determine the insecticidal activity of essential oils from oregano, Origanum onites L. (Lamiales: Lamiaceae), savory, Satureja thymbra L. (Lamiales: Lamiaceae), and myrtle, Myrtus communis L. (Rosales: Myrtaceae) against three stored-product insects. Essential oils from three species of plants were obtained by Clevenger-type water distillation. The major compounds in these essential oils were identified using gas chromatography-mass spectrometry and their insecticidal activity was tested against adults of the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae), the Indian meal moth Plodia interpunctella Hübner (Lepidoptera: Pyralidae) and the bean weevil Acanthoscelides obtectus Say (Coleoptera: Bruchidae). While the major compound found in oregano and savory was carvacrol, the main constituent of the myrtle was linalool. Among the tested insects, A. obtectus was the most tolerant species against the essential oils. However, the insecticidal activity of the myrtle oil was more pronounced than other oils tested against A. obtectus adults. The essential oils of oregano and savory were highly effective against P. interpunctella and E. kuehniella, with 100% mortality obtained after 24 h at 9 and 25 μl/l air for P. interpunctella and E. kuehniella, respectively. LC 50 and LC 99 values of each essential oil were estimated for each insect species.
Keywords | Acknowledgements
The authors thank Dr. Dogan Bulut for language improvement, and to the editors for critical review of the manuscript. | CC BY | no | 2022-01-12 16:13:45 | J Insect Sci. 2010 Mar 16; 10:21 | oa_package/47/87/PMC3014752.tar.gz |
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PMC3014753 | 20578886 | Introduction
Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is polyphytophagous, damaging numerous vegetables and field crops in China and many other Asian countries ( Shu 1959 ; Hill 1975 ; Shivayogeshwara 1991 ). S. litura is also known as the common or tobacco cutworm, or the cluster or tobacco caterpillar. Although it had been a sporadic pest of tobacco in northern China for many years, it has been becoming gradually a very important insect pest in recent years ( Guan and Chen 1999 ; Gao et al. 2004 ; Qin et al. 2004 ). It also becomes resistant to many commonly used insecticides, particularly pyrethroids and carbamates, resulting in failure of effective controls ( Wu et al. 1995 ; Kranthi et al. 2002 ; Ahmad et al. 2007 ; Huang and Han 2007 ).
Study of the effects of host plants on the biology of insects is important in understanding host suitability of plant-infesting insect species. There have been a number of studies on the biological parameters of S. litura on different host plants under different environmental conditions, particularly in India ( Patel et al. 1986 , 1987 ), Pakistan ( Ahmad et al. 2007 ), China ( Guan and Chen 1999 ; Zhu et al. 2000 ; Qin et al. 2004 ; Zhu et al. 2005 ), Korea ( Bae et al. 1997 ; Bae 1999a , b ; Bae and Park 1999 ), and other Asian countries ( Etman and Hooper 1979 ; Holloway 1989 ) where S. litura has been an important pest on various crops. However, not all of these studied the effects of the same host plants on development, survival, pupal weight and oviposition of S. litura under the same environmental conditions, and none studied the adaptation of S. litura on tobacco for two generations after they were reared on an artificial diet.
Thus, the objectives of this study were to determine (a) oviposition preference by the females and the development and survival of larval and pupal stages of S. litura on tobacco, Chinese cabbage, cowpea and sweet potato and (b) food utilization on the four host plants. | Materials and Methods
Host plants
Four host plants were used in this study, including tobacco ( Nicotiana tabacum L., variety ‘NC89’), Chinese cabbage ( Brassica rapa var. chinensis , variety ‘Shandong Fushen Baotou’), cowpea ( Vigna sinensis L. Walp. ssp. uniguiculata , variety ‘Zhijiang 28-2’), and sweet potato ( Ipomoea batatas L., variety ‘Yushu 10’). These plants were selected because they are the most important economic crops in northern China and are primary host plants of S. litura .
The four species of host plants were singly planted in plastic pots (19-cm in diameter and 14 cm in depth) in a greenhouse at Shandong Agricultural University at Taian, Shandong, China, and were maintained insecticide-free. The plants were used when they had 4–5 true leaves.
Insects
S. litura larvae were originally collected from cabbage fields ( Brassica oleracea var. capitata L.), and were subsequently reared on an artificial diet at the Institute of Pomology, Guangdong Academy of Agricultural Science, Guangzhou, Guangdong. The artificial diet contained the following ingredients: 137 g of corn flour, 10 g of yeast, 37.5 g of soybean flour, 3.5 g of multiple vitamins, 1 g of sorbic acid, 2 g of nipagin, 12.5 g of agar, 0.16 g of inositol, and 0.15 g cholesterol. Soybean flour and corn flour were sterilized for 40 min before they were used. The diet was prepared as described by Zhu et al. (2001).
Host preference for oviposition
This experiment was conducted in an airconditioned insectary at 26 ± 1° C, 12:12 L:D, and ≈70% RH. S. litura adults were developed from the larvae that had been fed with the artificial diet for three generations. Newly emerged adults were collected and released in each cage at a sex ratio of 1:1. The adults were fed with 10% sugar-water solution through a cotton ball in a small plastic container (3.5× 1.3 cm). The adults were allowed to mate in the cage for two days. Eight plants were placed in a screen cage (50×50×50 cm), and two plants from each species were randomly placed at one of the four corners. The plants were adjusted to the same height. On the third day, 10 pairs of adults (5 females and 5 males) were released in each screen cage containing eight plants, and again, 10% sugar-water solution was supplied for the adults. The females were allowed to oviposit for 2 days, and the number of egg masses and eggs in each egg mass was recorded for each plant. The experiment was replicated six times.
Larval development and adult reproduction
Newly hatched larvae reared on the artificial diet were transferred and reared separately in small containers (3.5×1.3 cm) on each of the four host plants until they reached the fifth instar. Newly exuviated fifth instars were individually reared in Petri dishes (9.0 × 1.5 cm) to avoid cannibalism. Each host plant treatment had 100 larvae. The larvae were monitored for development and mortality at 12-h intervals. In the meantime, the dishes were cleaned, and new leaf pieces were replaced as needed. Before pupation, a few pieces of paper tissue were placed on the dish bottom for larval pupation. Two-day old pupae were sexed and separately weighed. Newly emerged adults were placed in a container (6.5 × 12 cm) and were fed with 10% sugar-water solution. The females would readily oviposit on paper stripes (1 cm wide, 5–10 cm long) in the container. The adults were monitored daily for mortality and oviposition, and number of egg masses oviposited on the paper stripes by each female were collected and counted twice daily until the female died.
Food consumption and utilization
Newly exuviated sixth instar larvae that had been reared on each of the four host plant species for two generations were used in this study. Larvae of approximately the same size were selected and individually placed in small containers. The larvae were starved for 10 h, and then each larva was individually coded and weighed. Twenty larvae were used in each of the four host plant treatments. The larvae were equally divided into a control group and a treatment group. In the control group, the 10 larvae and 10 fresh leaves from each of the four host plants were individually weighed, dried in a drier at 80° C, and weighed again. The dry weights were used as the standard for all other treatments. In the treatment group, leaves detached from each of the four host plants were weighed and provided to the 10 larvae. The larvae were fed with the leaves for 48 h. The larvae were then starved for 6 h to allow the larvae to defecate. The larvae, leaf tissues, and feces in each dish were weighed and then dried in a drier at 80° C. The dried leaf tissues and larvae were weighed again. Food utilization rates were then calculated based on the formulas of Waldbauer ( 1968 ): Where A is the weight of dried leaf tissues in the control, B is the weight of the dried leaf tissue in each treatment, C is the weight of dried larvae in the control, D is the weight of dried larvae in each treatment, and E is the weight of dried feces in each treatment.
Data analysis
The life history parameters of S. litura were analyzed using one-way and factorial ANOVA ( SAS Institute 2008 ). Means associated with host plants for each variable were separated using the least significant difference test when significant values were obtained. | Results
Host plant preference for oviposition
In the choice test, numbers of egg masses oviposited by S. litura females on the four host plants differed significantly ( F = 17.73; df = 3, 23; p = 0.002) ( Figure 1 ). S. litura oviposited the most on Chinese cabbage (13.3 egg masses, 36.7%), followed by sweet potato (9.3 egg masses, 26.7%) and cowpea (9.0 egg masses, 24.8%), and the least on tobacco (4.7 egg masses, 12.8%).
Larval and pupal development
Overall larval development was significantly affected by host plants ( F = 26.26; df = 3, 15; p < 0.0001) ( Figure 2 ), and was longest on tobacco (23.2 d), followed by sweet potato (17.5 d), cowpea (15.8 d), and shortest on Chinese cabbage (13.3 d) ( Figure 2A ). Of the six instars, the first, third and fourth instars development took significantly longer on tobacco ( F = 58.01 - 91.68; df = 3, 15; p < 0.0001) ( Figure 2B ). In contrast, second instar development took significantly longer on sweet potato than on the other three host plants, and the for the fifth and sixth instars development time was not significantly different for tobacco and sweet potato ( Figure 2B ). Pupal development times on Chinese cabbage (10.9 d), cowpea (10.1 d) and sweet potato (10.1 d) were not significantly different, and were longer than on cowpea (9.5 d) ( F = 6.84; df = 3, 15; p < 0.05) ( Figure 2A ).
Larval and pupal survival
The survival rates of S. litura larvae varied on the four host plants ( Figure 3 ). The overall, accumulated survival rates of all larval stages on the four host plants differed significantly ( F = 26.43; df = 3, 15; p < 0.05) ( Figure 3A ) and was lowest on tobacco (49.0%), followed by that on sweet potato (66.2%), on Chinese cabbage (75.4%), and highest on cowpea (81.7%). Of the six larval stages, the survival rates of S. litura were significantly different in the first four instars ( F = 4.03 - 42.27; df = 3, 15; p = 0.0339 - 0.0001), but not in the two oldest ones ( F = 0.83 and 2.62; df = 3, 15; p = 0.0987 and 0.5018) ( Figure 3B ). Different larval instars responded differently on each of the four host plants. The survival rates of the first instar were highest on sweet potato (99.0%) and lowest on tobacco (80.1%), with intermediates on Chinese cabbage (90.0%) and cowpea (91.7%). The survival rates of the second instar were not significantly different on Chinese cabbage (90.3%), sweet potato (86.5%) and tobacco (80.6%), but were significantly higher on cowpea (98.9%) than on the other three host plants. The survival rates of the third instars were similar on Chinese cabbage (100%) and cowpea (95.5%), which were higher than those on sweet potato (86.8%) and tobacco (83.8%). The fourth instars survived less on tobacco (90.3%) than on the other three host plants (98.6 – 100%). The survival rates for prepupae were not significantly different ( F = 0.93; df = 3, 15; p > 0.05), but those for pupae were ( F = 4.56; df = 3, 15; p < 0.05).
Pupal weight
Pupal weights differed significantly depending on the host plants on which the larvae were fed and differed significantly between females and males when they fed on the same host plants and when larvae fed on different host plants (p < 0.005) ( Table 1 ). The female pupae on Chinese cabbage were heaviest, followed by those on cowpea and sweet potato, and lightest on tobacco, and the male pupae on Chinese cabbage were heavier than on the other three host plants. Female pupae were generally heavier than their male counterparts, except for those on tobacco, which were the same weight.
Sex ratio, adult longevity and oviposition
Sex ratios were biased, and more female adults emerged than male adults when their larvae were fed with the four host plants ( F = 19.52; df = 3, 15; p = 0.0001) ( Table 1 ). Of the six treatments, male ratios were higher on cowpea and sweet potato than on the other two host plants. The longevities of both female and male S. litura adults were also significantly affected by the host plants on which their larvae fed (female: F = 6.15; df = 3, 15; p = 0.0089; male: F = 10.6; df = 3, 15; p = 0.0014). Numbers of egg masses and total eggs oviposited by S. litura females on the four host plants differed significantly (egg masses: F = 16.61; df = 3, 15; p = 0.0001; total eggs: F = 31.13; df = 3, 15; p = 0.0001) ( Figure 4 ). S. litura oviposited similar numbers of egg masses and eggs per female on cowpea, sweet potato, and Chinese cabbage, but less on tobacco than on the other three host plants. Numbers of eggs per egg mass were similar on Chinese cabbage (283.5 eggs/egg mass) and sweet potato (282.1 eggs/egg mass), which were more than those on cowpea (224.9 eggs/egg mass) and tobacco (233.6 eggs/egg mass).
Food consumption and utilization
Food consumption and conversions of ingested and digested food by S. litura larvae varied considerably among the four host plants that the larvae consumed ( Table 2 ). The relative growth rates on tobacco (0.43), cowpea (0.43) and Chinese cabbage (0.40) were higher than that on sweet potato (0.32) ( F = 8.050; df = 3, 39; p < 0.001). The relative consumption rates were highest when the larvae fed on sweet potato (3.90), followed by that on cowpea (3.16), then on Chinese cabbage (2.28), and the lowest on tobacco (1.51) ( F = 56.19; df = 3, 39; p < 0.001). The efficiency of conversion of ingested food was highest on tobacco (29.75), followed by that on Chinese cabbage (17.85), then that on cowpea (14.04), and lowest on sweet potato (8.34) ( F = 74.59; df = 3, 39; p < 0.001). The efficiency of conversion of digested food was higher when the larvae fed on tobacco than when fed on the other three host plants ( F = 18.73; df = 3, 39; p < 0.001).. However, the larvae that fed on Chinese cabbage and cowpea were similar and lower than those on sweet potato and tobacco. The approximate digestibility of S. litura larvae on the four host plants differed significantly ( F = 63.56; df = 3, 39; p < 0.001) and were higher on Chinese cabbage and cowpea than on sweet potato and tobacco. | Discussion
The data clearly show that S. litura performed differently in oviposition, larval and pupal development and survival, pupal weight, and oviposition of emerged females when Chinese cabbage, cowpea, sweet potato and tobacco were offered as the food plants for their larvae. These results are supported by a number of studies, although direct comparison of these data can be difficult because different host plants and environmental conditions were used in these studies. Although the same insect was used, it differed in origins, and it could be different strains or biotypes. However, in a few studies, the same host plants (one or two) were used, including cowpea ( Qin et al. 2004 ; Zhu et al. 2005 ) and Chinese cabbage (Zhang et al. 1997). Qin et al. ( 2004 ) used the same variety of cowpea but found that the developmental time of S. litura larvae was 12.8 d at 29° C, which was 3 d shorter than in this study. Zhu et al ( 2005 ) found that S. litura larvae finished their development in 10.1 d on cowpea as compared with 15.8 d in the current study. This difference (5.7 d) could be caused by higher temperature (28.1° C) or different cowpea variety from what was used here (26° C; ‘Zhijinag 28-2’).
Larval development of S. litura varied greatly depending on host plants and temperature, and the development was prolonged under low or high temperatures ( Zhu et al. 2000 ; Chen et al. 2002 ; Seema et al. 2004 ). For example, on tobacco, larval development can range from 19.3 d at 26° C ( Chen et al. 2002 ), 23.2 d in this study at 26° C, to as long as 30 d at 23° C ( Rattan and Nayak 1963 ). Similarly, on cowpea, larval development ranged from 10.1 d at 28° C ( Zhu et al. 2005 ) to 15.8 d at 26° C in the present study.
It has been reported that pupal development was not affected by host plants on which their larvae fed ( Patel et al. 1986 ). However, our results show that pupae developed faster on cowpea than on Chinese cabbage, sweet potato and tobacco, although the difference was <1 d longer on sweet potato, 1 d longer on tobacco, and 1.5 d longer on Chinese cabbage. Bae and Park et al. ( 1999 ) found that temperature plays a vital role on pupal development. Bae and Park ( 1999 ) reported that the mean pupal developmental duration was as long as 13.8 d at 24° C on perilla to as short as 7.4 d at 32° C on soybean.
Larval survival or pupation rate of S. litura varied greatly on different host plants, ranging from 100% on Ricinus communis ( Patel et al. 1987 ) to 49.0% on tobacco in this study. Bae and Park ( 1999 ) found that pupation rates were positively correlated with high temperature, 30.0, 33.3 and 38.5% on sweet potato, perilla and soybean, respectively, at 24° C, and 57.5, 80.0 and 87.5% at 32° C on the same host plants, respectively.
These results show that larval food directly affects pupal size and weight, and the female pupae were heavier than male pupae on all four host plants ( Table 1 ). The pupal weights of S. litura in this study were 0.32 to 0.36 g, which were generally within a wide range on various host plants, from 0.28 g on sweet potato to 0.40 g on perilla and cowpea ( Bae and Park 1999 ; Qin et al. 2004 ). Pupal weights were not found to be significantly different among different temperature regimes ( Seema et al. 2004 ), but 27° C was considered the optimum temperature for larval and pupal growth and development. Bae and Park ( 1999 ) found that pupal weight tended to be 3 – 13% lower with increasing temperature from 24° C, to 28° C, then to 32° C.
In the present data, more than 91% of S. litura pupae successfully developed to adults. In contrast, Bae and Park ( 1999 ) reported significantly low emergence rates on four foods. They found that only 31.4% of pupae developed to adults at 24° C and 88.6% at 30° C, and their emergence rate increased 1.1 to 2.8 fold with temperature increase from 24° C to 32° C.
Numbers of eggs of S. litura oviposited by the females from the larvae that fed on the four host plants were generally within the range as reported on various host plants ( Figure 3 ). In the literature, oviposition by females varied greatly on different hosts under different environmental conditions ( Patel et al. 1986 ; Bae and Park 1999 ). Numbers of eggs laid by a single female ranged from 935 on soybean ( Bae and Park 1999 ) to 3,467 on cotton ( Patel et al. 1986 ). In contrast, Bae and Park ( 1999 ) reported that S. litura females oviposited an average of 803 eggs on an artificial diet, and Chu and Yang ( 1991 ) found that S. litura oviposited as many as 5,995 per female on a different artificial diet.
In the present study, male adults generally lived longer (7.4 – 8.8 d) than females (6.6 – 7.7 d), differing on different host plants ( Table 1 ). Similar results were reported by Bae and Park ( 1999 ), although the differences were generally less than 1 d (0.25 – 1.0 d). However, Patel et al. ( 1986 ) found that on cotton, male adults lived 6.3 d as compared with 12.3 d for female adults. It has been found that adult longevity became shorter as the temperature increased ( Bae and Park 1999 ). In the present study, the sex ratio was biased, and more females emerged than males with sex ratios of 1.0:0.64 – 0.75. This was supported by Seema et al. ( 2004 ). Differences in pupal survival, adult sex ratio, longevity, and fecundity may also be affected by temperature and other environmental conditions ( Zhu et al. 2000 ; Chen et al. 2002 ; Seema et al. 2004 ).
Our data show that all nutritional indices varied when S. litura fed on the four host plants. Food conversion efficiencies on different host plants vary considerably by S. litura larvae ( Balasubramanian et al. 1985 ) and by insects in general ( Scriber and Slansky 1991 ; Slansky and Scriber 1985 ). The current data show that S. litura had similar relative growth rates on Chinese cabbage, cowpea, and tobacco, but had the lowest relative consumption rate when feeding on tobacco compared with those for the other three host plants. However, the larvae were more efficiently converting tobacco tissues into their biomass than other plant tissues as shown by larvae fed on tobacco having the lowest approximate digestiblity and the highest efficiency of conversion of digested and ingested food. One cause of such variation may involve homeostatic adjustment of consumption rates and efficiency parameters such that an insect can approach its “ideal” growth rate even with foods of different quality. Zhu et al. ( 2005 ) found that S. litura larvae did not prefer feeding on banana leaves and had lower relative growth rate, relative consumption rate, and approximate digestibility, but it had a significantly higher efficiency of conversion of ingested food and an extremely higher rate of efficiency of conversion of digested food, indicating that the larvae are capable of compensating by more efficiently utilizing their limited banana leaf tissues than other host plants. The digestion rate is affected by the enzyme activities of various host plants, including trehalase, invertase, and others. In practice, however, it can be quite difficult to ascertain “cause” and “effect” responses with efficiency parameters. Does the insect eat more because digestibility is low, or is digestibility low because the insect is eating more? Efficiency parameters are so closely related physiologically that determination of “cause” and “effect” is not a trivial matter. Factors contributing to such changes are still largely unknown, but may include shifts in food selection, digestive physiology, metabolic rates, and body composition. Understanding of these basic principles of nutritional ecology can enhance our appreciation of insects' adaptation to new food resources.
As this study found that S. litura dose not prefer feeding on tobacco, why has it been able to cause severe damage on tobacco in China? Our data indicate that S. litura was able to adapt on tobacco in a relatively short period of time (Xue, unpublished data). For example, comparison of the development, survival, and fecundity parameters between the two generations clearly show that the larvae in the second generation developed faster, survived more, had heavier pupae, and oviposited more. These results may partially explain the facts that S. litura could adapt on tobacco and quickly become a severe pest. The outbreaks of S. litura can be affected by many biotic and abiotic factors. Gao et al. ( 2004 ) identified several factors that may cause outbreaks of S. litura in northern China. Those factors include increasing acreage of tobacco and many preferred crops (mainly vegetables), which provides abundant food sources; expanding of protected cultivations, which provides suitable sites for overwintering; more mild winter and warmer spring, which enable the pest to occur earlier and build up higher spring populations in the first generation; high temperature and less rainfall in summer; and misuse of pesticides that cause resistance to insecticides and less natural enemies. Monoculture could be another factor because it is more favorable for the pest, and irrigation may also contribute to the outbreaks of S. litura in China. The occurrence of S. litura is generally synchronous with the rapid growing period of tobacco, especially during summer, providing plenty of sources for oviposition and larval feeding. In India, Pandey ( 1977 ) considered a good rainfall after a long period of drought as the key factor causing S. litura outbreaks. S. litura have also become resistant to most commonly used insecticides ( Wu et al. 1995 ; Kranthi et al. 2002 ; Huang and Han 2007 ). In addition, culture practices, cropping systems, irrigation, and other weather conditions (i.e., typhoon) affect the outbreaks of S. litura on various cropping systems around the world ( Murata et al. 1998 ; Chaudhary and Bajpai 2006 ).
In conclusion, based on oviposition preference, larval development, survival, growth, pupal weight and duration, and emergence and fecundity of adults of S. litura , the preference and nutritional values of the four host plants were ranked as Chinese cabbage > cowpea > sweet potato > tobacco. Hence, the present study has shown the suitability of selected host plants for the development, longevity, and survival of S. litura. These findings will help to understand the biology of this particular pest and could help in its management and control, particularly on tobacco. Therefore, future studies should focus on testing a wider range of host plant species for the development of S. litura , and assessment of the chemical components of the host plant species would help to better understand the mechanism of host suitability. | Effects of four host plants, tobacco, Chinese cabbage, cowpea and sweet potato, on larval and pupal development and survival, and longevity and fecundity of adults of Spodoptera litura (F) (Lepidoptera: Noctuidae), were studied under laboratory conditions (26° C, 60–80% RH), as was the utilization of the four host plants and adaptation on tobacco. All of the biological parameters included in the study were affected by the host plants. In a choice test, S. litura females oviposited most on Chinese cabbage, least on tobacco, and intermediate on cowpea and sweet potato. S. litura larvae developed differently on the four host plants, from shortest to longest in the following order: Chinese cabbage, cowpea, sweet potato, and tobacco. Pupal development was shorter on cowpea than on the other three host plants, and males generally developed longer than females. More females than males were found among emerged adults, and male adults lived 1–2 d longer than females. Larvae survived best on cowpea (81.6%), followed by Chinese cabbage (75.5%), then sweet potato (66.1%), and worst on tobacco (49.2%). Pupal survival rates were relatively high (91.4 – 95.9%) in all four host plant treatments, although that on sweet potato was lower than those on the other three host plants. Pupal weights on tobacco and sweet potato were similar, but both were lower than those on Chinese cabbage and cowpea. Generally, male pupae weighed less than female pupae. Numbers of eggs oviposited by female S. litura were highest on sweet potato, followed by those on cowpea, Chinese cabbage, and lowest on tobacco. Relative food consumption rate was highest on sweet potato, followed by that on cowpea, Chinese cabbage, and lowest on tobacco. In contrast, S. litura larvae that fed on tobacco had higher efficiency of conversion of digested food, highest efficiency of conversion of ingested food, and lowest approximate digestibility as compared with larvae that fed on other host plants. The potential causes for S. litura outbreaks on tobacco are discussed.
Key words | Acknowledgements
We would like to thank Y. Zhang, L. Q. Wei and L. L. Li for technical assistance, and the Institute of Pomology, Guangdong Academy of Agriculture Science (Guangzhou, Guangdong, China) for providing the insects for this study. This work was financially supported by The State Tobacco Monopoly Administration, China Tobacco Corporation (2003579), and The Nature Science Foundation of China (30671379). | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Mar 23; 10:22 | oa_package/42/5c/PMC3014753.tar.gz |
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PMC3014758 | 20578955 | Introduction
Phenology is the seasonal timing of life history events of organisms. Proper timing of life events — such as emergence, dispersal and reproduction — is a keystone for individual survival and success. Therefore, the phenology must evolve to match the environment in a manner that optimizes fitness, and traits that determine timing are presumably under strong selection ( Wiklund and Fagerström 1977 ; Iwasa and Levin 1995 ; Jonzén et al. 2007 ). Evidence of the finetuning between phenology and environment would be the rapid alteration detected in the life cycles of a wide array of species in response to recent climate change ( Parmesan and Yohe 2003 ; Root et al. 2003 , 2005 ).
In contrast to the significant attention recently paid to the temporal changes in phenological traits, the spatial variability of phenology remains poorly studied. More knowledge is needed regarding both the description of spatial variation patterns of phenology and also the search for potential environmental, biological or evolutionary mechanisms shaping them. The asymmetry between the temporal and spatial focus of current phenological research may constrain the abilities to fully understand factors controlling phenology and to make accurate predictions about the impact of climate change on organisms' phenology. Therefore, a better knowledge of factors governing the spatial variability of phenology is essential for fully understanding how and why phenology is changing with time.
To date, insect macroecology has focused mainly on large-scale patterns of distribution ( Eyre et al. 2003 , 2005a ; Jiménez-Valverde et al. 2007 ) or diversity ( Hawkins and Porter 2003 ; Stefanescu et al. 2004 ; Baselga and Jiménez-Valverde 2007 ) of insect species. Meanwhile, large-scale patterns of other biological features of insects, such as their phenology and its causes, remain unexplored. Data requirements are one possible reason for the lack of research in this area. In fact, reliable results and sound conclusions can only be derived from huge datasets gathered by many people involved in perfectly coordinated sampling networks. Such collaborative efforts in Britain enabled the use of 1.5 million records collected between 1995 and 1999 ( Asher et al. 2001 ) in the exploration of spatial trends in the mean sighting dates of 29 resident butterfly species ( Roy and Asher 2003 ). The sighting dates showed marked latitudinal and longitudinal gradients through Britain for most species. However, some butterfly species did not show spatial patterns in their phenology. This result is striking because it is expected that the spatial variability of insect phenology should mirror gradients of the most influential environmental variables affecting the timing of insect lifecycles, such as temperature (e.g., Fielding et al. 1999 ). These species lacking obvious spatial patterns may require stronger environmental gradients than those gradients that occur in Britain in order to offer a measurable gradient of phenology. Alternatively, there may be other physiological or behavioural mechanisms for ensuring a macroscale synchronous phenology that could be more beneficial than regional differences in phenology. In any case, these results stress the necessity of more studies on spatial patterns of insect phenology and on the mechanisms that influence them ( Weiss et al. 1988 , 1993 ; Fielding et al. 1999 ).
Another example of the value of monitoring networks for the assessment of the spatial variability of phenology is offered by recent studies based on the EXAMINE project ( Cocu et al. 2005 ; Harrington et al. 2007 ). The capture of flying aphids in a network of suction traps spread over Western Europe allowed the determination of the geographical and environmental factors related to the spatial variability of both the numbers and the phenology of some aphid species. Regarding phenology, these studies demonstrated that differences in flying periods among trapping sites were due to differences in climatic conditions prevailing in them. This result was expected due to the well-known effect of climate over aphid activity ( Worner et al. 1995 ; Zhou et al. 1995 , 1996 ). However, spatial trends of aphid phenology through Europe were also explained by other environmental features, such as land use in the area surrounding each trap ( Cocu et al. 2005 ; Harrington et al. 2007 ). Moreover, the effect of climate, land use or geography on aphid phenology varied according to the working scale ( Cocu et al. 2005 ). This research on aphid phenology both provided insights into the complexities involved in spatial aspect phenology and also stressed the urgent necessity to devote more efforts toward this largely unexplored issue.
In a previous study, Gordo and Sanz ( 2006b ) demonstrated that the honey bee, Apis mellifera (L.) (Hymenoptera: Apidea), and small white, Pieris rapae (L.) (Lepidoptera: Pieridae), phenologies had significant latitudinal, longitudinal and altitudinal trends in the Iberian Peninsula. Unfortunately, variables such as latitude or longitude do not provide conclusions concerning the possible environmental or biological factors underlying the observable differences in the appearance schedule of the insects among study sites. The aim of Gordo and Sanz ( 2006b ) was to determine the temporal aspect of phenology, and the spatial variables were used as covariates in order to account for the possible confounding effects of spatial gradients on temporal trends across the Iberian Peninsula. Nonetheless, Gordo and Sanz ( 2006b ) suggest the existence of a spatially structured variability in the appearance dates of A. mellifera and P. rapae. Therefore, these species have become ideal candidates for a comprehensive study about the spatial variability of insect phenology. The aim of this study is to complete the temporal picture explored by Gordo and Sanz ( 2006b ) by further investigating the spatial patterns of the appearance dates of A. mellifera and P. rapae in Spain, together with an exhaustive search for the underlying environmental factors. Therefore, the present study is focused on the patterns and causes of phenological variability among localities. | Materials and Methods
Phenological data
Since the 1950s, the first appearance dates for A. mellifera and P. rapae have been recorded by volunteer observers in hundreds of localities throughout Spain. These observers applied the standardized protocolsproposed by the Spanish Intituto Nacional de Meteorología (for more details about this scheme, see Gordo and Sanz ( 2006a , b )). Both species are widespread in Spain and are well-studied due to their relevance in agriculture. A. mellifera is the main pollinator for orchards and most fruit trees, while the larval stages of P. rapae are important pests of cabbage crops. Adults of P. rapae also may act as pollinators of some entomophilous crops.
Records dated between 1952 and 2004 from original files of the Intituto Nacional de Meteorología were collected and digitized. A total of 7,263 records from 737 localities were gathered (see Figure 1 for details for each species). Each date was transformed into a Julian day scale (1 = 1 January). For leap years, one day was added after February 28 to take the extra day into account. Before performing any analyses, temporal trends of phenological data were removed by the regression of appearance dates against the quadratic function of the year for each species ( Gordo and Sanz 2006b ). Residuals obtained from temporal regression models were used as a measure of the phenological variation independent of the year from which these dates were recorded (see also Gordo et al. 2007a , b ). Temporally corrected dates were used thereafter.
The mean appearance date for all records from the same 100 km 2 (10 × 10 km) universal transverse mercator (UTM) cell was calculated for each species. Because some localities of the phenological network were in the same UTM cell, the final sample size available for calculations (i.e. different UTM cells) was smaller than the number of original localities (see Figure 1 ). Mean values for each UTM cell could be biased due to differences in the number of records. This possible dependence was tested by calculating Spearman rank correlations between mean values and number of records in each UTM cell ( A. mellifera: r s = 0.034, t 617 = 0.845, P = 0.395; P. rapae: r s = -0.011, t 440 = -0.231, P = 0.817). Because the mean appearance date was not dependent on the number of records, all UTM cells with available records for both species were used.
Explanatory variables A. mellifera
A total of 47 explanatory variables were classified into five categories (spatial, topographical, climatic, vegetation productivity and land uses; see Table 1 ) and used to model the appearance dates of A. mellifera and P. rapae throughout Spain. Seven topographic variables were obtained from a digital elevation model ( Clark Labs 2000 ) for each of the 100 km 2 UTM Iberian squares (n = 6063) using the IDRISI 32 Geographic Information System ( Clark Labs 2001a ). The mean, minimum and maximum altitude of each 100 km 2 UTM cell was calculated from all 1 km 2 pixels included in each 100 km 2 UTM cell. The altitude range, slope, aspect (the mean direction of the slope) and diversity of aspects were also obtained for each 100 km 2 UTM cell. Delayed appearance dates are expected in UTMs at higher elevations and/or with northern exposures ( Scott and Epstein 1987 ; Weiss et al. 1988 , 1993 ; Gutiérrez and Menéndez 1998 ; Cocu et al. 2005 ; Gordo and Sanz 2006b ; Harrington et al. 2007 ).
Eighteen climatic variables were provided by the Instituto Nacional de Meteorología for each of the 100 km 2 UTM Iberian squares. Climatic variables were rainfall and minimum, mean, and maximum temperatures during each season (spring, summer, autumn and winter), together with the annual temperature range and an aridity index. Spring was defined as April, May and June, summer as July, August and September, autumn as October, November and December, and winter as January, February and March for all seasonal variables. The aridity index was calculated as: where P is the annual precipitation, and T is the mean annual temperature. Later appearance dates were expected in UTMs with cooler temperatures due to the strong effect of this variable on the development and activity of insects ( Scott and Epstein 1987 ; Zhou et al. 1995 ; Sparks and Yates 1997 ; Fielding et al. 1999 ; Dell et al. 2005 ; Gordo and Sanz 2006b ). In the case of precipitation, later appearance of insects was predicted for the UTMs that were more moist, especially during the spring ( Cocu et al. 2005 ; Harrington et al. 2007 ). Therefore, the more arid areas (i.e., higher AI values) should be also those with the earliest appearance of bees and butterflies.
Vegetation productivity also was evaluated as a possible explanatory variable for spatial patterns of the spring appearances of A. mellifera and P. rapae in the Iberian Peninsula. This variable was measured as the normalized vegetation difference index (NDVI). The NDVI is the normalized difference between red (0.55 – 0.68 μm) and infrared (0.73 – 1.1 μm) reflectance, as measured by the National Oceanic and Atmospheric Administration's polar orbiting satellite's advanced very high resolution radiometer sensor ( Smith et al. 1997 ). The NDVI is determined by the degree of red wavelength absorption by chlorophyll, which is proportional to leaf chlorophyll density, as well as by the reflectance of near infrared radiation, which is proportional to green leaf density ( Tucker et al. 1985 ). Therefore, the NDVI correlates well with variables such as green leaf biomass, leaf area index, total accumulated dry matter and annual net primary productivity ( Nicholson et al. 1990 ). NDVI data were available from Clark Labs world images as monthly values from 1982 to 2000 at a spatial resolution of 0.1 degree ( Clark Labs 2001b ). A value of vegetation productivity for each 100 km 2 UTM of the Iberian Peninsula was calculated by averaging monthly images available for each season between 1982–2000. More productive areas were expected to support higher abundance and diversity of organisms of upper trophic levels, such as insects ( Hawkins and Porter 2003 ; Bailey et al. 2004 ; Seto et al. 2004 ). Larger populations of insects may enhance an early appearance simply due to increased chances for detection of early individuals ( Tryjanowski et al. 2005 ; Dennis et al. 2006 ). Alternatively, larger populations may indeed be related to an earlier appearance because of the greater genetic diversity and the consequent higher probabilities for early phenotypes.
Land use types were also included because features of the environment eventually determine the presence of insect species ( Eyre et al. 2003 , 2005b ). Therefore, habitat availability can be considered as another surrogate for the abundance of populations at a local scale, which, in turn, can affect detection (see above). Furthermore, land use has been demonstrated to have effects on the phenology of other insect taxa, such as aphids ( Cocu et al. 2005 ; Harrington et al. 2007 ). Consequently, it would be of interest to understand to what extent this evidence is applicable to different taxonomic groups, such as bees or butterflies. The distribution of 15 land use types for the Iberian Peninsula was obtained from Corine Land Cover 2000 at a 100 × 100 m resolution. The percentage of coverage in each category within each 100 km 2 UTM cell was calculated and used as 15 explanatory variables for the analyses (see Table 1 ). The heterogeneity of land use types within each UTM was summarized by the Shannon diversity index and included as another explanatory variable.
Finally, spatial variables were used to verify the existence of spatial gradients. They were defined as the central latitude and longitude of each UTM cell and were included in the analyses as a third degree polynomial ( Legendre and Legendre 1998 ). The nine terms of the spatial polynomial can help to incorporate effects of other historical, biotic or environmental variables not otherwise taken into consideration ( Legendre and Legendre 1998 ). Latitude and longitude were standardized (mean = 0, and standard deviation =1) as were topographic, climatic, and vegetation productivity variables in order to eliminate their measurement scale effects.
Statistical analyses
Multiple regression models implemented in the General Regression Models module of STATISTICA ( StatSoft 2001 ) were conducted to determine the relationship between response (appearance date of A. mellifera and P. rapae ) and explanatory (spatial, topographic, climatic, vegetation productivity and land use) variables. Models were built in three sequential steps. First, the relationship between insect appearance and each explanatory variable was explored one-by-one. For each predictor, linear, quadratic or cubic relationships were sought. The functions whose terms were statistically significant (p < 0.05) were selected. Only those environmental variables that were significantly related to appearance dates were included in further analyses. In the second step, the modelling ability of each type of explanatory variable (i.e. spatial, topographic, climatic, vegetation productivity and land uses) was explored by including significant variables belonging to the same category in a single regression model. A procedure of backward stepwise selection was applied in all cases to obtain simplified models that included only significant variables. The model with spatial variables allowed assessment of the spatial structure of phenological data, while models with environmental variables (i.e., the rest of the predictors) constituted the rationale for a biological interpretation of the spatial structure of dates that was identified. In the third and final analytical step, the best explanatory model of appearance dates was sought. For this purpose, two different regression models were carried out. One was with environmental explanatory variables and the other included environmental and spatial variables. The nine terms of the third degree spatial polynomial included in the latter model seek to account for the potential effect of other non-considered variables that are spatially structured. A backward selection procedure was applied in both models to include only significant (p < 0.05) predictors. Predicted scores of the best final regression model were mapped and examined.
Explanatory variables were correlated to each other (i.e., multicollinearity) due to the cofluctuation imposed by spatial and environmental gradients of the Iberian Peninsula (e.g., northern and high altitude regions have cooler and moister climates). This fact hinders the estimation of the true relevance of predictors (Quinn and Keough 2002). A hierarchical partitioning of variance procedure ( Birks 1996 ; MacNally 2000 , 2002 ) was implemented to determine the relative importance of each type of explanatory variable. The relative importance can be estimated as the average effect of including each type of variable in all possible models built with the remaining types of variable. Therefore, 2 k functions must be constructed for k types of explanatory variables. In our case, k = 5 (spatial, topographic, climatic, vegetation productivity and land uses) and thus 2 k = 32 different models.
Residuals from all multiple regression models were examined to check for spatial autocorrelation. If residuals are spatially autocorrelated, one or several important spatially structured explanatory variables are left out of the models ( Cliff and Ord 1981 ; Legendre and Legendre 1998 ). Moran's I autocorrelation coefficient with a Bonferroni-corrected significance level ( Sawada 1999 ) was calculated against ten classes separated by a lag distance of 60 km (from 60 to 600 km). | Results
Apis mellifera
Many localities with an early phenology (especially those with very early appearance dates, i.e., prior to mid-February) were located in the southern and coastal areas of Spain ( Figure 1a ). In contrast, late sites for bee phenology occur mainly in central Iberia (i.e. Northern Plateau) and some mountainous regions (e.g., Iberian System; see Figure 2 ). Nevertheless, this row pattern is blurred by variability in the smaller scale. There were large differences between neighbouring UTMs. This could explain why the assessment of the spatial structure of data (i.e., the spatial model) showed a moderate explanatory ability ( Table 2 ). The only variable included in the spatial model was the cubic function of the latitude. In agreement both with the prediction and the visual inspection of raw data, the spring appearance of A. mellifera is later in northern areas ( c . 3 days·degree of latitude -1 ).
The topographical model included only the minimum altitude, the cubic function of which was strongly fitted to appearance dates ( Table 2 ). A. mellifera appears later in those localities that are at higher elevations ( Figure 3a ). The effect was especially marked in those UTMs with a minimum altitude above 300 m.
The climatic model was the best environmental model ( Table 2 ), although just one variable — the spring minimum temperature — was included. The paucity of variables in this model is remarkable because all climate variables (except autumn rainfall, see Table 1 ) were each significantly related to A. mellifera appearance dates. UTMs with the earliest bee appearance were located in those areas with the warmest springs ( Figure 3b ).
The model obtained with vegetation productivity variables was the least explanatory ( Table 2 ). The quadratic function of the spring NDVI was able to capture just 5.55% of the variability in appearance dates. The appearance of A. mellifera was earlier in regions that are less productive during the spring.
Up to six variables were significantly included in the land-use model, although they accounted for a low proportion of the variability in appearance dates overall ( Table 2 ). The negative sign of all variables means that A. mellifera were recorded earlier in the UTMs with large coverage of urbanized (i.e. humanized) areas, vineyards, fruit trees, olive groves, croplands and wild vegetation.
The average relative explanatory capacity of each variable was notably lower than that obtained in previous models ( Table 2 ). This could have been because of the high degree of collinearity among the explanatory variables. Nevertheless, the types of explanatory variables that showed the best fitted models also accounted for greater fractions of variability themselves. Interestingly, the variance hierarchical partitioning procedure revealed that the topographic model explained, on average, 2.4% more of the variability than the climatic model (Wilcoxon Matched Pairs test: Z 16 = 3.154, p = 0.002).
The complete environmental model included four variables and captured a bit more variability in appearance dates than the topographic or climatic models ( Table 2 ). Minimum altitude and spring vegetation productivity had the same effect (i.e. sign) that was described above. The cubic function of the minimum altitude was the most relevant predictor of this model (partial R 2 = 22.81%). One climatic and one land use variable that were not included in their respective models were included in the final model. Although they did not show the best explanatory capacity within their groups of variables, they likely worked on a different part of the variability of dates. Consequently, summer precipitation and cover of dry farming were not overridden by altitude effects. According to the positive sign of summer precipitation and percentage of dry farming, localities with an early appearance of A. mellifera were related to regions with little precipitation during the summer and with a low proportion of dry farming. Spatial terms were not included in the complete model after the backward selection procedure, and, consequently, the model remained equal. Hence, no spatial structure remained in the phenological data after modelling with these environmental variables. This agrees with the absence of spatial autocorrelation in residuals of the complete model ( Figure 4 ).
When predicted appearance dates from the complete model were mapped, the pattern of bee appearances in Spain was clearer than that offered by raw data ( Figure 5 ). A. mellifera were observed for the first time on the southeastern Mediterranean coast of Spain. Other markedly early regions that were predicted included the southern regions and the Mediterranean coastal regions. Immediately later, A. mellifera appeared during the first 10 days of March in most of Spain. Finally, the late appearance of A. mellifera (from April 1st onwards) was expected in some central (Northern Plateau) and mountainous (Iberian System, Sierra Nevada or Pyrenees) regions of the Iberian Peninsula as a consequence of the high altitude.
Pieris rapae
A visual examination of raw data offered more evident spatial patterns for P. rapae appearances than observed for A. mellifera ( Figure 6 ). Regarding the appearance of these insects, Spain can be unambiguously divided into two regions: one early area in the southern half of the Iberian and a late area in the northern half. Coastal areas both along the Mediterranean and along the Cantabrian seas also showed early appearance dates. Such patterns support a spatially well-structured P. rapae phenology. Indeed, all regression models for this species were better fitted (i.e., had a higher R 2 ) than those obtained for A. mellifera ( Table 2 ). This is especially striking because the explanatory variables included in the models were the same in most cases. Therefore, differences in the models' fitting between A. mellifera and P. rapae are likely not due to a different composition of the included explanatory variables. The spatial model showed a strong cubic latitudinal gradient (partial R 2 for latitude = 21.82%). The appearance of P. rapae was delayed almost one month from the southernmost point of Spain to the Cantabrian Mountains ( Figure 3c ). In the small latitudinal range from the Cantabrian Mountains to the Cantabrian coast, appearance dates tended to be earlier. Such a gradient was likely a result of the milder conditions due to the low altitude and to the proximity to the Atlantic Ocean.
The minimum altitude also played an important role in the spatial variability of P. rapae phenology ( Table 2 ). Those UTMs with higher minimum altitudes showed later appearance dates. The effect of minimum altitude was especially pronounced at higher altitudes, as was stressed by the quadratic relationship (see Figure 3a ).
The climatic model was also the best model for P. rapae , although in this case, the explanatory capacity was markedly higher than the topographic model ( Table 2 ). Temperature was the best explanatory variable. The linear relationship of appearance dates against mean spring temperatures accounted for 41.18% of the variability of P. rapae phenology. P. rapae is recorded earlier in areas that are warmer during the spring. The quadratic function of the summer rainfall was also included in the climatic model. This variable notably modeled appearance dates ( R 2 = 26.2%; Figure 3d ). The earliest appearances were linked to the driest areas. The early appearance in the moistest UTMs is linked to the latitudinal gradient. The moistest areas of the Iberian Peninsula are located on the Cantabrian coast, which has unusually mild conditions due to its proximity to the sea. The strong explanatory capacity of temperature and precipitation models did not add up as a result of the strong collinearity of both variables. Consequently, the full climatic model fit slightly more than previous models with a single climatic variable ( Table 2 ). Nevertheless, the mean spring temperature was the most important variable in the climatic model (partial R 2 = 38.50%).
Vegetation productivity variables produced the worst explanatory model ( Table 2 ). Spring patterns of productivity were related to appearance dates in a quadratic form. Early dates were linked to regions with low NDVI values during the spring.
The land use model included a large number of variables ( Table 2 ) and showed a moderate explanatory capacity of P. rapae phenology ( Table 2 ). The first butterflies are recorded in areas with large coverage of olive groves and fruit trees, but with a low coverage of dry farming, coniferous forests, moorlands and transitional areas from shrublands to forests and grasslands. This result concurs with the spatial meaning of previous models. Fruit trees and olive groves are located mainly in southern Spain and on the Mediterranean coast. However, dry farming dominates landscapes from the Northern Plateau, and coniferous forests, moorlands and grasslands are typical for mountainous regions with high elevations, low temperatures and moist climates.
The average percentage of variability that accounted for each type of variable (see relative R 2 adj in Table 2 ) was much lower, considering the high degree of collinearity among explanatory variables. Nevertheless, the relevance order was maintained. Topographic and climatic variables accounted for greater fractions of variability by themselves. Interestingly, true differences in the explanatory capacity of topographical and climatic variables were not too large, according to the relative R 2 values. On average, vegetation productivity and land use captured a negligible part of the variability.
The best complete model was built with climatic variables, minimum altitude and the coverage of dry farming ( Table 2 ). The addition of minimum altitude and coverage of dry farming to climatic variables minimally increased the explanatory capacity previously achieved by the climatic model. The inclusion of spatial terms improved explanatory capacity of the model. This was due to the replacement of climatic variables with latitude. Residuals from both complete models (with and without spatial variables) did not show significant autocorrelation scores at any lag distance ( Figure 4 ). Therefore, all spatially structured variations were included in P. rapae models.
Predicted date scores from the best complete model showed plainly differentiated phenological regions ( Figure 5b ). The earliest appearances of P. rapae were in the southern coast of Spain on both the Atlantic and the Mediterranean sides. However, the latest appearance dates were in the inner regions of the Northern Plateau and the Iberian System, as well as in the mountainous regions of the Pyrenees and Sierra Nevada. Intermediate regions also show a clear latitudinal gradient with later appearance dates in the Cantabrian coast and the Ebro valley than the dates in the Levant coast and southwestern Spain. | Discussion
Spatial patterns of A. mellifera and P. rapae appearance dates were strongly related to environmental gradients, especially those related to temperature. In fact, temperature effects were both direct (see Figure 3 ) and indirect. The indirect effect was through other environmental and geographical gradients, such as altitude (see Fig. 3 ) and latitude (see Figure 3c ). This strong effect of temperature on insect phenology was already demonstrated for the interannual variability of spring appearance phenology of the same species in the same area ( Gordo and Sanz 2006b ). Therefore, temperature was the most important factor in the year to year fluctuations in appearance dates ( Sparks and Yates 1997 ; Roy and Sparks 2000 ; Dell et al. 2005 ; Gordo and Sanz 2005 ), but was also the most important factor in the site to site variability. Such strong effects of temperature were expected because insects are poikilotherms, and, thus, their activity, development, and vital cycles are heavily influenced by weather.
The best complete models showed a good ability to model regional differences in phenological dates (especially in the case of P. rapae ). In a previous study ( Gordo and Sanz 2006b ), it was found that the appearance of A. mellifera and P. rapae had significant latitudinal, longitudinal, and altitudinal gradients. The results for latitude and altitude were confirmed here. Significant effects of longitude were not found, but as was pointed out by Gordo and Sanz ( 2006b ), the effect of this variable was negligible. In any case, the explanatory capacity of the models in the present study performed better than previous models suggested by Gordo and Sanz ( 2006b ), which were focused on temporal (i.e., interannual) variability rather than on spatial (i.e., intra-annual) variability. They did not further explore the biological meaning of spatial gradients that they found.
One of the most interesting peculiarities of these models (see Table 2 ) is the small number of explanatory variables included in them, despite the plethora of predictors employed (see Table 1 ). For example, just three variables were necessary to explain more than half of the spatial variability of P. rapae. Most explanatory variables were ruled out during the stepwise selection due to their redundancy. This strong collinearity was expected because all variables belong to a common spatial scenario (i.e. the Iberian Peninsula), and, consequently, all of them depicted the same environmental gradients. Furthermore, residuals from complete models did not show spatial autocorrelation, and thus there was no remaining spatial variability in the phenological data at this working scale. Therefore, the explanatory capacity of complete models could not be improved by adding other types of variables. In any case, the included variables and their effects in all types of models (i.e. climatic, topographical, etc) pointed toward the same spatial gradients: insects appear later in cooler areas, which correspond to high altitude and northern regions that receive higher amounts of summer precipitation and are dominated by dry farming. The effect of dry farming is probably due to the influence of the Northern Plateau region (see Figure 2 ). This is a high altitude region with a marked continental climate in the northern half of Iberia, where land use is dominated by extensive cereal areas.
Both species have very similar appearance dates ( A. mellifera appears just one week earlier than P. rapae ), and thus both are under very similar ecological influences, such as photoperiod and spring temperatures. This opens the possibility of determining to what extent spatial variability is under common environmental constraints or is dependent on species' biology peculiarities ( Gordo et al. 2008 ). Both species showed models composed of similar predictors, but the explanatory capacity and predicted maps of the complete models (see Figures 5 and 7 ) were different. Differences in explanatory capacity could be due to differences in the strategies used by each species to survive during the winter. A. mellifera hibernates as adults, while P. rapae hibernates as quiescent pupae. A. mellifera may react faster than P. rapae to increasing temperatures during the spring. A. mellifera only must stop its lethargy, while P. rapae must complete part of its development ( Gordo and Sanz 2006b ). Consequently, A. mellifera may be more influenced by climatic conditions on a microscale level (e.g., beehive located in a southern exposure), which would be unable to be modelled at this work resolution and extent. Such local environmental peculiarities would add white noise to the macroscale gradients of the Iberian Peninsula. However, P. rapae requires the constancy of raised temperatures to complete its development. As a consequence, spatial variability of P. rapae appearance phenology would fit much better with the spatial gradients of temperature during the spring. Nevertheless, almost half of the variability among localities remains to be explained for P. rapae, which could also be evidence of the effect of microclimatic conditions at each study site.
Land-use and the NDVI explanatory variables showed a poor ability to model insect appearance (see relative R 2 adj values in Table 2 ). In fact, landscape composition and vegetation productivity were not associated with any strong phenological gradient shown by bees or butterflies in the Iberian Peninsula. These results agree with the main findings of Cocu et al. ( 2005 ) for aphid phenology. The main predictor of aphid flying dates was climate (see also Harrington et al. 2007 ), while differences in land use had a negligible effect. One possible reason for the poor modelling ability of land use could be purely methodological. The present distribution of land use was used, while phenological data have been recorded during five decades. Land use has changed during the last five decades ( Pavón et al. 2003 ; Calvo-Iglesias et al. 2006 ). Thus, the present landscape may be not fully representative of the past landscape structure, when part of the data set was recorded. Nonetheless, this possibility can be ruled out because landscape changes occur on a rather small spatial scale without any coherent spatial pattern (e.g., afforestation is a generalized phenomenon). The most plausible hypothesis for the lack of land use effect is the working extent. The largest differences in appearance dates were imposed by large scale trends (e.g., south-north), which are beyond the local or regional scale where land use patterns exist. In the case of the NDVI, the poor modelling performance suggests that the proposed hypothetical link among vegetation productivity, insect abundance, and phenology could be ruled out.
Among climate variables, summer rainfall had a noticeable effect ( Figure 3d ). This merits interest because insect appearance occurs during the spring. The “advanced” effect of this variable has been demonstrated for other spring phenological events, such as the arrival of migratory bird species ( Gordo et al. 2007a , b ). This generalized effect of summer rainfall over a wide range of species suggests that organisms arrange their life cycles according to constraints imposed by aestival conditions in the Mediterranean. Alternatively, the summer rainfall effect may be due to the strong collinearity between summer rainfall and spring temperature patterns ( r = -0.72 for the 6,063 UTM cells of Spain). Those areas with the warmest spring temperatures became, several months later, those areas with the driest summer. High temperatures during the spring also encourage an early phenology. This would allow all spring processes (e.g., reproduction) to finish prior to the arrival of harsh summer conditions. Therefore, the advance of phenology in response to scarce summer rainfalls could be mediated by high temperatures during the spring. Unfortunately, the correlative approach in this study does not allow for differentiating the true acting mechanism of summer rainfalls over phenology.
Comparing the spatial patterns of insect appearance with the arrival patterns of migratory birds revealed that the environmental determinants in each case are different. Insect phenology is driven by climatic gradients, while the arrival of birds is strongly influenced by geographical configuration (such as mountains, valleys, etc.), which shapes the optimum progression routes through the Iberian Peninsula ( Gordo et al. 2007a , b , 2008 ). As a consequence of these differences, insect appearance patterns (see Figures 5 and 7 ) depict a different picture with regard to migratory birds ( Gordo et al. 2007a , b , 2008 ). In fact, the above mentioned differences between A. mellifera and P. rapae patterns become of minor relevance when insect patterns are compared to bird patterns. Such differences are paramount because most migratory birds rely on insects to feed. The earliest appearances of insects occur in the southern coast of Spain or even in the southeastern coast, as in the case of A. mellifera (see Figure 5 ). However, Gordo et al. ( 2007a , b , 2008 ) found that the earliest arrivals of birds occur in southwestern Spain, while the southeastern corner of the Iberian Peninsula showed a delayed arrival due to the geographical difficulties in being reached from the Straits of Gibraltar. Therefore, the phenological scenario found by migratory birds in different regions of Spain is not comparable. For instance, birds arrive later to southeastern Spain than to southwestern Spain, while insect phenology shows similar dates in both regions. Therefore, birds breeding in southeastern Spain found a more advanced insect phenological scenario. Similarly, a relatively late arrival of birds with regard to insect appearance also occurs at most coastal sites. Hence, spatial patterns found for insects support the geographical constraint hypothesis proposed by Gordo et al. ( 2007a ) for migratory birds. Unfortunately, this reasoning has two weaknesses: 1) spatial patterns were explored for only two insect species ( A. mellifera and P. rapae ), which are not necessarily representative of all insects, and 2) insectivorous migratory bird species may rely on insect prey other than bees and butterflies. Nevertheless, the strong dependence of both studied insect species on temperature gradients, which has been found in other insect taxa (e.g., aphids: Cocu et al. 2005 , Harrington et al. 2007 ; spittlebugs: Fielding et al. 1999 ), suggests that the spatial patterns found here are probably representative of the spatial gradients in the phenology of other insect communities. When organisms are altering their life cycles in response to climate change, it is of paramount importance to know the extent that species' phenological responses will maintain the phenological matching with their environment ( Visser and Both 2005 ). However, the potential constraint imposed by the differential role of determining environmental factors in the spatial variability in each species has been skipped.
The Spanish phenological network was created with an applied intent. In fact, A. mellifera and P. rapae were selected by their relevance for agriculture. The results also offer information of applied interest. For example, more severe damage to cabbage crops by P. rapae larvae can be predicted in the southern and warmer areas of Spain due to an earlier initiation of their life-cycle (up to three months). Consequently, individuals may complete more cycles (i.e., multivoltinism) there. Unfortunately, no data exists concerning damage caused by P. rapae to evaluate this hypothesis. In the same way, A. mellifera resume their activity with a noteworthy spatial variability, which should be taken into account for entomophilous crops. A correct selection of plant species or varieties with a suited phenology would be necessary to ensure fertilization of flowers by this pollinator species and an optimum crop yield. | Associate Editor: Timothy Meehan was editor of this paper
The spatial patterns of the variability of the appearance dates of the honey bee Apis mellifera L. (Hymenoptera: Apidea) and the small white Pieris rapae (L.) (Lepidoptera: Pieridae) were investigated in Spain. A database of more than 7,000 records of the dates of the first spring sightings of each species in more than 700 localities from 1952–2004 was used. Phenological data were related to spatial, topographical, climate, land use, and vegetation productivity explanatory variables by means of multiple regression models in order to search for the environmental mechanisms underlying the observable phenological variability. Temperature and altitudinal spatial gradients accounted for most of the spatial variability in the phenology of the studied species, while vegetation productivity and land use had low relevance. In both species, the first individuals were recorded at those sites with warmer springs and dry summers, at low altitudes, and not covered with dry farming (i.e., cereal crops). The identity and magnitude of the effect of the variables were almost identical for both species and closely mirrored spatial temperature gradients. The best explanatory models accounted for up to half of the variability of appearance dates. Residuals did not show a spatial autocorrelation, meaning that no other spatially structured variable at our working resolution could have improved the results. Differences in the spatial patterns of phenology with regard to other taxa, such as arrival dates of migratory birds, suggest that spatial constraints may play an essential role in the phenological matching between trophic levels.
Keywords | Acknowledgements
This study is dedicated to the incommensurable task of hundreds of volunteers of the Spanish phenological network. We thank the INM for access to the phenological data.
Abbreviations
aridity index;
normalized difference vegetation index;
universal transverse mercator | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 15; 10:34 | oa_package/70/49/PMC3014758.tar.gz |
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PMC3014759 | 21209803 | 1. Introduction
ID of the PCo-A refers to funnel-shaped widenings with a diameter less than 3 mm at the junction of the posterior communicating artery and the internal carotid artery [ 1 – 3 ]. ID of the PCo-A was regarded as normal anatomical variants with a low risk of rupture [ 4 ]. However, with increasing reports of ruptured ID of the PCo-A, bleeding is also found to occur in cases of ID of the PCo-A. Aggressive treatment (i.e., surgical clipping) is preferred for most patients presenting with hemorrhage [ 2 – 6 ], but little is known about the endovascular treatment of this condition. We report a case of ruptured ID of the PCo-A successfully treated by endovascular embolization. | 3. Discussion
ID of the PCo-A is not a rare clinical entity. However, the prevalence of it on carotid angiography ranges from 5% to 17% [ 6 – 9 ] or even higher, as shown in other studies [ 10 , 11 ]. The natural history of ID of the PCo-A remains unclear; it is generally recognized as an anatomical variant resulting from incomplete retrogression of vessels during embryonic development. ID of the PCo-A usually does not rupture or bleed [ 6 , 8 ]. However, a number of studies have reported fatal SAH caused by ID of the PCo-A rupture or its progression to aneurysms [ 3 , 4 ]. Hemorrhage due to the rupture of the ID of the PCo-A is surgically treated (i.e., surgical clipping) to prevent disastrous consequences due to rebleeding [ 5 , 6 , 8 , 12 – 20 ]. In most IDs of the PCo-A, PCo-A is not well developed and has few perforating branches; so, the origin of the ID of the PCo-A is clipped in an orientation parallel to the internal carotid artery to prevent rebleeding [ 12 – 18 ].
However, in our patient, although the right PCo-A was underdeveloped, neurosurgical clipping treatment was not selected, and stent-assisted endovascular coil embolization was performed using a combined approach of anterior and posterior circulation. This approach was considered because we found that the ID of the PCo-A had a wide junction with the internal carotid artery, making full clipping quite difficult similar to wide-necked aneurysms [ 21 ]. Moreover, a previous study has shown that the bleeding points in the ruptured ID of the PCo-A were mostly located at the distal side of the ID, close to the internal carotid artery [ 22 ]. Under such circumstances, blood could still flow into the inside of the ID along the PCo-A even when the origin of the ID is clipped.
In the current study, enhancement of the PCo-A was not revealed in the DSA of posterior circulation, but it appeared in the angiography of the compressed internal carotid artery. Blood flowed reversely into the internal carotid artery ( Figure 2(b) ) indicating the possibility of rebleeding if the neck of the ID was clipped and the ruptured bleeding point was left untreated. However, clipping the bleeding points of the ID of the PCo-A might result in new bleeding sites near the previous ones, due to the continuous impact of blood flow; there is also the probability that the ID of the PCo-A will progress to an aneurysm [ 22 , 23 ]. Therefore, at least 2 aneurysm clips are needed for isolation of the ID of the PCo-A in order to prevent rebleeding. It is difficult to place two aneurysm clips because of the narrow operating space around the posterior communicating artery. On the contrary, stent-assisted coil embolization by means of endovascular approach is relatively safe in comparison to neurosurgical clipping. Therefore, we preferred to perform endovascular treatment.
Although the patient experienced two SAH episodes in this study, the CTA on admission and first the DSA did not reveal the aneurysm but a conical expansion of the infundibulum at the origin of the right posterior communicating artery. At that time, ruptured ID of PCo-A could not be confirmed because profound SAH might have resulted in vasospasms that hide a very small aneurysm. In addition, the condition of the patient was critical. If we had attempted to treat the expansion of the infundibulum, disastrous result might have occurred or the aneurysm might have been missed. The better alternative was to wait until the blood was reabsorbed. After 20 days of conservative treatment, the repeated CT revealed reabsorption of blood along with an improvement in the condition of the patient. The DSA was performed and confirmed the diagnosis of ruptured ID of the PCo-A. Endovascular treatment lead to a good outcome. The follow-up DSA did not show the recurrence of ID of the PCo-A.
To date, no report on endovascular intervention treatment for ruptured ID of the PCo-A is available. We made an attempt to provide a new strategy for its treatment. While considering endovascular embolization for eligible patients with ID of the PCo-A, the following two factors need to be taken into account. First, the ipsilateral internal carotid artery needs to be compressed when conducting vertebral angiography so as to fully assess the hemodynamic status of the ID of the PCo-A. Therefore, rupture due to incomplete filling and blood supply from the posterior circulation can be prevented. Second, stent-assisted embolization is required because of the wide neck of the ID of the PCo-A. When full blocking is not possible through the internal carotid artery of the anterior circulation alone, it is preferable to carry out the blocking through the combined approach of anterior and posterior (via the posterior communicating artery) circulation. | 4. Conclusion
Stent-assisted endovascular coil embolization through a combined approach of anterior and posterior circulation provides a new strategy for treatment of hemorrhage due to rupture of the ID of the PCo-A. | Academic Editor: Aaron S. Dumont
Hemorrhage due to the rupture of the infundibular dilatation of the posterior communicating artery (ID of the PCo-A) occurs infrequently. The preferred treatment of such hemorrhages is surgical clipping through craniotomy. There are few reports about endovascular coil embolization in such cases. We report such a case treated by endovascular embolization. A 35-year-old man, who had experienced 2 episodes of subarachnoid hemorrhage (SAH), was found to have a ruptured ID of the PCo-A by head computed tomography angiography (CTA) and digital subtraction angiography (DSA). We performed stent-assisted endovascular coil embolization through a combined anterior and posterior circulation approach. Postembolization angiography showed absence of contrast filling of the ID of the PCo-A and nonleakage of the contrast agent. The patient recovered well with no complications. SAH recurrence was not recorded during the 1-year followup. The postoperative angiographic result was good. To our knowledge, this is the first case of hemorrhage due to ruptured ID of the PCo-A that was treated by such a technique. | 2. Case Presentation
A 35-year-old man was admitted with sudden onset of headache and vomiting. At admission, he was in relatively good condition. Hunt-Hess scale was grade III, and the GCS score was 13 at presentation. A CT scan of the head revealed SAH mainly in the right suprasellar cistern with a Fisher grade of 3 ( Figure 1(a) ). Dehydration therapy was performed immediately. Nine hours after the onset of symptoms, the patient lapsed into a coma; Hunt-Hess grade progressed to IV, and the GCS score deteriorated to 8. A repeated head CT examination revealed rebleeding, acute hydrocephalus, hematocele at bilateral posterior horns of the lateral ventricle, and a Fisher grade of 4 ( Figure 1(b) ). A CTA was performed together with a CT and revealed a conical expansion of the infundibulum at the origin of the right posterior communicating artery with a diameter of less than 3 mm. The thin right posterior communicating artery arose from the apex of the infundibulum. In addition, no aneurysm was noted in angiography (arrow in Figures 1(c) and 1(d) ). DSA and 3-D reconstruction were then performed, and these findings were similar to those seen in the CTA (arrow in Figures 1(e) , 1(f) , and 1(g) ). After these imagings, conservative treatments including dehydration therapy were administered to allow the reabsorption of SAH. Nimodipine was given for antivasospasm treatment. In the meantime, lumbar drainage was performed to drain bloody cerebrospinal fluid.
After 20 days of conservative treatment, the patient's condition got better, and he could follow simple commands. A repeated head CT showed that SAH was reabsorbed, and the size of the ventricles became normal ( Figure 1(h) ). DSA was performed again and yielded the same result as the previous one (arrow in Figure 1(i) ). Thus, ruptured ID of the PCo-A was diagnosed, and we planned to perform coil embolization. Because of the wide neck of the ID, stent-assisted occlusion was required. As a result, clopidogrel and aspirin were administrated orally for one week.
At day 27 after the bleed, endovascular coil embolization was performed under general anesthesia. The right femoral artery was punctured using the Seldinger technique to introduce a vascular sheath. Heparin was administered. Vertebral angiography with carotid compression was performed before vascular embolization so as to observe the blood supply to the posterior circulation of the ID of the PCo-A. The result showed that blood flow reversed into the internal carotid artery through the posterior communicating artery (Figures 2(a) and 2(b) ). Subsequently, endovascular embolization of the ID of the PCo-A was conducted. After a suitable working angle was selected, a Neuroform3 stent (4.0 mm × 20 mm) was successfully released using an Echelon-14 microcatheter and Transend-300 microguidewire to completely cover the neck of the ID of the PCo-A. Subsequently, an Echelon-10 microcatheter was delivered into the ID of the PCo-A using Transend-300 microguidewire through the stent mesh. After angiography, a HydroCoil 10 coil (2 mm × 2 cm, MicroVention, Aliso Viejo, Calif) was released into the ID of the PCo-A. However, the coil was not detached due to the unfavorable location ( Figure 2(c) ). The left femoral artery was punctured for introduction of a vascular sheath, and then an Echelon-10 microcatheter was placed into the PCo-A ID using a Transend-300 microguidewire via the posterior communicating artery ( Figure 2(d) ). Four HydroCoil 10 coils were sequentially released (2 mm × 2 cm, 2 mm × 2 cm, 2 mm ×1 cm, 2 mm × 1 cm) to fill the ID of the PCo-A ( Figure 2(e) ). No enhancement of the ID of the PCo-A was shown in the angiography of anterior or posterior circulation, and leakage of the contrast agent did not occur (Figures 2(f) and 2(g) ). The coil placed in the internal carotid artery was detached ( Figure 2(h) ). After catheters and sheaths were removed, the procedure was completed.
Postoperative course was uneventful. The patient was treated with clopidogrel and aspirin. During a 1-year followup, the patient recovered well and could care for himself. His GOS score was 4, and SAH did not recur. The follow-up DSA showed no signs of ID of the PCo-A (Figures 3(a) and 3(b) ). | Acknowledgment
The authors thank Medjaden Bioscience Limited for assisting in the preparation of this paper. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 27; 2010:210397 | oa_package/dc/5e/PMC3014759.tar.gz |
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PMC3014760 | 20578887 | Introduction
Species of Tamarix (Caryophyallales: Tamaricaceae) are native of Eurasia and Africa and, due to their extraordinary biological features and adaptive capabilities ( Fornasari 2004 ), have been introduced and established in 44 countries throughout the world.
At least four species of Tamarix ( T. gallica , T. ramosissima , T. chinensis and T. parviflora ) and their hybrids occur in Argentina ( Gaskin and Schaal 2003 ; Natale et al. 2008 ). They were introduced to cover human necessities, mostly as windbreaks, to fix dunes, to control soil erosion, to supply shade, and in some desert places tamarisks are the lone surviving woody plants ( León 2006 ).
However, tamarisk is now an alien plant that invaded the Pampean grasslands, mostly in coastal dunes or in riparian habitats, where it is more invasive, spreading over natural or semi-natural ecosystems ( Zalba and Villamil 2002 ). In addition, tamarisk became a noxious weed in Llancanelo and Guanacache lagoons, both protected RAMSAR sites in Cuyo region, because its invasiveness produces soil salinization, water obstructions, and habitat modifications altering the riparian/wetland trophic structure ( Sosa 2003 ). Consequences of tamarisk's invasiveness, such as water uptake and low diversity of dependent wildlife, have made it the target of classical biological control in the United States ( Fornasari 1997 , 2004 ).
Few insects are cited as affecting Tamarix spp. in Argentina: Ceroplastes sp., C. formicarius Hempel and Coccus hesperidum (L.) (Hem: Coccidae), Automeris aspersa (Felder) (Lep: Saturniidae), Oiketicus platensis Berg (Lep: Psychidae), Bostrichopsis uncinata Germ. (Col: Bostrichidae), and Praxithea deroudei (Chabrillac) (Col: Cerambicidae)( Cordo et al. 2004 ). | Methods and Materials
The geographic range of wild populations of Tamarix spp. cover most of Argentina, except the northeast ( Natale et al. 2008 ). Seven out of 12 provinces where Tamarix spp occurs were surveyed. All the specimens were collected using entomological nets and manual aspirators. Specific identification was made using the keys provided by Oman ( 1936 ) and Linnavuori ( 1959 ), using both external and male genitalia's characters. Voucher specimens of O. stactogalus resulting from this study are deposited in the collections of the Museo de Ciencias Naturales de La Plata, Buenos Aires (MLPA) and Fundación e Instituto Miguel Lillo at San Miguel de Tucumán (IMLA), Argentina. | Results
Gonatocerus tuberculifemur Ogloblin (Hym: Mymaridae) is an egg parasitoid candidate for the control of Homalodisca vitripennis (Hem: Cicadellidae) in the United States ( Jones et al. 2005 ). During the process of selecting leafhopper species for the study of their host range, abundant populations of a small leafhopper were found on Tamarix sp. in several sites in the Pampas and Monte provinces (Neotropical region, Chacoan subregion), and central and subandean patagonia provinces (Andean region, patagonian subregion) ( sensu Morrone 2001 ); it was identified as the Holartic species Opsius stactogalus Fieber, 1866 (Hemiptera: Cicadellidae) known as the “Tamarix leafhopper”.
Opsius stactogalus (Deltocephalinae: Opsiini) is a leafhopper native to Europe and is strongly associated with the shrubby tree tamarisk ( Wiesenborn 2001 , 2002 ). The distribution is primarily Paleartic, but now nearly cosmopolitan, and always associated with tamarisk. Linnavouri & De Long ( 1977 ) found this species associated with Casuarina sp. in Chile. This species was previously mentioned by Metcalf ( 1967 ) for “Argentina” without locality or region reference, but the known distribution that was published by several authors subsequent to the Metcalf catalogue ( Linnavuori 1959 ; Linnavouri and De Long 1977 ; Evans 1977 ; Oman et al. 1990 ; Zanol 2006 ) did not mention Argentina. The Tamarix leafhopper is a sap-feeder, and the aggregate feeding imposed by their populations can reduce tamarisk's growth (Liesner 1971, as cited by Wiesenborn 2002 ). Given the plant's pervasiveness and its undesirable qualities, new distributional data were obtained.
Examined material
ARGENTINA: Rio Colorado (Rio Negro province) (38° 59′ 45.1 S 64° 06′ 07.6 W, elevation: 91 MASL), 21.II.07, 3 females, 1 nymph, Virla & Logarzo cols. (MLPA); Choele Choel (Rio Negro prov.) (39° 17′ 22.6 S 65° 40′ 07.4 W, 126 MASL), 4 females, 1 male, Virla & Logarzo cols. (MLPA); Chimpay (Rio Negro prov.) (39° 10′ 08.7 S 66° 08′ 48.8 W, 154 MASL), 22.II.07, 3 females, 2 males, Virla & Logarzo cols. (MLPA); Neuquén city (Neuquén prov.) (38° 57′ 18.2 S 68° 08′ 42.9 W, 274 MASL), 22.II.07, 5 males, 3 females, 2 nymphs, Virla & Logarzo cols. (MLPA); Zapala (Neuquén prov.) (38° 59′ 4 S 70° 04′ 5 W, 1020 MASL), 10.II.08, 2 females, 4 males, 2 nymphs, Virla col. (IMLA); Villa E1 Chocón (Neuquén prov.) (39° 15′ 43.4 S 68° 46′ 39.1 W, 412 MASL), 02.II.08, 3 females, 3 nymphs, Virla col. (IMLA); Santa Isabel (La Pampa prov.) (36° 22′ 27.2 S 67° 04′ 06.8 W, 303 MASL), 18.II.08, 3 males, 10 females, Logarzo col. (IMLA); Algarrobo del Aguila (La Pampa prov.) (36° 24′ 23.9 S 67° 08′ 22.3 W, 301 MASL), 10.II.08, 2 males, 1 female, 1 nymph, Virla col. (IMLA); San Rafael (Mendoza prov.) (34° 45′ 51 S 68° 24′ 41.4 W, 674 MASL), 11–12.II.08, 8 males, 12 females, 14 nymphs, Virla col. (IMLA); Aimogasta (La Rioja prov.) (28° 34′ 18 S 66° 48′ 03 W, 847 MASL), 24.XII.07, 3 females, 3 males, 2 nymphs (IMLA), Virla col.; Villa Mazán (La Rioja) (28° 39′ 57.9 S 66° 31′ 24.2 W, 652 MASL), 10.II.08, 24 females, 65 males, 46 nymphs, Virla col. (IMLA); Chumbicha (Catamarca prov.) (28° 48′ 56 S 66° 14′ 50 W, 506 MASL), 21.XII.07, 4 females, 2 males, 3 nymphs, Virla col. (IMLA). | Discussion
Opsius stactogalus was found in all provinces sampled, except Córdoba (National Road n°7 km 526, near Gral. Levalle), in 12 out of the 13 sites surveyed. In all sites O. stactogalus was very abundant, however damage produced by this leafhopper on the sampled plants was not observed. Therefore, the potential usefulness of this hopper as a biocontrol agent for the invasive tamarisk populations established in Argentina is doubtful. Opsius stactogalus was also recorded in the United States of America, and again it did not cause significant damage to Tamarix spp. ( De Loach 2001 ).
These findings encourage exploration for possible natural enemies of O. stactogalus , either natives of Argentina or others that arrived with this leafhopper, in order to better understand the community history of tamarisk in Argentina. | Editor: Eugene Hall was editor of this paper
The paleartic tamarix leafhopper, Opsius stactogalus Fieber (Hemiptera: Cicadellidae), can reduce the growth of tamarisk due to the aggregate feeding imposed by their populations. The species was mentioned for Argentina in Metcalf's catalogue ( 1967 ) without locality or region reference, and the contributions on Cicadellidae published by many authors after Metcalf omitted this distributional data. Populations of O. stactogalus on Tamarix sp. were found in 12 sites between 28° 48′ to 39° 17′ S and 64° 06′ to 70° 04′ W, located in both the Neotropical and Andean biogeographic regions.
Keywords | Acknowledgements
The authors would like to thank Miss G. Rios de Fanjul for review of an early draft of the manuscript. We also are grateful to the reviewers for providing valuable suggestions.
Abbreviations
Fundación e Institute Miguel Lillo at San Miguel de Tucumán, Argentina;
Museo de Ciencias Naturales de La Plata, Buenos Aires;
meters above sea level | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Mar 23; 10:23 | oa_package/c1/7e/PMC3014760.tar.gz |
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PMC3014761 | 20569134 | Introduction
In rural Africa communities, thermal expulsion and direct burning of aromatic plants before sleeping continue to play a very important role in household protection against mosquito vectors of dangerous diseases such as malaria, yellow and dengue fever, elephatiasis and lymphatic filariasis ( Norbert et al. 2003 ; Seyoum et al. 2003 ). Leaves of Suregada zanzibariensis Verdc. (Angiospermae: Euphobiaceae) are used for this purpose by people of the coastal region of Tanzania, and they are used also in traditional medicine for treating skin diseases, asthma, abdominal pains, and malaria ( Atal et al. 1978 ; Hedberg et al. 1983 ; Omulokoli et al. 1997 ). The plant, known as ‘mehungwa pori’ (meaning “wild citrus”) in Kiswahili language, has leaves that resemble those of citrus plants which exhibits insecticidal ( Al Dakhil and Morsy 1999 ; Ezeonu et al. 2001 ) and repellent ( Oshaghi et al. 2003 ) activities against various mosquito species. Suregada zanzibariensis is well distributed in tropical Africa and grows as trees of about 9 m height ( Redcliffe-Smith 1987 ). No follow up phytochemical study and characterization of bio-active constituents of the plant has been reported. The goal of this bioprospecting initiative was to search for plants and their phytochemicals that could be useful in mosquito control. Towards this end, the constituents of the essential oil of the plant were characterized and were studied, individually and in blends, for their repellency against Anopheles gambiae s.s. Giles (Diptera: Culicidae). | Materials and Methods
Plant materials and extraction
Leaves of S. zanzibariensis were collected from Pugu Forest Reserve in Pwani region, Tanzania. The plant materials were authenticated at the Herbarium of the Department of Botany, University of Dares-Salaam, where voucher specimens were deposited. The leaves were dried in the shade for 4–6 days and then hydro-distilled using a Clevenger-type apparatus for 8 h. The distilled oil was separated from the aqueous layer, dried over anhydrous Na2SO4, and stored at 4° C.
Gas chromatography and gas chromatography-mass spectrometry
Analysis of the essential oil was carried out on a Hewlett Packard 5890A gas Chromatograph (GC) equipped with a flame ionization detector and a Hewlett Packard 3396 series II integrator. A cross-linked methyl silicon capillary column (50 m × 0.2 mm id × 0.33 μm film thickness) was used for separation of the essential oil components. Nitrogen was used as the carrier gas at a flow rate of 0.84 ml/min. The injector and detector temperatures were maintained at 250° C and 270° C respectively. The temperature program consisted of an initial temperature of 40° C, which was raised at a rate of 10° C/min to 140° C where it was maintained for 15 min. The temperature was then increased 10° C/min to 180°C and maintained for 15 min, and then increased at 10° C/min to 280° C and maintained for 15 min. Identification of the essential oil components was carried out on a Hewlett Packard 5790A series GC coupled to the VG Masslab 12–250 mass spectrometer (Micromass, Waters Inc., www.waters.com ) with mass range m / z 1-1400. The spectrometer was equipped with a computerized data system running using MassLynx (Waters) software with Wiley Version 6 and NIST Version 1.0 MS libraries. The spectrometer was operated in the EI mode at 70 eV with temperature of the source held at 180° C, multiplier voltage at 1350V, scan cycle of 1.5 s (scan duration of 1 s and inter-scan delay of 0.5 s), and scan range of m/z 38–650. The instrument was calibrated using heptacosafluorotributyl amine, [CF3(CF2)3]3N, (Apollo Scientific Ltd., www.apolloscientific.co.uk ). The column and temperature program used for GC-MS was the same as for GC analysis except for the carrier gas, which was helium in this case. Where possible (depending on availability), identities of the essential oil components were confirmed by GC coinjections with authentic samples obtained from Sigma Aldrich Chemical Co. ( www.sigmaaldrich.com ) or Fluka (Sigma). Identification of the other compounds that were not commercially available was based on detailed comparison of their mass spectra with those in the libraries.
Mosquitoes
Mated female adult An. gambiae s.s. mosquitoes used in the study were obtained from a colony reared according to World Health Organization (WHO) ( 1996 ) protocol at International Centre of Insect Physiology and Ecology insectary (initially cultured from specimens obtained from Ifakara in Tanzania in 1998). The larvae were reared at 32–36° C on a diet of TetraMin® (Tetra GmbH, www.tetra.de ). The adult mosquitoes were maintained in an insectary at 26–28° C and 70–80%, RH. on a 6% glucose solution. Females fed on humans three times per week. Female mosquitoes used in the experiments were 5–7 days old, initially maintained on human blood but changed to glucose (6% solution) a day before the bioassays and then starved for 18 h before use.
Ethical clearance and volunteer safety
As the experiment required sources of human blood for mosquitoes and human landing catches, local volunteers were recruited with informed consent. A research protocol was submitted to the International Centre of Insect Physiology and Ecology, based at Duduvile-Nairobi, and to the Kenya National Ethical Review Committees, based at the Kenya Medical Research Institute. Ethical clearance was obtained from the Kenya National Ethics Board. The discomfort and potential risks of mosquito bites was explained to the volunteers. The individuals had previously participated in similar studies, had good knowledge of malaria transmission, and showed mild or no allergic reaction to mosquito bites or the essential oil. Five adult volunteers (3 male and 2 female) were involved in the experiments, and they did not object to being identified for publication. A parasite-free environment was ensured through regular screening of the volunteers' peripheral blood for Plasmodium . Sulphadine-pyriproxyfen prophylaxis was provided to each volunteer.
Repellency assays
The repellency assay was performed in a dark room with red light as the only source of illumination ( WHO 1996 ). The room temperature and humidity were controlled at 28 ± 2° C and 75 ± 5% RH respectively to mimic the feeding conditions for female An. gambiae s.s. mosquitoes. Cages (50 × 50 × 50 cm) made of aluminium sheet at the bottom, Pyrex window screen on sides and top, and a cotton stockinet sleeve for access on the front, were used in the dose response assays. Different concentrations (0.01–10%) w/v) of the essential oil, selected constituents, and blends of these were prepared by dissolving 1 g of each sample in 10 ml of analytical grade acetone (99.95%)), followed by successive ten-fold dilutions with acetone to obtain the other concentrations. (1 S )-(-)-verbenone (17) and other compounds whose repellency against An. gambiae had previously been reported ( Omolo et al. 2004 , 2005 ; Odalo et al. 2005 ) were not evaluated singly, but as constituents of blends. The blend of the four main constituents of the oil (phenylacetaldehyde (5), verbenone (17), artemisia ketone (11), and geranyl acetone (24)) in relative amounts present in the essential oil (14:12:10:9 gram ratios), as well as four blends with one of each of these constituents missing, were prepared. Acetone acted as a blank in all experiments, and DEET acted as a positive control. Fifty test mosquitoes were used in each of five replicates involving five different adult volunteers for each concentration of a sample. The volunteers had no contact with any lotion, perfume, oil, or perfumed soap on the day of the bioassay. The forearm (average area of 696.6 cm2) of each volunteer from the elbow to the hand was washed with water and left to dry. The test sample (1 ml) was spread as evenly as possible on one of the forearms of a volunteer from the wrist to the elbow. The rest of the hand was covered with a glove. Acetone (1.0 ml) was dispensed on the other forearm to serve as control. The control and treated arms were interchanged regularly to eliminate bias. The control arm was first introduced into the cage for 3 min immediately after introduction of the mosquitoes. The number of mosquitoes that landed on the arm was recorded, and the insects were shaken off before they imbibed any blood. This was followed by exposure of a volunteer arm first to the lowest concentration (0.01% w/v) of the test sample followed by sequential exposures to progressively higher concentrations (0.1, 1, 10% w/v) of the sample, each time to fresh mosquitoes in a clean cage. The test arm of the volunteer was washed using a non-perfumed soap and tap water and allowed to dry naturally for at least 20 min before dispensing the subsequent concentration. Only one compound/sample was tested per day.
Data analysis
Percentage protective efficacy (PE) was calculated using the formula PE = (C-T/C) × 100%, where C and T are the mean numbers of mosquitoes that landed on the control and test arm, respectively ( Sharm and Ansari 1994 ; Matsuda et al 1997). Means were subjected to analysis of variance (ANOVA) and compared by the Student-Newman-Keuls test ( SAS 2000 ). Probit to compute repellency concentration that caused 50% response of the test mosquitoes (RC50) was done using the Lackfit inversel of the SAS programme ( SAS 2000 ). | Results and Discussion
Chemical composition of essential oil from S. zanzibariensis
The yield of essential oil from the dried leaves of S. zanzibariensis was 0.004%. Table 1 shows the composition of the essential oil and the compounds (representing ∼80% of all constituents of the essential oil) that were identified. Phenylacetaldehyde (5) (14.4%), artemisia ketone (11) (10.1%), (1 S )-(-)-verbenone (17) (12.1%), and geranyl acetone (24) (9.4%) were the main constituents. Ketone compounds accounted for 56.5% of all identified components. Methyl ketone terpenes included 6-methyl-5-hepten-2-one (4), cis -6-methyl-3,5-heptadien-2-one (8), α-ionone (23), geranyl acetone (24), β-ionone (27), pseudoionone (29), 6, 10, 14-trimethyl pentadecan-2-one (31) and farnesyl acetone (32) ( Figure 1 ).
Repellency activity of S. zanzibariensis essential oil, compounds and blends
Among the compounds tested in this study, methyl ketone terpenes exhibited good repellency against An. gambiae s.s. ( Table 2 ), which is consistent with previous studies that reported that aliphatic methyl ketones exhibit good mosquito repellency activities ( Barton 2003 ; Roe 2004 ; Innocent 2008 ). Repellency activity of geranyl acetone, β-ionone, and farnesyl acetone ( Table 2 ) compared well with other plant-based mosquito repellents (Dethier 1947; Grayson 2000). Repellency activity of β-ionone (27) was higher than its acyclic counterpart geranyl acetone (24). Likewise, farnesyl acetone (32) was more repellent than geranyl acetone (24). 6-Methyl-5-hepten-2-one (4), which has been identified as a defensive allomone of many insects such as ants, termites, and cockroaches ( Blum 1996 ), was not commercially available and could not be assayed in this study. Interestingly, 6-Methyl-5-hepten-2-one (4) and geranyl acetone (24) previously identified in the headspace of fresh and incubated sweats of humans, were shown to elicit response to the neurons innervating a grooved peg sensillum of a female An. gambiae , and were electro-attennogram active ( Meijerink et al. 2000 , 2001 ).
The repellent activity of constituents in the oil was dose dependent (p < 0.05). At concentration of 1% w/v, the blend of the four major constituents in the proportion found in S. zanzibariensis essential oil was higher than that of the crude essential oil (p < 0.05) indicating additive or synergistic effects of the compounds. The presence of artemisia ketone (11) caused a significant increase in the activity of the blends ( Figure 2 ). Although none of the assayed constituents exhibited repellency comparable to that of DEET, the repellency of some of the blends at 1% w/v was comparable to that of this synthetic repellent, suggesting that further screening of different blends may lead to the discovery of more repellent combinations. | Results and Discussion
Chemical composition of essential oil from S. zanzibariensis
The yield of essential oil from the dried leaves of S. zanzibariensis was 0.004%. Table 1 shows the composition of the essential oil and the compounds (representing ∼80% of all constituents of the essential oil) that were identified. Phenylacetaldehyde (5) (14.4%), artemisia ketone (11) (10.1%), (1 S )-(-)-verbenone (17) (12.1%), and geranyl acetone (24) (9.4%) were the main constituents. Ketone compounds accounted for 56.5% of all identified components. Methyl ketone terpenes included 6-methyl-5-hepten-2-one (4), cis -6-methyl-3,5-heptadien-2-one (8), α-ionone (23), geranyl acetone (24), β-ionone (27), pseudoionone (29), 6, 10, 14-trimethyl pentadecan-2-one (31) and farnesyl acetone (32) ( Figure 1 ).
Repellency activity of S. zanzibariensis essential oil, compounds and blends
Among the compounds tested in this study, methyl ketone terpenes exhibited good repellency against An. gambiae s.s. ( Table 2 ), which is consistent with previous studies that reported that aliphatic methyl ketones exhibit good mosquito repellency activities ( Barton 2003 ; Roe 2004 ; Innocent 2008 ). Repellency activity of geranyl acetone, β-ionone, and farnesyl acetone ( Table 2 ) compared well with other plant-based mosquito repellents (Dethier 1947; Grayson 2000). Repellency activity of β-ionone (27) was higher than its acyclic counterpart geranyl acetone (24). Likewise, farnesyl acetone (32) was more repellent than geranyl acetone (24). 6-Methyl-5-hepten-2-one (4), which has been identified as a defensive allomone of many insects such as ants, termites, and cockroaches ( Blum 1996 ), was not commercially available and could not be assayed in this study. Interestingly, 6-Methyl-5-hepten-2-one (4) and geranyl acetone (24) previously identified in the headspace of fresh and incubated sweats of humans, were shown to elicit response to the neurons innervating a grooved peg sensillum of a female An. gambiae , and were electro-attennogram active ( Meijerink et al. 2000 , 2001 ).
The repellent activity of constituents in the oil was dose dependent (p < 0.05). At concentration of 1% w/v, the blend of the four major constituents in the proportion found in S. zanzibariensis essential oil was higher than that of the crude essential oil (p < 0.05) indicating additive or synergistic effects of the compounds. The presence of artemisia ketone (11) caused a significant increase in the activity of the blends ( Figure 2 ). Although none of the assayed constituents exhibited repellency comparable to that of DEET, the repellency of some of the blends at 1% w/v was comparable to that of this synthetic repellent, suggesting that further screening of different blends may lead to the discovery of more repellent combinations. | Associate Editor: Sonny Ramaswamy was editor of this paper.
In traditional African communities, repellent volatiles from certain plants generated by direct burning or by thermal expulsion have played an important role in protecting households against vectors of malaria and other diseases. Previous research on volatile constituents of plants has shown that some are good sources of potent mosquito repellents. In this bioprospecting initiative, the essential oil of leaves of the tree, Suregada zanzibariensis Verdc. (Angiospermae: Euphobiaceae) was tested against the mosquito, Anopheles gambiae s.s. Giles (Diptera: Culicidae) and found to be repellent. Gas chromatography (GC), GC-linked mass spectrometry (GC-MS) and, where possible, GC-co-injections with authentic compounds, led to the identification of about 34 compounds in the essential oil. About 56% of the constituents were terpenoid ketones, mostly methyl ketones. Phenylacetaldehyde (14.4%), artemisia ketone (10.1%), (1 S )-(-)-verbenone (12.1%) and geranyl acetone (9.4%) were the main constituents. Apart from phenylacetaldehyde, repellent activities of the other main constituents were higher than that of the essential oil. The blends of the main constituents in proportions found in the essential oil were more repellent to An. gambiae s.s. than was the parent oil (p < 0.05), and the presence of artemisia ketone in the blend caused a significant increase in the repellency of the resulting blend. These results suggested that blends of some terpenoid ketones can serve as effective An. gambiae s.s. mosquito repellents.
Keywords | Acknowledgements
This study was funded through a grant from the WHO Special Programme for Research and Training in Tropical Diseases (Grant No. U19A145511-01). EI is grateful to the Singenberg Foundation for a research grant through ICIPE. We thank Mr. FM Mbago from the Herbarium of the Botany Department at the University of Dar-es-Salaam, Tanzania, for the identification of the investigated plant species and Mr. PM Njeru of ICIPE for technical support.
Abbreviations
gas chromatography;
mass spectrometry; | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Jun 9; 10:57 | oa_package/56/df/PMC3014761.tar.gz |
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PMC3014762 | 20578954 | Introduction
The grape berry moth Lobesia botrana Denis and Schiffermüller (Lepidoptera: Tortricidae), is a major pest of European and Mediterranean vineyards. The yield reductions caused by this insect are due to both damage done by the larvae and further attack by fungi. Most growers control this pest with traditional chemical pesticides, however, mating disruption and microbiological insecticides are used as alternatives in a few areas ( Coscollá 1997 ). Considerable effort is being directed toward reduced use of traditional pesticides and increased use of integrated pest management techniques, emphasizing the joint use of natural enemies and selective pesticides, an alternative compatible with the protection of non-target organisms and the environment. Azadirachtin, a tetranotriterpenoid, is the most active insecticidal compound found in the seeds and leaves of the neem tree, Azadirachta indica A. Juss (Sapindales: Meliaceae) ( Koul et al. 1990 ; Schmutterer 1990 ). This compound exerts several biological effects on insects (e.g. antifeedant, insect growth regulator and repellent) and has low mammalian toxicity ( Schmutterer 1990 ; Ascher 1993 ; Mordue and Blackwell 1993 ; Mordue et al. 1998 ; Mulla and Su 1999 ; Koul and Wahab 2004 ; Isman 2006 ). Pesticides containing azadirachtin are reported to provide broad-spectrum control of more than 200 species of insect pests ( Ascher 1993 ) and to be less toxic to natural enemies of insect pests compared with synthetic, chemical pesticides ( Hoelmer et al. 1990 ; Schmutterer 1997 ). Although the safety of azadirachtin for numerous beneficial insects has been recently questioned ( Spollen and Isman 1996 ), applications of formulated azadirachtin had no undesired effects on important predators such coccinellids ( Banken and Stark 1998 ), lacewings ( Medina et al. 2004 ) and Phytoseiid mites ( Castagnoli et al. 2005 ; Brito et al. 2006 ). Thus, pesticides derived from the neem tree seem to be promising for use in integrated pest management programs to control various pest species ( Isman 2006 ).
Grape growers use large amounts of traditional chemical pesticides, and, therefore, the development of strategies that minimize pesticide use is needed. According to the European Directive 98/8/EC, “proper pesticide use includes application at an efficacious concentration and minimization of use of biocidal products where possible.” To ensure sufficient management of pests, registered pesticides are normally applied at rates above the upper asymptote of the concentration/response curve. Thus, in theory, it should be possible to achieve control of insect pests and, at the same time, reduce the effects on non-target insects by using dosage rates below these maxima, with the species-specific degree of selectivity strongly influenced by the applied concentration ( Poehling 1989 ). Published studies of dose reduction from recommended field rates demonstrated adequate levels of control and improved control exerted by natural enemies without sacrificing yield or inflating cost ( Smith et al. 1985 ; Poehling 1989 ; Acheampong and Stark 2004 ).
In the only published study of azadirachtin on L . botrana , Ioriarti et al. ( 1992 ) obtained 30% mortality on 3-day-old eggs and 52.6% mortality on larvae, using doses of 500 – 600 mg litre -1 . Studies are needed to determine the most efficient usage against this pest. Herein the effects of azadirachtin on adult fecundity, fertility and longevity are described. This study also reports the effects of azadirachtin on the development and mortality of L. botrana eggs and larvae. Finally, the possibility of incorporating azadirachtin into programs for management of the European grape berry moth are discussed. | Materials and methods
Insects
A stock culture of L. botrana was established from larvae collected in an organic vineyard in La Rioja, Spain during May 2000, and the culture was augmented with new individuals once each year. The insects were maintained in a growth chamber at 24±1°C, 60±10% RH and 16:8 L:D photoperiod, following the method described by Del Tío ( 1996 ) and modified by Sáenz-de-Cabezón ( 2003 ). All bioassays were conducted under these uniform conditions.
Chemicals
Formulated azadirachtin (ALIGN® EC, 32 g. azadirachtin litre -1 ) was obtained in Spain from Sipcam Inagra.
Effect on adult fecundity, fertility and longevity
To assess the activity of azadirachtin on L. botrana adults, three pairs of recently emerged adults were introduced into a 33 ml plastic container placed upside-down on the base of a 90 mm diameter Petri dish. Azadirachtin was administered orally via a water trough that contained 1, 5, and 10 mg litre -1 of azadirachtin mixed in a 10% honey solution. Water troughs were changed every 3 days to avoid fungal proliferation. Every day, the glasses with the laid eggs were replaced, and the eggs were then transferred to plastic boxes (12 cm diameter, 5 cm high) until emergence. Fecundity and fertility were counted daily during the oviposition period. Mortality was recorded during the entire life period. Five replicates were used for both the treatment and the control (10% honey solution). Embryonic development was followed using a stereoscopic lupe (Olympus SZH-10).
Ovicidal bioassays
To test topical ovicidal effects, ten pairs of L. botrana adults were introduced into an oviposition chamber consisting of a cylindrical plastic body (9 cm diameter, 13 cm high) covered inside with a transparent plastic film on which females could lay their eggs. The bottom was covered with a Petri dish, and the top was covered with filter paper. Moths were provided with water. Eggs laid during the next 24h were removed and then treated using a manually loaded Potter spray tower, with 5.5 ml in the tank at 20 kPa pressure producing a deposit of 0.05±0.01 ml cm-2. The concentrations that were used were obtained by preliminary assays ranging from 1 to 768 mg litre -1 . Eggs of different age classes (0–24, 24–48, 48–72 and 72–96 h old) were collected and treated in the same manner. Treatments were made on each of the age groups. The concentration used was the LC 50 obtained in the previous assay. Egg hatch was recorded 5 days after treatment. There were five replicates per concentration/treatment, in addition to the water treated control.
Ovicidal effects of Align were also examined using a contact bioassay. Surfaces were treated by dipping four plastic glasses in a 1:1 solution of LC 50 contact concentration, and then air-drying for 5 min. Five pairs of adults were introduced and allowed to lay eggs for 24 h. Larval emergence from eggs was checked at the sixth day of treatment. Four replicates were used for the treatment and the control, which was dipped in distilled water.
Larvicidal bioassays
To determine the effect of Align on the 1st, 3rd and 5th instars, ten larvae were introduced to a Petri dish (5 cm diameter) containing treated diet at concentrations ranging from 0.75 to 3.8 mg litre -1 for 1 st instars and 8.0 to 32.0 mg litre -1 for 3rd instars. The concentrations used were established by preliminary assays. 5th instars were not treated because of delayed effects in development at ≥ 30 mg litre -1 . Five replicates were used for each dose and control. The mortality (larvae that failed to molt) was measured when control larvae reached the next stadium (i.e., the 4th and 3rd day after treatment for 1st and 3rd instars, respectively). Sublethal effects of Align exposure were measured during the larval stage by providing 1st instar larvae ad libitum diet containing low concentrations of Align (i.e. 0.15, 0.3, 0.6, 1.2 mg litre -1 ). Mortality and adult emergence were checked daily. Five replicates, of 10 individuals each, were used for each concentration and the control.
Statistical analysis
Estimates of LC 50 and LC 90 for different stages and their 95% fiducial limits were obtained using the POLO program ( Russell et al. 1977 ) based on Finney ( 1971 ). The criterion used to estimate the differences between LC ratios was non-overlap of their 95%) confidence intervals ( Robertson and Presley 1992 ). The significance of results (fecundity, fertility, egg mortality, and larval mortality) were tested by ANOVA, and means were separated by an LSD multiple range test (p < 0.05). Percentage values were arcsin transformed, and, in all cases, untransformed means are presented. Abbott's formula was used to correct mortalities ( Abbott 1925 ). Percentage of sterility was calculated using the formula of Toppozada et al. ( 1966 ): where F t = fecundity of treated females; Fe t = fertility of treated females; F c = control fecundity; and Fe c = control fertility | Results
Effects on adults
L. botrana adults fed Align in a honey solution showed decreased fecundity and fertility at each concentration tested ( Table 1 ). Total eggs laid per female were significantly (p < 0.05) less than the control, and fertility was significantly (p < 0.05) decreased in L. botrana adults. In addition, eggs laid by Align-treated females showed the same toxic symptoms on the embryo as spray-treated eggs (see below). Longevity was not affected by the treatment of any concentration of Align.
Effects on eggs
The parameters obtained for the probit-log concentration regression line on eggs treated with azadirachtin and less than 24 h are given in Table 2 . Contact treatment was significantly (p < 0.05) more effective than surface treatment (with 41.4 ± 4.3 and 30.0 ± 7.1 corrected mortalities, respectively). Treatment with Align significantly (p < 0.05) reduced hatching for all egg classes but the last (72–96 h old). The reduction was greater for eggs less than 24 h old than for other egg classes ( Table 3 ). High concentrations of Align (768 mg litre -1 ) interrupted the normal development of the embryo. Treated eggs reached the final black-head stage but had mandibular and cephalic deformities.
Effects on larvae
Table 2 shows probit-log concentration regression line parameters obtained for the different stages tested. Repellent effects of Align were evident during the course of preliminary assays, when death due to starvation was evident in feeding experiments with concentrations higher than 12 mg litre -1 . Younger larvae were more susceptible than older larvae. Effects on larval development were observed and included the inability to shed the old cuticle and cephalic capsule. In addition, larvae that successfully molted exhibited mouthpart deformities, integument injuries and malformations. Treatment of 5th instars with concentrations higher than 32 mg litre -1 of Align caused larvae to prematurely enter a wandering state and molt the pupal stage. Hardening the cuticle sclerites and other abnormalities, such as larval-pupal intermediates, were also observed. When treated with reduced concentrations of Align, there was a high percentage of larval mortality in the 5th stadium for all concentrations tested except 0.15 mg litre -1 . Mortality on the next stadia reached >85% in all concentrations but 0.15 mg litre -1 . Almost 100% of the individuals died without reaching adulthood at concentrations above 0.15 mg litre -1 . The lowest concentration reduced adult elosion up to 50%. Mortality occurred mostly during the last larval stage ( Figure 1 ). | Discussion
Align®, a commercial formulation of azadirachtin, has larvicidal and ovicidal activity and significantly affected the reproduction of L. botrana. At low concentrations total adult eclosion was reduced to 0%.
In a previous study with this insect, Iorriati et al. ( 1992 ) measured mortalities of 30% for 3-day-old eggs and 52.6% for larvae using concentrations between 500 and 600 mg litre -1 . In the present study the effects of Align on eggs, larvae and adult L. botrana have been assessed, emphasizing the time and mode of treatment.
Effects on adult fertility, fecundity, and longevity
Align strongly affected the reproduction of L . botrana by producing a high percentage of sterility at the concentrations tested. Fecundity was severely reduced, and no eggs were laid at the highest concentrations tested. Lower concentrations significantly reduced the fertility of L. botrana. Eggs laid by treated females showed embriocidal effects, which suggest pesticide transference through the gravid female. It has been demonstrated that adult exposure to azadirachtin by topical, surface or ingestion treatments, reduces fecundity and/or fertility among different insect orders, including Lepidoptera ( Schmutterer 1990 ; Ascher 1993 ; Mordue and Blackwell 1993 ; Mordue 2004 ; Seljansen and Meadow 2006 ). It is known that azadirachtin causes profound effects on reproductive processes of both male and female insects. Studies showed that insects treated with azadirachtin have degenerate ovaries and a high degree of yolk resorption ( Rembold and Seiber 1981 ; Koul 1984 ; Dorn et al. 1987 ; Schlüter 1987 ; Medina et al. 2004 ). Azadirachtin also interferes with the synthesis of vitellogenin by the fat body and its uptake by the eggs, resulting in reduced fecundity and sterility. Such effects could be due to the disruption of juvenile hormone levels and ovarian ecdysteroid production ( Rembold and Seiber 1981 ; Feder et al. 1988 ; Tanzubil and McCaffery 1990 ).
Ovicidal effects
Align was not a potent ovicide of L. botrana eggs (LC 50 of 231.5 mg litre -1 for eggs < 24 h). Ovicidal activity was also affected by age and treatment. Younger eggs were more susceptible than older ones, and surface treatment was less effective than spray treatment. These findings are in agreement with those from previous studies showing that azadirachtin has low ovicidal activity on L. botrana ( Iorriati et al. 1992 ) and on insects of different orders ( Schmutterer 1990 ; Ascher 1993 ). The results from the present study differ from previous studies ( Ioriartti et al. 1992 ) that obtained a greater susceptibility on 3-day-old eggs.
Larvicidal effects
L. botrana larvae were highly susceptible to diet containing Align, with younger stages being more susceptible than older ones. These results agree with those from previous studies ( Mordue and Blackwell 1993 ) of other lepidopteran insects showing larvicidal activity inversely proportional to larval age. In addition to increased mortality, Align acted as an insect growth regulator and an antifeedant: 1st instars did not fed when concentrations were higher than 12 mg litre -1 (data not shown) ( Scmutterer 1990 ; Ascher 1993 ; Mordue and Blackwell 1993 ). Also, 5th instars displayed premature wandering and cessation of feeding, possibly due to Align's effects on release of prothoracicotropic hormone. As described for other species, an increase in the levels of prothoracicotropic hormone is necessary to begin the transformation into pupa. Without this increase, the larvae remain in a state of wandering and die ( Schmutterer 1990 ; Mordue 2004 ). In a study on Tripanosoma cruzi , the complete elimination of azadirachtin did not occur until 50 days after the treatment. Thus, this compound can interrupt the insect development for a long time ( González and Garcia 1992 ). Low concentrations of Align resulted in elevated mortality during the last larval stage, probably as a result of compound accumulation reaching concentrations that have activity in older instars.
Integrated Pest Management
The main impediment for the implementation of integrated pest management is to provide growers with a convincing reason to adopt its tactics, such as economic incentives (Reitz et al. 1999). Integrated pest management often increases net profits for growers who adopt it; however, many growers still hold the perception that it does not offer short-term economic advantages compared with conventional control. However, in a previous study ( Sáenz-de-Cabezón 2003 ), Align was as effective as the conventional organophosphate fenitrothion, even when applied at a concentration (50 cc/hl) lower than that recommended on the label (125 cc/hl; data not shown). Thus, proper pesticide-use tactics reduce the cost of pesticide use and further the implementation of integrated pest management programs.
More laboratory, small plot and field testing is required before the insecticidal potential of azadarachtin against L. botrana will be fully understood. The activity observed in this study suggests that the combination of the acutely toxic and reduced concentrations could lead to the incorporation of this compound in integrated pest management programs against the grape berry moth. Sprays for the second and third generations should be applied at the beginning of flight, to cover the oviposition period and egg hatching. Resistance studies are needed in order to know how azadarachtin resistance works in L. botrana populations using different levels of selection pressure. | Associate Editor: James Ottea was editor of this paper
Azadirachtin, derived from the neem tree, Azadirachta indica A. Juss (Sapindales: Meliaceae), seems promising for use in integrated pest management programs to control a variety of pest species. A commercial formulation of azadirachtin, Align®, has been evaluated against different developmental stages of the European grape berry moth, Lobesia botrana Denis and Schiffermüller (Lepidoptera: Tortricidae). When administered orally, Align reduced the fecundity and fertility of adults treated with 1, 5, and 10 mg litre -1 . At the highest doses, fecundity and fertility were zero, but longevity was not affected. An LC 50 of 231.5 mg litre -1 was obtained when Align was sprayed on eggs less than 1 day old. Hatching of all egg classes was significantly reduced, and this reduction was more pronounced for eggs less than 24 h old. LC 50 values of 2.1 mg litre -1 for first instars and 18.7 mg litre -1 for third instars were obtained when Align was present in the diet. Larvae reared on a diet containing different concentrations of Align did not molt into adults at the highest concentrations (0.3, 0.6, 1.2), and 50% molted at the lowest concentration (0.15). Phenotypic effects included inability to molt properly and deformities. The combination of acute toxicity and low, effective concentrations of Align observed in this study could lead to the inclusion of insecticides containing azadirachtin in integrated management programs against this pest.
Keywords | Acknowledgements
The authors thank the Comisión Interministerial de Ciencia y Tecnología (CICYT) and Fondo Europeo de Desarrollo Regional (FEDER) for funding this research. We also thank Christina Pease for reviewing the English version of the manuscript. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 10; 10:33 | oa_package/57/55/PMC3014762.tar.gz |
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PMC3014772 | 20578951 | Introduction
Many viruses express glycoproteins in the form of an inactive precursor and then utilize furin, a host-cell enzyme,to cleave the precursor into its mature functional form ( Garten et al. 1994 ). Furin localizes primarily to the trans-Golgi network and the plasma membrane; thus, viral proproteins are cleaved by furin during their transit through the trans-Golgi or after arrival at the cell surface ( Thomas 2002 ). Furin cleaves proproteins at a site immediately following four residues ordered in a basic-X-basic-basic (bxbb) motif ( Hosaka et al. 1991 ). Although the bxbb motif represents the core furin cleavage signal, the residue immediately adjacent to this motif (+1 position) influences furin-mediated cleavage of a range of viral proprotein substrates in vertebrate cells ( Toyoda et al. 1987 ; Morrison et al. 1993 ; Heidner and Johnston 1994 ; Horimoto and Kawaoka 1995 ; Fujii et al. 1999 ). Specifically, cleavage of the viral proproteins is strongly inhibited in vertebrate cells when residues at the +1 position contain branched aliphatic side chains (isoleucine, leucine, valine). Furin-like enzymes produced by insect cells also recognize the consensus bxbb motif ( De Bie et al. 1995 ; Chen et al. 1996 ; Cieplik et al. 1998 ). It is not known if residues at the +1 position influence insectderived furin enzymes' cleavage of proproteins.
Alphaviruses are relatively simple, enveloped, positive strand RNA viruses that belong to the Togaviridae virus family. Alphaviruses have a global distribution and many members of this genus cause significant disease in humans, ranging from fever, arthralgia and rash, to lethal encephalitis ( Griffin DA 2001 ). Sindbis virus (Group IV: Togaviridae: alphavirus) is the prototype member of the genus, and its structural and biological properties have been studied extensively (for review: Strauss and Strauss 1994 ). The natural alphavirus maintenance cycle involves alternating infections of vertebrate and arthropod hosts with mosquitoes serving as the most common insect vector. The alphavirus glycoproteins are synthesized as components of a polyprotein that is cleaved into proteins designated as PE2, 6K, and E1 by the host cell signal peptidase enzyme in the rough endoplasmic reticulum ( Strauss and Strauss 1994 ). PE2 and El proteins associate into PE2/E1 heterodimers and then are processed and transported through the exocytic pathway of the host cell ( Erwin and Brown 1980 ; Rice and Strauss 1982 ). The PE2 protein is cleaved by a host cell endoprotease as the heterodimers are transported through a trans or post-Golgi compartment ( Jones et al. 1974 ; De Curtis and Simmons 1988 ). The major product of PE2 cleavage is the E2 glycoprotein which, together with E1, forms the glycoprotein spikes that project from the surface of mature virions. Cleavage of PE2 occurs immediately downstream of a bxbb motif ( Rice and Strauss 1981 ). Furin has been shown to mediate PE2 cleavage during alphavirus replication in vertebrate cells ( Watson et al. 1991 ; Moehring et al. 1993 ; Zhang et al. 2003 ; Ozden et al. 2008 ). The cellular enzyme that cleaves PE2 in mosquito cells has not been identified but is likely to be an arthropod homolog of the vertebrate furin enzyme, as mutations that block access to the bxbb sequence and restrict PE2 cleavage in vertebrate cells also restrict PE2 cleavage and virus replication in mosquito cells ( Presley et al. 1991 ; Heidner et al. 1996 ). In addition, deletion of the bxbb sequence prevents PE2 cleavage in vertebrate cells and restricts virus replication in cultured mosquito cells and within living mosquitoes ( Davis et al. 1995 ; Turell et al. 1999 ).
Cleavage of PE2 in vertebrate cells is profoundly influenced by the residue immediately following the bxbb motif ( Heidner and Johnston 1994 ). Specifically, cleavage of PE2 proteins containing isoleucine, valine, or leucine was greatly reduced compared to PE2 substrates with other amino acids at the +1 position. Interestingly, when Sindbis virus variants containing valine or leucine were grown in cultured mosquito cells, PE2 was cleaved efficiently and the virus replicated with normal kinetics ( Heidner et al. 1996 ). The distinct PE2 cleavage phenotypes in vertebrate and mosquito cells could result from differences in the substrate specificities of the vertebrate and arthropod furin enzymes. Alternatively, they could result from differences in the carbohydrate-processing phenotypes of arthropod and vertebrate cells, which could differentially influence access of the furin enzyme to the PE2 cleavage site. Specifically, the N-linked oligosaccharides synthesized in vertebrate cells typically consist of complex or hybrid structures ( Kornfeld and Kornfeld 1985 ). In contrast, Nlinked oligosaccharides synthesized in insect cells are typically restricted to low-mannose and high mannose forms ( März et al. 1995 ). Consequently, the structures of N-linked oligosaccharides on alphavirus glycoproteins differ markedly depending on the host cell (vertebrate vs. arthropod) used to propagate virus, and these host-specific differences can have a profound influence on the biological properties of the virus ( Hsieh et al. 1983 ; Boehme et al. 2000a; Boehme et al. 2000b; Klimstra et al. 2003 ). The objective of this study was to differentiate between these two alternatives.
Virus replication and PE2 cleavage phenotypes were compared in a Chinese hamster ovary cell line (CHO-K1) and in a CHO-K1-derived furin-negative cell line (RPE-40) engineered to express the Dfurin1 enzyme from Drosophila melanogaster Meigen (Diptera: Drosophilidae). The use of Dfurin1 was based on the evolutionary relatedness of mosquitoes (Diptera: Culicidae) and D. melanogaster and on the likelihood that the furin enzymes derived from both flies share similar genetic and functional properties. Dfurin1 is expressed abundantly in adult flies and correctly processes substrates containing the consensus sequence for mammalian furin enzymes ( Roebroek et al. 1993 ; De Bie et al. 1995 ). This cell culture system made it possible to evaluate and compare the substrate specificities of representative vertebrate and arthropod furin enzymes under conditions where the glycosylation properties of the PE2 substrate remained constant. | Materials and Methods
Viruses and cells
The parental Sindbis virus (strain AR339), TRSB, has been described previously ( McKnight et al. 1996 ). The genetic and phenotypic properties of the TRSB-derived mutant viruses, TRSB-NE2G216, TRSB-E2S1, TRSB-E2L1, TRSB-E2V1, TRSB-E2F1, TRSB-E2N1, TRSB-E2D1, and TRSB-E2H1 also have been described ( Heidner and Johnston 1994 ; Heidner et al. 1996 ). In this report, the mutant viruses are referred to as NE2G216, E2S1, E2L1, E2V1, E2F1, E2N1, E2D1, andE2H1, respectively.
BHK-21 cells were obtained from the American Type Culture Collection. The CHOK1 and RPE.40 cell lines have been described ( Moehring and Moehring 1983 ; Spence et al. 1995 ). BHK-21, CHO-K1 and RPE.40 cells were maintained at 37° C in alpha minimum essential medium supplemented with 10% donor calf serum, 10% tryptose phosphate broth, and antibiotics. C6/36 cells were originally derived from Aedes albopictus Skuse (Diptera: Culicidae) larvae ( Igarashi 1978 ). C6/36 cells were maintained at 28° C in alpha minimal essential medium supplemented with 10% fetal calf serum, 10% tryptose phosphate broth, and antibiotics.
Plasmid constructions
Construction of a vector for stable expression of Dfurin1 in eukaryotic cells required the use of several shuttle vectors. The cDNA sequences of Dfurin1 were derived from a phagemid designated pIP63 ( Roebroek et al. 1993 ). First, the entire Dfurin1 sequence was amplified by PCR using pIP63 DNA as template and oligonucleotide primers that incorporated an XbaI restriction site (5′) and an ApaI restriction site (3′). The amplicon product was digested with XbaI and ApaI and ligated into a plasmid called SINrep5 ( Bredenbeek et al. 1993 ) from which a corresponding XbaI/ApaI fragment had been removed. The resulting construct was designated pSIN-Dfur1. Second, the 549 5′ terminal base pairs of the Dfurin1 gene were amplified by PCR using pIP63 DNA as template and oligonucleotide primers that incorporated an XbaI restriction site and a consensus Kozak translation initiation sequence (5′), and which flanked a unique BamHI restriction site at nucleotide 549 of the Dfurin1 coding sequence (3′). The PCR product was digested with XbaI and BamHI and ligated into a plasmid designated pH3′2J1 ( Hahn et al. 1992 ) from which a corresponding Xbai/BamHI fragment had been removed, to produce pH3/Dfur5′. Third, the remaining Dfurin1 sequences were transferred from pSIN-Dfur1 by subcloning of a BamHI/XhoI fragment into pH3/Dfur5′ to produce pH3/Dfur1. Finally, the entire Dfurin1 coding sequence was subcloned from pH3/Dfur1 into the eukaryotic expression vector pcDNA3.1 (Invitrogen, www.invitrogen.com ) by transfer of a XbaI/NotI fragment. This transfer placed the Dfurin1 coding sequences under transcriptional control of the human cytomegalovirus immediate early promoter. The resulting construct, pc3.1/Dfur1 was sequenced across the entire Dfurin1 region to confirm the sequence of the DNA insert.
Generation of stably transformed RPE.40 cells
Plasmid DNAs were purified by use of the Wizard Purefection Kit (Promega, www.promega.com ). RPE.40 cells were transformed with the pcDNA3.1 control plasmid or with the pc3.1/Dfur1 plasmid using the Calcium Phosphate Transfection Kit (Invitrogen). pcDNA3.1-based plasmids confer neomycin resistance, thus, cultures of stably transformed cells were selected in the presence of G418 sulfate (400 μg/ml) over a two week period. Cells were plated onto 150 mm culture dishes at a low density and individual cells were isolated using sterile cloning rings. Isolated cells were then expanded into clonal cell lines under constant G418 sulfate selection. In all subsequent experiments using transformed RPE.40 cell lines, G418 sulfate was included in the growth medium at a concentration of 400 μg/ml.
Indirect immunofluorescence staining of cells
CHO-K1, RPE.40, and transformed RPE.40 cell lines were grown to approximately 50% confluence in 8-well chamber slides. Cells were fixed in 4% paraformaldehyde (in PBS), and permeabilized with 0.1% Triton X-100 (in PBS). Cells were blocked with 10% bovine serum albumin (in PBS) and then probed for 1 hour with a non-immune rabbit serum or with a polyclonal rabbit antiserum raised against a Dfurin1/glutathione S-transferase fusion protein diluted in 25% donor calf serum (in PBS). Cells were washed extensively with 10 mM glycine/.05% Tween-20 in PBS and then probed with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich, www.sigmaaldrich.com ). Cells were incubated for 1 hour, washed with 10 mM glycine/0.05% Tween-20 in PBS. Cells were then treated with 4′,6-diamidino-2-phenylindole (DAPI) (10 μ g/mL, Sigma-Aldrich) to stain nuclei and then analyzed by fluorescence microscopy (Axioskop, Carl Zeiss, www.zeiss.com ).
Kinetics of viral growth in transformed and non-transformed cell lines
The kinetics of virus growth was determined for various viruses in CHO-K1, RPE.40, and Dfurin1-transformed RPE.40 cell lines. Infections were performed on duplicate monolayers of cells grown in 60 mm Petri dishes (2 × 10 6 cells/dish) and were initiated by infection with free virus at a multiplicity of infection of 10 plaque forming units (pfu) per cell. Virus was adsorbed to cells for 30 minutes, and, then, remaining virions were removed by repeated washes. Cells were overlaid with medium and maintained at 37° C. Supernatant samples were collected at regular intervals post-infection, clarified by microcentrifugation, and stored at -70° C. Infectious virus in each sample was quantified by plaque assay on BHK-21 cells. Virus titers are reported here as the average of the duplicate samples.
Polyacrylamide gel analysis of [ 35 S]methionine-labeled viral proteins
Virions were metabolically radiolabeled with [ 35 S]-methionine during growth in CHO-K1, RPE.40, transformed RPE.40 cells lines, and C6/36 cells as described ( Heidner et al. 1994 ). Radiolabelled virions were purified from cell supernatants by isolation on discontinuous potassium tartrate gradients (20% / 35%) followed by banding on continuous potassium tartrate gradients (20% to 35%). Potassium tartrate solutions were made in TNE buffer (0.5 M Tris-HCl (pH 7.2), 0.1 M NaCl, and 0.001 M EDTA). Banded virions were collected and pelleted through sucrose cushions (20% in TNE) by ultracentrifugation. Due to the instability of PE2-containing virions derived from RPE.40 cells, viruses were purified by a simple pelleting technique in which infected cell supernatants were clarified of cell debris by high speed spin, passed through a .45 micron filter, and then pelleted by ultracentrifugation. The virus pellet was then washed once with TNE buffer, re-pelleted by ultracentrifugation, and harvested. Radiolabelled virion preparations were quantified by liquid scintillation counting and equal quantities of each were resolved by SDS-PAGE (10% acrylamide) prepared as described ( Laemmli 1970 ).
Comparison of Dfurin1 with mosquito-derived proprotein convertase enzymes
To identify furin homologs in the Aedes aegypti L.(Diptera: Culicidae) mosquito, a BLASTP search was performed with the default parameter setting, using the protein sequence of D. melanogaster Dfur1 (accession number AAA28549) as a query sequence against the VectorBase ( http://www.vectorbase.org/index.php ). Conserved domains/motifs were identified by searching the Pfam protein family database ( Finn et al. 2008 ). Multiple alignments were generated using the T-coffee program ( Notredame et al. 2000 ), followed by manual inspection and editing. | Results
Generation of RPE.40 cells stably expressing Dfurin1
RPE.40 cells were permissive for Sindbis virus replication but failed to cleave PE2 due to genetic mutations within both furin alleles ( Watson et al. 1991 ; Spence et al. 1995 ). These phenotypes were confirmed by comparing the PE2 cleavage phenotypes of two Sindbis viruses, E2S1 and NE2G216, during growth in CHO-K1 and RPE.40 cells. E2S1 was used in place of TRSB because it encodes a PE2 glycoprotein with an optimal cleavage site for the furin enzyme expressed in cultured hamster cells ( Heidner and Johnston 1994 ; Klimstra et al. 1999 ). As predicted, E2S1 virions derived from CHOK1 cells contained E2 and E1 glycoproteins, and virions derived from RPE.40 cells contained uncleaved PE2 and E1 ( Figure 1 ). NE2G216 is defective for PE2 cleavage in all cell types due to the placement of an N-linked oligosaccharide adjacent to the furin cleavage site ( Heidner et al. 1994 ), and NE2G216 virions retained PE2 in place of E2 when grown in both cell types ( Figure 1 ).
RPE.40 cells were transfected with pc3.1/Dfur1 plasmid DNA or with the control plasmid pcDNA3.1, and stably transformed cells were isolated under G418 sulfate selection. Individual cells from the pc3.1/Dfur1 transfection were expanded into clonal cell lines (R-Dfur1); however, cells transformed with the control plasmid (R-3.1) were not cloned further. Based on the results obtained from pilot virus growth assays, cell lines R-Dfur1#11 and R-Dfur1#22 were selected for further study. To confirm that these cells expressed the Dfurin1 enzyme, CHO-K1, RPE.40, R-Dfur1#11 and RDfur1#22 cell lines were probed with nonimmune rabbit serum or with a polyclonal rabbit antiserum raised against a Dfurin1/glutathione S-transferase fusion protein in an indirect immunofluorescence assay. Staining was not observed in any cell line probed with the non-immune antiserum or in RPE.40 and CHO-K1 cells probed with the Dfurin1-specific antiserum (data not shown). In contrast, bright staining was observed in RDfur1#11 and R-Dfur1#22 cells probed with the Dfurin1-specific antiserum, but not in the parental RPE.40 cell line ( Figure 2 ). Staining localized to the perinuclear region which is consistent with the Golgi-specific localization that is predicted for the Dfurin1 enzyme.
The effects of Dfurin1-expression on Sindbis virus replication
The kinetics of viral growth were assessed in the CHO-K1, R-3.1, R-Dfur1#11 and RDfur1#22 cell lines following infection with TRSB or NE2G216. TRSB replicated to similar titers and with similar kinetics in the CHO-K1, R-Dfur1#11, and R-Dfur1#22 cell lines ( Figure 3A ). TRSB titers from these cell lines were 2–3 log 10 higher than those produced in R-3.1 cells ( Figure 3A ). The reduced titer of TRSB grown in R-3.1 cells is consistent with previous reports, and has been shown to result from a decrease in virion infectivity associated with retention of PE2 in virions grown under these conditions, and not from decreased virus yield from these cells ( Watson et al. 1991 ; Moehring et al. 1993 ; Heidner et al. 1994 ). As expected, expression of Dfurin1 in RPE.40 cells had no detectable effect on the growth of the PE2 cleavage-defective virus, NE2G216, which grew to comparable titers in all four cell lines ( Figure 3B ). NE2G216 contains a mutation at E2 residue 216 (glutamic acid to glycine) that facilitates normal replication of this virus in the absence of PE2 cleavage ( Heidner et al. 1994 ). These results suggested that the increased titers of TRSB in the R-Dfur1#11 and R-Dfur1#22 cell lines was due to increased virion infectivity associated with Dfurin1-mediated cleavage of PE2 in these cells. To investigate PE2-cleavage in these cells, E2S1 virions were grown in each cell line and analyzed by SDS-PAGE ( Figure 4 ). As predicted, PE2 was not cleaved during viral replication in R-3.1 cells, but was cleaved efficiently in the CHO-K1, R- Dfur1#11 andR-Dfur1#22 cell lines.
Substrate specificities of vertebrate and arthropod furin enzymes
Generation of the Dfurin1-expressing RPE.40 cells made it possible to compare the substrate preferences of model vertebrate ( Cricetulus griseus Milne-Edwards (Rodentia: Cricetidae)) and arthropod ( D. melanogaster ) furin enzymes under conditions that eliminated host-specific differences in glycoprotein processing. To accomplish this, the PE2 cleavage phenotype of TRSB and of seven TRSB-derived mutants was determined in the CHO-K1 and R-Dfur1#22 cell lines. In addition, PE2 cleavage was assessed following growth of these viruses in C6/36 cells. Each of the viruses contained a different amino acid at the +1 position. Viral proteins were radiolabeled during growth in each cell line, purified from cell supernatants, and analyzed by SDS-polyacrylamide gel electrophoresis. Essentially complete cleavage of PE2 was detected in all three cell lines when the infecting viruses contained arginine (TRSB), serine (E2S1), phenylalanine (E2F1), histidine (E2H1), asparagine (E2N1), or aspartic acid (E2D1) at the +1 position (data not shown). In contrast, obvious differences in PE2 cleavage efficiency were observed between the cell lines when the infecting viruses contained valine (E2V1) or leucine (E2L1) at the +1 position ( Figure 5 ). Consistent with previous reports, PE2 substrates containing leucine or valine at the +1 position were cleaved much more efficiently, albeit not completely, by the arthropod enzymes than by the vertebrate enzyme ( Figure 5 ). As expected, viral glycoproteins derived from C6/36 cells migrated faster than cognate viral glycoproteins derived from the vertebrate cells due to differences in their carbohydrate structures. | Discussion
The objective of this study was to determine the basis for the alternative cleavage fates of select Sindbis virus PE2 substrates in cultured vertebrate and arthropod cells. By evaluating PE2 cleavage in CHO-K1 and Dfurin1 expressing RPE.40 cells, it was possible to compare the cleavage site preferences of a model vertebrate ( C. griseus ) and arthropod ( D. melanogaster ) furin enzyme under conditions where the glycosylation properties of the PE2 substrate remained constant. The results indicated that the alternative PE2 cleavage phenotypes were not linked to differences in the carbohydrate processing phenotypes between the two cell types. The study did establish that the vertebrate and arthropod enzymes are differentially influenced by the amino acid occupying the +1 position relative to the bxbb cleavage signal and that this difference accounts for the alternative cleavage fates of the PE2 proteins in vertebrate and arthropod cells. Specifically, PE2 proteins containing valine or leucine residues at the +1 position were largely resistant to cleavage by the vertebrate furin enzyme, but were cleaved efficiently (albeit not 100%) by Dfurin1. These same substrates were cleaved efficiently in cultured C6/36 cells (albeit not 100%) , which suggests that mosquito cells produce a furin-like enzyme with cleavage site preferences similar to Dfurin1.
It is generally accepted that PE2 is cleaved by a furin-like enzyme during virus replication in mosquito cells. However, to our knowledge, this study is the first to directly evaluate the ability of an arthropod-derived furin enzyme to support alphavirus replication through proteolytic processing of PE2. The vertebrate and arthropod furin-like enzymes belong to the proprotein convertase family within the subtilisin superfamily of serine proteases. The proprotein convertase enzymes share common structural features, including an N-terminal prodomain that is removed by autoproteolytic cleavage, a subtilisin-like catalytic domain with an active site composed of an aspartate, histidine, serine catalytic triad, a P domain which is essential for enzymatic activity, and, in some cases, a cystein-rich domain of unknown function near the C-terminus ( Thomas 2002 ). During the completion of this project, the genomic sequence of the A. aegypti mosquito was completed and shown to encode several furin-like enzymes that were predicted to share these features ( Nene et al. 2007 ). Two of these enzymes (accession numbers AAAEL003652 and AAEL010725) display a particularly high degree of sequence and predicted structural similarity to Dfurin1 ( Figure 6 ). The most closely related of these (AAEL003652), shares 82% sequence identity with Dfurin1 within both the catalytic domain and the P domain ( Figure 6 ). This enzyme is nearly identical to an A. aegypti enzyme previously identified as vitellogenin convertase (accession number AAC37262). It was cloned from a cDNA library, and the expression properties and enzymatic activities of the enzyme were characterized ( Chen and Raikhel 1996 ). Vitellogenin convertase plays an important role in vitellogenesis ( Chen and Raikhel 1996 ) by cleaving its pro-vitellogenin substrate downstream of a bxbb motif (RYRR↓D) ( Sappington and Raikhel 1998 ). It is expressed to high levels in the fat body, and its expression is induced following the ingestion of a blood meal ( Chen and Raikhel 1996 ). Vitellogenin convertase probably is capable of cleaving PE2 and, based on sequence considerations, is a good candidate for the PE2-processing enzyme in mosquitoes. However, it is not known if it is expressed in mosquito tissues relevant to Sindbis virus replication and transmission, such as the salivary gland, or if it plays a role in processing viral proproteins during natural infections of mosquitoes by alphaviruses or flaviviruses. The second furin-like proprotein convertase identified in the mosquito genome project (AAELO 10725) is predicted to share common proprotein convertase structural features with Dfurin 1, but displays a lower level of sequence identity (64% sequence identity with the catalytic domain and 47% identity within the P domain) ( Figure 6 ). The tissue distribution and enzymatic properties of this protein have not been studied.
The cyclic nature of alphavirus replication places unique selective pressures on the virus as viral proteins and genetic elements must maintain their ability to functionally interact with the cellular components of both evolutionarily diverged hosts. As a consequence, alphaviruses are thought to evolve compromise genotypes that are not optimally adapted to either host ( Greene et al. 2005 ). Indeed, repeated virus passage within a single cell type (vertebrate or arthropod) leads to the generation of host range mutants that display increased virus fitness in the cell type used for passage and a concomitant fitness decrease in the cell type that was bypassed ( Weaver et al. 1999 ; Cooper and Scott 2001 ; Greene et al. 2005 ). Similar results were obtained when an alphavirus (Venezuelan equine encephalitis virus) was repeatedly passaged through mosquitoes or mice ( Coffey et al. 2008 ). Results from this study suggested that the distinct substrate preferences of the arthropod and vertebrate furin enzymes would influence evolution of the residue immediately downstream of the PE2 cleavage site. A comparison of viral sequences revealed that most alphaviruses encode a serine residue at the +1 position ( McKnight et al. 1996 ), and this residue appears to be optimal for furin cleavage in vertebrate cells ( Heidner and Johnston 1994 ; Klimstra et al. 1999 ). Presumably, if alphaviruses containing valine or leucine at this position were to arise during the mosquito phase of the maintenance cycle, they would likely be selected against in the vertebrate host due to inefficient cleavage of PE2 by the vertebrate furin enzyme and the adverse effect that this phenotype has on viral replication in the vertebrate host ( Heidner and Johnston 1994 ). Like the alphaviruses, members of the flavivirus genus (family Flaviviridae ) utilize furin to cleave a glycoprotein precursor, prM, immediately downstream of a bxbb motif ( Stadler et al. 1997 ). Serine also occupies the +1 position in the prM proprotein of nearly all insect vectored members of the flavivirus genus ( Keelapang et al. 2004 ). Interestingly, the prM protein of Kamiti River virus contains alanine at the +1 position, and the Culex flavivirus contains valine at this site ( Crabtree et al. 2003 ; Hoshino et al. 2007 ). Kamiti River virus and Culex flavivirus are insect-only flaviviruses and, therefore, are not subjected to any selective pressure in vertebrate cells. | Associate Editor: Jan Veenstra was editor of this paper
Alphaviruses replicate in vertebrate and arthropod cells and utilize a cellular enzyme called furin to process the PE2 glycoprotein precursor during virus replication in both cell types. Furin cleaves PE2 at a site immediately following a highly conserved four residue cleavage signal. Prior studies demonstrated that the amino acid immediately adjacent to the cleavage site influenced PE2 cleavage differently in vertebrate and mosquito cells (HW Heidner et al. 1996 . Journal of Virology 70: 2069–2073.). This finding was tentatively attributed to potential differences in the substrate specificities of the vertebrate and arthropod furin enzymes or to differences in the carbohydrate processing phenotypes of arthropod and vertebrate cells. To further address this issue, we evaluated Sindbis virus replication and PE2 cleavage in the Chinese hamster, Cricetulus griseus Milne-Edwards (Rodentia: Cricetidae) ovary cells (CHO-K1) and in a CHO-K1-derived furin-negative cell line (RPE.40) engineered to stably express the Dfurin1 enzyme of Drosophila melanogaster Meigen (Diptera: Drosophilidae). Expression of Dfurin1 enhanced Sindbis virus titers in RPE.40 cells by a factor of 10 2 – 10 3 , and this increase correlated with efficient cleavage of PE2. The PE2-cleavage phenotypes of viruses containing different amino acid substitutions adjacent to the furin cleavage site were compared in mosquito (C6/36), CHO-K1, and Dfurin1-expressing RPE.40 cells. This analysis confirmed that the substrate specificities of Dfurin1 and the putative mosquito furin homolog present in C6/36 cells are similar and suggested that the alternative PE2 cleavage phenotypes observed in vertebrate and arthropod cells were due to differences in substrate specificity between the arthropod and vertebrate furin enzymes and not to differences in host cell glycoprotein processing pathways.
Keywords | Acknowledgements
The authors thank Charles M. Rice for providing the SINrep5 and pH3′2J1 plasmids and Tom J. Moehring for providing the RPE.40 and CHO-K1 cell lines. The authors also thank Wim J.M. Van de Ven and Anton J.M. Roebroek for making the pIP63 phagemid available for use through the American Type Culture Collection and for providing polyclonal rabbit antiserum to a Dfurin1/glutathione S-transferase fusion protein. We thank Yufeng Wang for performing the sequence analyses and comparisons between Dfurin1 and mosquito proprotein convertase enzymes, and we thank Janet Sunter for her assistance with cell staining and microscopy procedures. This work was supported by grant S06 GM 08194 from the National Institutes of Health Minority Biomedical Research Service and NIH/NIAID grant R29 AI40937.
Abbreviations
basic-X-basic-basic;
Chinese hamster ovary;
plaque forming unit | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 Apr 4; 10:29 | oa_package/6f/d4/PMC3014772.tar.gz |
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PMC3014773 | 20572787 | Introduction
The Western Ghats has many streams and large rivers and is one of the biodiversity hotspots for terrestrial and freshwater organisms ( Meyers et al. 2000 ; Dudgeon 1999 ). The biota of Western Ghats streams has been little studied, except for some groups of aquatic insects such as mayflies ( Sivaramakrishnan et al. 1996 ) and dragonflies ( Subramanian and Sivaramakrishnan 2002 ). There are few investigations of environmental influences on aquatic macroinvertebrate distribution in tropical areas, and most of them are quite recent. They include the influence of seasonal variation in a headwater stream ( Julka et al. 1999 ; Anbalagan et al. 2004 ), temporal variation of functional feeding groups ( Anbalagan 2005 ); habitat and microhabitat distribution patterns ( Subramanian and Sivaramakrishnan 2005 ), and effects of land use ( Subramanian et al. 2005 ). The Trichoptera are not a well-studied group in streams of Western Ghats ( Dudgeon 1999 ). Studies on Trichoptera started only after the middle of the 19th century, and later study mainly focused on taxonomical, rather than ecological, aspects of this group ( Dudgeon 1999 ). Only one study concerned ecological aspects ( Dinakaran 2004 ). No studies have been performed in the Western Ghats that examined factors affecting large scale caddisfly distribution. In contrast, several examples have been done in Europe ( Leuven et al. 1987 ; Wiberg-Larsen et al. 2000 ), North America ( Ross 1963 ), and South Africa ( de Moor 1992 ). The objective of the present study was to examine spatial and temporal dynamics of caddisfly communities in 29 streams of southern Western Ghats. | Materials and Methods
Sampling sites
All totaled, 29 streams were surveyed across the three states (Kerala, Tamil Nadu and Karntaka) of southern Western Ghats. The mountains intercept the rain-bearing southwest monsoon winds and are consequently an area of high rainfall. The important east flowing rivers of southern Western Ghats are the Tampiraparani (east), Vaigai, Cauvery, Tungabhadra, Bhima and Krishna rivers. The Kallidai, Tampiraparani (west) and Kallar rivers are west-flowing. The present study was carried out in streams of seven river basins of southern Western Ghats, namely the Tampiraparani (Honey Falls, Shenbagadevi Falls, Five Falls and Chinna Kuttalam), Kallidai (Palaruvi streams 1–4 and the Kallidai river), Vamanapuram (Kallar), Vaigai (Thadaganachiamman Stream, Ayyanar Falls, Kumbakkarai Falls, Pampar Cascade downstream, Thalayar, Silver Cascade downstream, Kurusedi and Moolayaru), Tungabhadra (Thanikode, Boklapura halla, Sringeri Stream and Theerthahalli), Bhima (Sanjitha nathi, Kalgi and Kakini river) and Krishna river basins (Bonal, Upper Krishna and Kadarattar) ( Figure 1 ). Monthly samples were collected from the Thadaganachiamman stream (Vaigai river basin) of Sirumalai Hills, Tamil Nadu from May 2006 to April 2007. Sampling sites included a variety of river types and reaches and riparian communities with and without structured vegetation.
Sampling procedure
Air and water temperatures were recorded in the field. Dissolved oxygen, total dissolved solids, conductivity and pH were measured using a water analysis kit (Naina Solaris Limited, www.indianindustry.com ). Latitude, longitude, elevation and basin location were determined by global positioning, GPS. Substrates were classified ( Jowett et al. 1991 ) using the following criteria: <0.5 mm for mud/silt, 0.5–2 mm for sand, 2–64 mm for gravel, 65–256 mm for cobbles, and >256 mm for boulders. For statistical analyses, substrate composition was converted to a substrate index ( Suren 1996 ): The average stream width and depth were calculated from three measurements with a calibrated stick from one transect across the channel. Current velocity of the stream was obtained by a flow meter. Canopy cover was measured using a densitometer, and dominant species of riparian vegetation were recorded for each sampling site.
For each study site and sampling occasion, three 50 × 50 cm benthic samples were taken at random locations from each riffle and pool. In riffle samples, caddisflies larvae were collected using 180-μm-mesh kick-nets, and 500-μm-mesh dip nets were used for pool sampling. Soon after collection, the specimens were preserved in 70% ethanol. All caddisfly larvae were identified to the lowest possible taxonomic level using available keys ( Dudgeon 1999 ).
Data analysis
Observations of the physical and chemical characters were recorded for each site on each sampling date, and the richness and density of the Trichoptera taxa were summarized as mean values, standard deviation and coefficient variation. Dissolved oxygen, conductivity and temperature were graphically presented to illustrate the seasonal changes in water quality. In each sampling station, diversity indices were estimated. Alpha diversity indices of the Shannon-Wiener diversity index and the Simpson diversity index, species richness index of Margalef, and evenness of index Pielou were calculated according to Ludwig and Reynolds ( 1988 ). Similarities in taxonomic composition were quantified using Jaccard's index ( Sneath and Sokal 1973 ; Magurran 1988 ) based on a presence-absence matrix for the insect fauna of each stream. More specifically, similarity (S ij ) between any pair of sites i and j is given by where a is the number of taxa shared in common, b is the number of taxa in site i but not site j, and c is the number of taxa in site j but not site i. Two-way Bray-Curtis analysis was performed using the results of Jaccard's index. Canonical correspondence analysis (CCA) was calculated, measuring the relationship between 11 environmental variables, taxa richness of caddisflies, and seasonal changes on caddisfly taxa among sampling sites ( Legendre and Legendre 1998 ). | Results
Physico-chemical characteristic of the study streams
Physical parameters of 29 streams and rivers of southern Western Ghats are given in Table 1 . Average temperature among all the sampling sites was 24.8° C. Thalayar had the maximum temperature, and Silver Cascade had the minimum temperature. The mean pH and conductivity were 6.59 and 0.11μ mhos, respectively. Average dissolved oxygen concentrations were 8.62 mg/L. Total dissolved solids levels were low, and mean value was 56 mg/L. Particulate organic matter (leaf litter) ranged from 0% to 50% mg/m 2 . Stream width ranged from 0.25 m to 15 m. The average substrate index was 5.6, being lowest at Kallidai river (2.8) and highest at Palaruvi (7.6) ( Table 2 ). Seasonal variation of the physico-chemical parameters was analyzed in the Thadaganachiamman Stream of Sirumalai Hills between May 2006 and April 2007, and the physico-chemical parameters are given in Table 3 . Annual water discharge was high during northwest monsoon season (October and November), and this was gradually reduced and conspicuously absent during summer (May and June).
Taxa distribution and diversity analysis
All totaled, 20 caddisfly taxa were obtained, and the community of taxa differed among sampling sites. Kurusedi had the maximum number of taxa, whereas the Bonal, Kakini River, Kalgi and Sangitha Nadhi of Karnataka harboured only two taxa each. Other sites harbored from 3 to 5 each. Hydropsyche had the widest distributional range, whereas Georgium and Helicopsyche had the narrowest distribution range ( Figure 2 ). Diversity indices showed that the Kurusedi stream had higher diversity and species richness ( Table 4 ). Shannon and Simpson indices indicated that the highest diversity occurred during the month of October in the Thadaganachiamman stream, but the Margalef index showed higher species richness during the month of July ( Figure 3 ). Stream sites 5 to 8; 14, 26, 28, 4, and 12 had the highest faunal similarity than other stream sites. Higher similarity occurred between Anisocentropus and Lepidostoma ( Figure 4 ).
Spatial patterns of distribution
Results of the CCA analysis are given in Tables 5 and 6 . Eigen values for F1 and F2 axes were 0.007 and 0.003. Cumulative variance was 67.9% for axis F1 and 97.78% for axis F2. Total inertial values were 5.93 (F1) and 2.61 (F2). CCA analysis revealed that among 29 sampling sites, 2 sites represent 4 and 5 taxa each, 12 sites exhibit 3 taxa, 6 sites had 2 taxa, and the seven remaining sites had one. Elevation was an important factor in the F1 axis of CCA. Substrate, pH, and stream order were important in axis F2 ( Figure 5 ).
Temporal patterns of distribution
The effect of seasonality on caddisfly distribution was analyzed. Figure 6 shows changes in caddisfly taxa among seasons. Although the first canonical axis explained only 4.2%) of caddisfly variability, the Jolliffe cut-off test indicated that all canonical axes were significantly related with seasonality (0.0199, p < 0.05). Hydropsyche and Stenopsyche were present frequently in all seasons, whereas Lepidostoma and Anisocentropus were present only in winter and autumn, and not in summer. | Discussion
In large scale studies performed in other areas in the world, geomorphological, and other large scale variables such as climate and altitude have been considered as the major factors responsible for macroinvertebrate distribution ( Ross 1963 ; Corkum 1989). Our results are in accord with these findings and suggest that large scale variables were responsible for structuring caddisfly communities. The multivariate analysis suggested that some of the variables (substrate, pH, and stream order) examined in this study influenced the caddisfly distribution and abundance in streams of southern Western Ghats. Apart from these variables, elevation was an important factor.
For example, the stream of Kurusedi, which is located at high elevation, had the highest diversity and species richness. The abundance and distribution of lotic invertebrates has been attributed to a variety of factors (Hynes 1970), many of which vary as a function of altitude and thus may be responsible, directly or indirectly, for zonation patterns, Some species were restricted to the headwaters, others to middle or lower reaches, and a few species occurred over wide altitude ranges (Ward 1981), Similarly, Georgium and Helicopsyche were found at the headwater stream, and Hydropsyche was distributed in all stream reaches, This may have been due to flow regime and allochthonous food availability, The present study revealed that seasonality determined the assemblage of caddisfly communities observed during October (northeast monsoon) and July (southwest monsoon), Similar results were observed in the Danubian floodplains in lower Austria ( Waringer and Graf 2002 ), The shredder community of caddisfly ( Lepidostoma and Anisocentropus ) was present only in winter and autumn; this may have been due to a substantial amount of leaf litter fall during this season. A similar trend was observed in the lentic water system in south India (Dinakaran et al. 2008).
Physical disturbances and natural environmental gradients were the most important factors regulating the abundance of caddisflies in streams of southern Western Ghats. Physical disturbance in lotic systems was inextricably linked to environmental dynamics and resulting ecosystem and biotic interactions ( Allan 2004 ). The upstream and downstream gradient was an important parameter describing benthic community variation in streams of southern Western Ghats (Dinakaran and Anbalagan 2008). These upstream areas had a higher number of species and acted as colonizing sources for a variety of taxa occurring in the upper study reach that were not found in the lower portions of the study area because of the increased distance from the colonizing sources and anthropogenic impacts. Furthermore, non-point source impacts associated with land use patterns, as well as disturbances by recreational and commercial watercraft, and increases in downstream direction may contribute to environmental disturbances on caddisfly communities. Angradi ( 1996 ) observed faunal differences between habitats in Applachian streams. A similar trend was observed for caddisfly communities in streams of southern Western Ghats.
These findings suggest that considerable spatial and temporal dynamics exist in abiotic and caddisfly variability in streams of southern Western Ghats. Multivariate analysis revealed that elevation of the stream played a vital role in the existence and assemblage of caddisfly species. Kurusedi (one of the 29 sampling streams, located among the tourist spots of Palni hills) consisted of rare species of Helicopsyche and Georgium (not even a single species of Georgium was previously recorded in India). Since it lies within a tourist area, this area faces a threat from bathing and washing clothes and vehicles, and it needs to be protected. For example, an ephemeropteran species Isca is completely absent due to anthropogenic impacts, but was once recorded in Alagar hills, which is a pilgrimage spot to worship the local deity Theerthakarai (sacred bathing) Raakayee Amman (deity) of Eastern Ghats ( Dinakaran and Krishnan 1997 ). Further studies on the strategies employed with altitudinal distribution and anthropogenic impacts on caddisfly species' ability to persist and maintain their populations in the water body are needed to enhance knowledge of how they survive in unstable and stressed conditions in streams. | Associate Editor: Yves Carriere was editor of this paper
The dynamics of physico-chemical factors and their effects on caddisfly communities were examined in 29 streams of southern Western Ghats. Monthly samples were collected from the Thadaganachiamman stream of Sirumalai Hills, Tamil Nadu from May 2006 to April 2007. Southwest and northeast monsoons favored the existence of caddisfly population in streams. A total of 20 caddisfly taxa were collected from 29 streams of southern Western Ghats. Hydropsyche (Trichoptera: Hydropsychidae) were more widely distributed throughout sampling sites than were the other taxa. Canonical correspondence analysis showed that elevation was a major variable and pH, stream order, and stream substrates were minor variables affecting taxa richness. These results suggested that habitat heterogeneity and seasonal changes were stronger predictors of caddisfly assemblages than large-scale patterns in landscape diversity.
Keywords | Acknowledgements
We are grateful to the University Grants Commission (UGC), New Delhi, for funding this study through research grant F. No. 31-216/2005(SR). We also thank Mr. G. Muralidharan and Mr. R. Mahendran for field assistance. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 May 12; 10:46 | oa_package/79/90/PMC3014773.tar.gz |
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PMC3014791 | 21209804 | 1. Introduction
Peripheral T-cell lymphoma (PTCL), accounting for less than 15% of non-Hodgkin's lymphoma worldwide, derives from natural killer or mature T cells (NK/TCL) and often involves the skin primarily or secondarily [ 1 ]. PTCL has a relatively higher morbidity rate in Asia and Central/South America, especially in populations infected with human T-cell lymphoma/leukemia virus-1 or Epstein-Barr virus (EBV) [ 2 ]. Diagnosis in PTCL has improved with the development of molecular, immunologic, and genetic techniques. However, there is still no consensus about standardized therapy in PTCL, although CHOP (cyclophosphamide, pirarubicin, vincristine, and prednisone) or CHOP-like chemotherapy have been thought as the major regimen [ 3 ]. Progress in treatment of PTCL is much slow mainly due to disease rarity, biological heterogeneity, geographic variation, and limited recognition of the disease. To date, the prognosis of most PTCL types is extremely poor with a 5-year survival of 15–30% in the majority of series [ 1 ]. In recent years, bortezomib as a novel proteasome inhibitor, has shown good tolerance and therapeutic effect in PTCL besides defined indications for multiple myeloma and mantle cell lymphoma, which is bringing new light to PTCL patients [ 4 , 5 ].
Here, we present a case of peripheral NK/TCL of the skin with positive EBV infection, in which bortezomib plus CHOP chemotherapy were used and led to rapid improvement and then complete remission (CR) for 11 months until local relapse. We also discussed the diagnosis, prognosis, and bortezomib- or plus CHOP-based treatment of PTCL according to this case and previously published data. | 3. Discussion
As for most PTCL patients with skin involvement but no B symptoms, cutaneous lesions are always significant enough to be initially identified and treated as common dermatitis at a dermatology clinic such as in this case. Detailed physical examination, especially lymph node examination, and prompt tissue biopsy are judicious choices prior to any medical management in order to avoid misdiagnosis and delay of correct treatment. Histologically, PTCL contains multiple subtypes including PTCL unspecified (PTCL-U), cutaneous TCL (CTCL), angioimmunoblastic TCL (AILT), anaplastic large-cell lymphoma (ALCL) noncutaneous, and hepatosplenic TCL (HSTCL) [ 2 ]. Adequate immunophenotyping is essential to exclude B-cell lymphomas and establish the specific type of PTCL by immunohistochemistry or flow cytometry. In our case, the immunostaining results of strongly positive CD3, CD43, CD56, and Ki67, but negative CD20, CD30, and Bcl-2, definitely supported the diagnosis of peripheral NK/TCL of the nasal type, although the negativity for perforin is indeed unusual [ 6 ].
Up to now, there is no standard efficient therapy for PTCL [ 1 ]. CHOP-like treatment, as the most commonly used first-line regimen for PTCL patients, is largely ineffective with low CR and high relapse rate except in anaplastic-lymphoma-kinase- (ALK)-positive ALCL [ 3 , 7 ]. Some emerging evidence demonstrated that CHOP might not be appropriate as a chemotherapy backbone for PTCL patients since anthracyclines perhaps cannot influence the outcome, at least of PTCL-U [ 1 , 8 – 10 ]. Previous data also suggested that expression of P-glycoprotein in PTCL possibly results in multidrug resistance to conventional systemic therapy, at least partly [ 11 ]. Other regimens that are more intensive than CHOP did not show any significant improvement in the overall survival of PTCL patients yet, with the exception of ALCL [ 12 ].
Bortezomib exerts an antitumor activity mainly through affecting the ubiquitin-proteasome pathway subsequently leading to inhibition of NF- κ B and stabilization of proapoptotic proteins such as P53, Bcl-2, Bim, Bik, and Noxa [ 13 ]. It has been reported that NF- κ B plays a critical role in the pathogenesis of CTCL, and that bortezomib can significantly cause NF- κ B down-regulation and CTCL cell apoptosis [ 14 ]. Therefore, bortezomib may play a special therapeutic role and, at the same time, be safe from the emergence of acute toxicity in some types of PTCL due to its basic function mechanisms. As reported, an advanced HSTCL patient of gamma/delta type remained almost in CR for 27 months after 4 cycles of bortezomib plus the modified high-dose CHOP prior to autologous peripheral blood stem cell transplantation [ 15 ]. A phase II clinical trial has confirmed that bortezomib indeed is safe and has significant single-agent activity in inducing remission (67% overall response rate, ORR), even CR (17%), in patients with relapsed or refractory CTCLs including mycosis fungoides and PTCL-U with isolated skin involvement [ 4 ]. The mentioned study also indicated that bortezomib leads to higher or at least comparable ORR compared with published data on gemcitabine, pegylated liposomal doxorubicin, 2-chlorodeoxyadenosine, or pentostatin. Another recent phase I study demonstrated that PTCL patients consisting of PTCL-U, NK/TCL, ALCL, and angioimmunoblastic lymphoproliferative disease gained profits from the bortezomib plus CHOP therapy, with 61.5% ORR and unconfirmed CR rates [ 5 ]. The combined regimen just caused grade-4 neutropenia associated with febrile episode in one patient and grade-one peripheral sensory neuropathy in three patients, which indicated a good tolerance.
It has been reported that PTCL-U presenting in the skin has an unfavorable prognosis [ 16 ]. The outcome of PTCL appears to be associated with the International Prognostic Index (IPI), Prognostic Index for PTCL-U (PIT), and the International peripheral T-cell lymphoma Project score (IPTCLP), which are used for risk classification and response and survival prediction [ 3 , 12 , 17 , 18 ]. In addition, EBV is positive in about 40% of PTCL, and some case series have reported that EBER-positive tumors have a worse outcome [ 3 , 19 ]. Our patient belongs to the subgroup of high risk with bad prognosis because of poor prognostic indexes due to multiple risk factors: >60 years of age, ECOG performance status >2, late stage, elevated serum LDH, number of extranodal sites of involvement >1, high Ki67 expression, and positive EBV infection [ 20 , 21 ]. Anyway, the adopted regimen showed enough safety and quickly led to satisfied therapeutic effects in this case. Therefore, the bortezomib plus CHOP chemotherapy is safe and efficient in peripheral NK/TCL according to our case and the literature review, although further large-sized clinical trials with long-period followup are still needed. | Academic Editor: Yolanda T. Becker
Peripheral T-cell lymphoma (PTCL) is rare and difficult to treat for its high relapse rate. The authors report a case of PTCL of the skin, regarding which clinical and pathological features, treatment, and prognosis were discussed. A 66-year-old woman was admitted with complaints of enlarging erythematous noduloplaques on the right anterior tibial skin for one year and similar lesions on the left for 6 months. Surgical resection of right leg lesion and biopsy of enlarged inguinal lymph nodes histologically indicated a PTCL of the nasal type. The patient was treated by CHOP plus bortezomib, reached complete remission just after two courses of chemotherapy and then received another two as consolidation. The patient remained in remission for 11 months until local relapse. As for cutaneous lesions, detailed lymph node examination and prompt tissue biopsy are judicious choices prior to any medical management. The chemotherapy consisting of bortezomib and CHOP is safe and efficient in PTCL of the skin. | 2. Case Report
In October 2007, a 66-year-old woman firstly visited a dermatology clinic with chief complaints of erythematous noduloplaques with mild tenderness and pitting edema on the right anterior tibial skin. She was diagnosed with dermatitis and continuously improved under infrared irradiation until April 2008, when similar dermatic lesions occurred on the left anterior tibial area. In September 2008, the reddish-violet noduloplaques on both legs increased to a size of ahen's egg, and the lesion on right leg even ulcerated and bled. The woman was then admitted to our hospital without a history of fever or weight loss. Physical examination showed multiple painless swollen lymph nodes of moderate hardness and limited motion in bilateral inguinal areas, which were assessed by color Doppler ultrasound with the largest one being 19 × 6 micrometer in size. A comprehensive metabolic profile displayed an elevated serum level of lactate dehydrogenase (LDH) of 256 U/L (normal: 60 to 240 U/l). Computerized tomography scan of the chest and abdomen was normal except for hepatic cysts.
The surgical resection and dermatoplasty of right leg lesion and biopsy of homolateral inguinal lymph nodes were carried out by plastic surgeons. Both the sections of invaded skin and lymph nodes histologically indicated peripheral NK/TCL of the nasal type with strongly positive CD56, CD43, CD3 and Ki67, but negative CD20, CD30, Bcl-2, and perforin by immunohistochemistry (Figures 1 and 2 ). EBV-encoded RNA (EBER) was positive in skin specimen by in situ hybridization. Serum anti-EBV capsid antigen (CA) IgM and antiearly antigen IgG were negative, but antinuclear antigen-1 IgG, anti-CA IgG, and anti-CA IgA were significantly positive.
The patient underwent a combination chemotherapy consisting of classical CHOP-21 plus bortezomib (1.3 mg/m 2 ), which were administered by bolus injection on days 1, 4, 8, and 11, every 21 days. Simultaneously, acyclovir was intravenously used to protect against mucocutaneous infection with herpes simplex virus. The disease was quickly and significantly improved, which mainly displayed in diminishing noduloplaques, shrinking erythemas and swollen lymph nodes, decreasing edema, elimination of tenderness, and normalization of local skin temperature. She reached CR with mild brown pigmentation just after two courses of chemotherapy and received another two courses as consolidation ( Figure 3 ). The therapy was well tolerated with only grade-two toxicities of neutropenia and thrombocytopenia. The patient remained in remission for 11 months but relapsed locally and became lost to followup at last. | Conflict of Interests
The authors did not receive any grants or financial support and have no financial interest in the products presented in this work. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 26; 2010:403237 | oa_package/35/c9/PMC3014791.tar.gz |
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PMC3014792 | 20569135 | Introduction
Being a mechanical vector of pathogenic microorganisms, Chrysomya megacephala (F.) (Diptera: Calliphoridae) is a blowfly of considerable medical and veterinary importance ( Guimarães et al. 1978 ; Furlanetto et al. 1984 ; Laurence 1986 ; Lima et al. 1991 ; Gabre et al. 2005 ). C. megacephala is able to cause facultative myiasis in humans and animals, has an expanding geographic distribution, and was unintentionally introduced in Brazil in the 1970s ( Zumpt 1965 ; Guimarães et al. 1983 ; Laurence 1986 ; Gabre et al. 2005 ). Moreover, the genus Chrysomya has been increasingly deployed in forensic studies for the determination of human postmortem intervals ( Greenberg 1991 ; Catts and Goff 1992 ; Arnaldos et al. 2005 ; Gomes and Von Zuben 2005 ).
The larval phase of C. megacephala is deemed to be a critical developmental period in which intense limitation of resources frequently occurs ( Levot et al. 1979 ; Goodbrod and Goff 1990 ; Reis et al. 1994 ). This limitation is conducive to dynamic competitive processes, wherein each larva attempts to feed off the available resources, scrambling to exploit the feeding substrate before the depletion of the food resource ( Ullyett 1950 ; de Jong 1976 ; Lomnicki 1988 ; Von Zuben et al. 2001 ). Therefore, such exploitative conditions are embedded in interrelated processes that take place at the individual and population levels ( Wijesundara 1957 ; Herzog et al. 1992 ; Von Zuben et al. 2001 ; Tammaru et al. 2004 ).
Regarding the nutritional ecology of immature blowflies, it has been suggested that the competition for food is influenced, concomitantly, by larval density and availability of food. Hence it is very useful and important to investigate the crowding level of immature individuals on the feeding resources by means of simultaneous variations of larval densities and amounts of food ( Von Zuben et al. 2000 ; Ireland and Turner 2006 ).
The length of the larval stage of C. megacephala represents a useful variable for the adequate comprehension of its nutritional ecology as a whole. The importance of this parameter is strongly connected with the fact that individuals that require relatively long periods of larval development in order to reach their adult phases may undergo harsh feeding conditions ( Von Zuben et al. 2001 ). Moreover, the accurate measurement of the larval phase duration may substantially contribute to the determination of the most cost-effective relationships between initial larval density and amount of available food in relation to feasible mass rearing techniques ( Von Zuben et al. 2001 ; Tammaru et al. 2004 ).
The outcomes of exploitative competition for feeding resources determine the dynamics of population parameters such as survival, fecundity, weight, and size of the emergent adults ( Von Zuben et al. 1993 ; Papandroulakis et al. 2000 ; Von Zuben et al. 2000 ; Ireland and Turner 2006 ). In relation to the resultant individuals, Von Zuben et al. ( 1993 ) regard the number of emerging adults as a variable that tends to decrease with an increase in the number of immature individuals of C. megacephala.
Under natural environmental conditions, there are several difficulties in conducting experiments and collecting suitable data on population bionomic features of C. megacephala ( Gabre et al. 2005 ). Most natural situations do not allow researchers to obtain representative sample sizes or detailed descriptions of every developmental phase, whereas in laboratory conditions, immature specimens of C. megacephala feeding on a wide variety of substrates are able to develop into more advanced stages in an effective manner ( Roy and Dasgupta 1971 ).
The complexities of the nutritional ecology of blowflies could be clarified and detailed by the deployment of appropriate modelling techniques such as artificial neural networks, which are mathematical tools widely applied to the resolution of complex biological problems. A notable feature of artificial neural networks is their independence from any assumptions about the theoretical distribution shape of the data used. Also, the performance of artificial neural networks over linear or non-linear regression-based statistical models is at least interesting, as the dimensionality and/or the non-linearity of the systems increase ( Schultz and Wieland 1997 ; Haykin 1999 ; Schultz et al. 2000 ).
Neural networks algorithms are founded on the construction of models that consist of a great number of simple processing units called neurons (or nodes) that possess several connections between them and are lined up in layers. The number of neurons in the input layer corresponds to the variables that will be used to feed the neural network and should be the most relevant variables to the problem in question ( Jang 1993 ; Haykin 1999 ).
Artificial neural networks were conceived with the aim of imitating the human brain functionality. Thus part of the terminology used in the area of artificial neural networks, namely neurons, synapses, learning, layers, etc., is due to such a fact.
However, it is important to emphasise that these terms are only associated with mathematical functions or the method of utilising them.
Regarding the use of neural networks in entomology, considerable improvements have been achieved in recent years. Obach et al. ( 2001 ) predicted the abundance of selected water insects in a small stream in central Germany by means of neural models. Using environmental data that were collected as part of a study of microhabitat use by butterflies, Bryant and Shreeve ( 2002 ) highlighted the potential for utilising neural networks in developing predictive models of microhabitat temperature. Several multidisciplinary studies have described the development of scale independent models, based around coupling artificial neural networks with climate-hydrological process models in order to simulate species' distribution, including insect species ( Pearson et al. 2002 ; Pearson and Dawson 2003 ; Harrison et al. 2006 ). Worner and Gevrey ( 2006 ) used a self-organising map, which is an artificial neural network model, with the purpose of identifying global pest species assemblages and potential invasive insects, including dipteran species.
Furthermore, neural models were assessed in combination with a set of well-known ecostatistics in order to conduct function approximation (function fitting) and documentation based on invertebrate data (99 invertebrate families), including immature and adult specimens ( Zhang and Barrion 2006 ). Howe et al. ( 2007 ) used neural networks to describe and predict insect body temperatures and insect behaviour in relation to environmental variables. Zhang and Zhang ( 2008 ) deployed neural models with the purpose of assessing the effectiveness of neural networks in modelling survival process and mortality distribution of a holometabolous insect ( Spodoptera litura , tobacco cutworms) at different temperatures. Zhang et al. ( 2008b ) utilised various neural networks in order to fit and recognise spatial distribution patterns of grassland insects. Nonetheless, apart from the present study, neural models have not been directly employed in the modelling of the nutritional ecology of blowflies.
Therefore, the principal aim of this work is to use well-known neural networks to ascertain whether these tools are able to outperform a classical statistical method (multiple linear regression) in the prediction of the number of resultant adults of experimental populations of C. megacephala , based on initial larval density (number of larvae), amount of available food, and duration of immature stages (in hours). Results obtained through statistical methods were compared with those derived from artificial neural networks algorithms for the purpose of achieving such a goal. Additionally, some basic concepts of neural models are outlined in order to permit entomologists to assess the potential of using artificial networks in the nutritional ecology of other insect species. | Materials and Methods
Formation of the laboratory generations
Adult individuals of both sexes were collected in Campinas (22° 49′ 9.52′′ S and 47° 4′ 12.54′′ W), São Paulo, Brazil. They were then identified and kept in nylon net cages (30 × 30 × 48 cm). Decaying organic matter, such as rodent and fish carrions, was utilised as bait. Prior to identification, the individuals were anaesthetised in a freezer at -18° C for 30 s. These insects were provided with water and refined sugar ad libitum and taken as the parental generation of this study. The cages were kept in a controlled temperature room (25 ± 1° C) at a relative humidity of 60 ± 10% and a photoperiod of 12:12 light:dark. In order to induce the development of the gonotrophic cycle, females were supplied with fresh macerated beef liver as a source of protein. For the formation of the next generations, ovipositions were obtained using small pots containing macerated decaying beef that were put into the cages in order to stimulate egg laying.
Different larval densities were formed using glass pots (8 cm in height × 7 cm in diameter) that contained four amounts (15, 30, 60, and 90 g) of an artificial diet (feeding resource) proposed and described by Leal et al. ( 1982 ). Such larval densities were based on F 2 individuals (second laboratory generation), and five different proportions of larvae to amount of food were considered: 5, 10, 20, 30 and 40 larvae/g. Thus the larval densities utilised were: 75, 150, 300, 450 and 600 larvae, each in pots containing 15g of food; 150, 300, 600, 900 and 1200 larvae growing in pots that contained 30 g of food; 300, 600, 1200, 1800, and 2400 larvae in pots with 60 g of food; and 450, 900, 1800, 2700, and 3600 larvae in pots containing 90 g of the artificial diet.
The larvae counts were done using newly hatched individuals. Artificial diet pots were covered with organza and kept in a climatic room at 25 ± 0.2° C, 60 ± 10% relative humidity and 12:12 L:D. After the complete exhaustion of the food substrate, the pots were put into bigger plastic pots (20 cm in height × 12 cm in diameter) with a 5 cm-thick sawdust layer (pupation substrate). Organza was removed from the small pots in order to enable the larvae to move through these pots toward the sawdust. The bigger plastic pots were covered with organza and kept at identical experimental conditions.
Variables
In this study, the number of survivors (resultant adults) was the dependent variable (output), and the others were considered the independent or explanatory variables (inputs), namely larval density (initial number of larvae), amount of available food (in grams), and duration of immature stages (from the first instar larval stage to the final pupal stage, in hours). Such variables were primarily chosen based on their biological significance for the comprehension of the nutritional ecology of blowflies. Combinations of the three input variables provided 40 values (sample size) concerning the output variable.
The whole data set ( n = 40) provided a coefficient of determination of 0.59 ( F = 30.94, p < 0.001) prior to splitting the original data set into smaller subsets by means of neural network techniques. A stepwise linear regression (forward selection) revealed that the joint contribution of larval density and duration of immature stages to the linear regression was equal to 2%. This low value, albeit statistically significant (p < 0.001), did not represent the practical importance of such variables because it is biologically inconceivable that the number of resultant adults could vary only as a function of the available amount of food, irrespective of variations in larval density and duration of immature stages ( Levot et al. 1979 ; Getz 1984 ; Von Zuben et al. 1993 , 2000 ; Ireland and Turner 2006 ).
Each independent variable (input) produced distinct output responses ( Figure 1 ). On the whole, such explanatory attributes generated non-linear variation in the number of survivors. Additionally, an overall lack of strong linear relationships between input and output variables was observed ( Figure 2 ), and the accuracy of conventional regression methods may have been significantly reduced in the presence of non-linearity ( Neter et al. 1996 ; Schultz and Wieland 1997 ). Moreover, the p-values concerning the significance of the input variables were based on underlying statistical assumptions ( Neter et al. 1996 ). On the other hand, artificial neural networks are able to model complex nonlinear systems, even when the exact nature of any relationships is unknown ( Schultz and Wieland 1997 ; Schultz et al. 2000 ; Bryant and Shreeve 2002 ; Howe et al. 2007 ; Zhang et al. 2008b ). From a biological point of view, all the explanatory variables are considerably important, and neural models permitted the utilisation of such variables, irrespective of their statistical significance.
Basic concepts of the utilised neural network models
Neural networks technology utilises a multilayered approach to approximating complex mathematical functions in order to process data. It consists of many processing elements (nodes or neurons) that work in a parallel manner. Neurons are connected to each other in layers that are interconnected. The connections between neurons weight the data transformation process of each neuron, sending the information to the next node or output layer. Such connections are known as synaptic strengths or weights. The training process starts by furnishing the neural network with a variety of examples (called training sets). The data sets normally contain input and output data. The neural model creates connections and is able to learn patterns based on the relationship between input and output data sets via the adaptation of the synaptic weights to changing inputs.
Three well-known neural networks, namely Multi-Layer Perceptron (MLP) ( Haykin 1999 ), Radial Basis Function (RBF) ( Wasserman 1993 ), and Adaptive Neural Network-Based Fuzzy Inference System (ANFIS) ( Jang 1993 ), were used to compare their outcomes with those derived from the deployment of multiple linear regression (a classical statistical method). The coefficient of determination (R 2 ), which lies within the interval 0 ≤ R 2 ≤1, was utilised for comparing such outcomes. A zero R 2 indicates that the predictive model does not explain the variance of the actual data set.
The statistical assumptions of the multiple linear regression model and its feasibility followed the detailed descriptions in Neter et al. ( 1996 ). A basic description of the neural models will be made in the following sections.
Multi-Layer Perceptron (MLP)
MLP is the most widely employed type of feed-forward neural network ( Haykin 1999 ). MLP networks consist of an input layer, one or more hidden layers, and an output layer ( Figure 3 ).
Each layer has a number of processing units, and each of them is fully interconnected with units in the subsequent layer by means of weighted connections in a “cascade” manner, without any connections between neurons in the same layer. The number of neurons in the input layer ( X ) is equal to the number of variables of the problem in question. Also, there is an output layer ( Y ) in which the network response is made available and the number of neurons is equal to the desired number of quantities derived from the inputs. With respect to a regression-based analysis, there is a single neuron in the output layer. The layers between input and output layers are called hidden layers ( H ).
The computation of final output values is conducted in a layer-by-layer manner. Accordingly, each neuron of a specific layer other than the input layer computes a linear combination of outputs of the previous layer. Secondly, the resultant values are multiplied by the weight of the connection ( w ). Finally, such products arrive at each hidden neuron and are summed. Furthermore, there is a possibility of incorporating a shift called bias ( b ) into the neuron inputs. Each neuron calculates its output value by means of the corresponding activation function ( f ). Then, those values are continuously propagated toward the next layer, thereby reaching the output layer. Activation and output functions that are used frequently encompass linear (identity) functions, sigmoidal (S-shaped) functions, such as the logistic function, and the Heaviside thresholding function.
The training process is conducted as follows ( Haykin 1999 ): a pattern is presented to the inputs. This pattern is transformed during its passage through the layers of the network toward the output layer. Then, the outputs of the network, as they are in this phase, are compared with the outputs that ideally would have been encountered if this pattern had been exactly stated. Based upon such comparisons, all the connection weights ( w ) are modified to some degree to ensure that the same pattern could be presented to the inputs. The differences (errors) between the actual outputs and the desired outputs are propagated backward from the top layer to lower layers in order to modify the connection weights. Generally, steepest descent techniques would achieve a suitable performance if local minima were relatively distant. On the other hand, they require a lot of iterations to converge when minima are near. A number of different types of back-propagation learning algorithms have been proposed, such as the Levenberg-Marquardt ( Hagan and Menhaj 1994 ), with the aim of finding an optimum solution to a minimisation problem. It utilises an approximation to the Hessian matrix updating Newton-like weight.
Radial Basis Function (RBF)
RBF networks were introduced into the literature in the late 1980s ( Broomhead and Lowe 1988 ; Poggio and Girosi 1990 ). Such neural models are non-linear hybrid networks that represent an approach to universal function approximation, and they were first used to solve multivariate interpolation problems ( Poggio 1994 ). Furthermore, they are able to approximate a wide class of non-linear multidimensional functions. These neural networks are deemed to be a special class of multilayer feed-forward networks. They consist of a fully connected architecture with an input layer, a hidden layer, and an output layer ( Figure 4 ).
There is one neuron in the input layer for each predictor variable. The input neurons (X i ) act as an input data buffer and do not execute any processing. The hidden layer has a variable number of neurons that should be determined by the training process. The neurons in the hidden layer contain the gaussian function as an activation function, and their outputs are inversely proportional to the distance from the centre of the neuron. Each neuron consists of an RBF centred on a specific point that possesses a number of dimensions that should be equivalent to the quantity of predictor variables. The spread (radius) of the RBF function may be different for each dimension. When this network is presented with the X vector of input values derived from the input layer, a hidden neuron computes the Euclidean distance from the test case to the central point of the neurons and then applies the RBF kernel function to this distance, using the spread (σ). The resultant value is transferred to the output layer. A hidden neuron is more sensitive to data points near its centre. Regarding the gaussian RBF, such neuron sensitivity may be tuned by adjusting the spread, whereby larger spreads indicate less sensitivity. In the output layer, the neurons implement a weighted sum of hidden unit outputs (linear combination of hidden functions).
During the training process, the parameters of the RBF are the number of neurons in the hidden layer, the coordinates of the centre of each hidden-layer RBF function, the radius (spread) of every RBF in each dimension, and the weights that were applied to the RBF function outputs in the summation layer.
There are several training algorithms for the RBF networks. In contrast to other networks, the training process is divided into two phases: (1) determination of the parameters of the basis functions, and (2) computation of the output weights. During the first phase, clustering methods for finding central positions of the radial basis function can be used with a specific number of hidden units. Regarding fixed basis functions in the second phase, the weights have been traditionally optimised by the least mean square algorithm or by other optimisation methods. The number of hidden layers is dynamically adapted in response to the output error. Further information concerning RBF learning algorithms may be found in Schwenker et al. ( 2001 ).
Adaptive-Network-based Fuzzy Inference System (ANFIS)
A fuzzy inference system is a framework that entails fuzzy logic, fuzzy decision rules, and fuzzy reasoning ( Takagi and Sugeno 1985 ). It consists of a left-half side (also called “if” or “antecedent” side) and a right-half side (also called “then” or “consequence” side). Conventionally, each linguistic state variable has one or more fuzzy sets that are represented by a linguistic “value.” Such fuzzy sets are characterised by associated membership functions over the Universe of Discourse of a specific variable. A state membership value (μ(X)) represents the “degree of membership” of the state variable x in a fuzzy set (linguistic value), or the “degree of truth” of x , taken as an actual value. The outcome is a number within the interval [0,1], in which 1.0 signifies a full membership. Each rule may include every combination of state (μ(x)) and input memberships (μ(u)) on the left-half side and must incorporate a state membership value calculation (μ(x)) into the right-half side, indicating how an alteration in a specified state could occur.
An ANFIS is a first-order Sugeno-type fuzzy inference system where the membership function parameters are fitted with a specific data set by a hybrid-learning algorithm ( Jang 1993 ). Its structure ( Figure 5 ) consists of a first layer (values layer), in which the nodes represent sets of each state variable (input). At this same layer, membership values are computed, and the membership functions are deemed to be adaptable. The second layer (rules layer) implements the t-norms for modelling the logical fuzzy “AND” operator and computing the rule matching factor. The third layer (normalisation layer) acts to scale the firing strengths (matching factors).
The output of the fourth layer (function layer) is comprised of a linear combination of the inputs multiplied by the normalised firing strength w : where p and r are adaptable parameters. The fifth layer (output layer) is a simple summation of the outputs of the fourth layer. The adjustment to modifying parameters is a two-step process. First, information is propagated forward through the network to the fourth layer, where the parameters are identified by a least-square estimator. Then the parameters in the second layer are modified using gradient descent backpropagation. The number of membership functions in the Universe of Discourse for each input and the output training information must be specified at this phase.
Experimental procedures for analysing the data set
All the tests and results were derived from programming Matlab 6.5®. An optimum network and parameter configuration for each of the three networks deployed was established by trial and error. The input layer of every network utilised in this study consisted of three input nodes, representing the independent variables (initial number of larvae, amount of food, and duration of immature stages). The output of the network models was the dependent variable (number of survivors).
It has been suggested that a one-hiddenlayer MLP permits the approximation of any continuous function, provided that an adequate number of nodes in this layer are found; two hidden layers are sufficient to furnish a “best” approximation for any nonlinear mapping ( Cybenko 1989 ; Haykin 1999 ). The introduction of additional hidden layers in the network architecture could allow the resolution of more complex problems. Nevertheless, such an introduction reduces the generalisation ability of the network, while the training time increases ( Foody 1995 ). Haykin ( 1999 ) stated that the number of hidden nodes should be as small as possible while still allowing the network to retain a performance close to the optimum. Because the prediction of the number of survivors (output) may be adequately represented by a continuous function ( Figure 2 ), the MLP network was deployed with one hidden layer.
The number of nodes in the hidden layer of the MLP network and the stopping criteria were optimised with the purpose of obtaining precise and accurate output values. The hidden layer consisted of five neurons. The activation function of the hidden layer was the hyperbolic tangent sigmoid function, and the linear function was used for the output neuron. The Levenberg-Marquardt training algorithm ( Hagan and Menhaj 1994 ) was selected. During the training processes, the stopping criteria fixed the number of epochs at 2000. The target error for this phase was set at 0.001.
The number of hidden nodes of the RBF network was automatically found to be 25. The outputs of the hidden layer neurons were determined by the Euclidean distance between the network input and the centre of the basis function. The spread of the gaussian function was empirically set at 0.78. The RBF network output was formed by the weighted sum of the hidden layer neuron outputs and the unity bias. Additionally, the target error deployed was the same used with the MLP network.
Regarding the ANFIS model, three bellshaped membership functions (low, medium, and high) were determined for the number of larvae and amount of available food, whereas four triangular membership functions (very short, short, long, and very long) were set for the duration of immature stages. The optimisation method used in the training of the input membership function parameters was the Backpropagation learning algorithm. The output membership functions were linear zeroth and first-order Sugeno-type system. The number of epochs was fixed at 500, and 0.001 was taken as the target error; the other parameters were determined by trial and error in order to reach the best performance. The membership functions were learned from an adaptive neuro-fuzzy inference system. Furthermore, a set of 36 fuzzy rules was implemented, and their weights were adjusted in order to feasibly model the training data.
The entire data set was split into two subsets that were used for training and testing each neural network. Accordingly, the whole data set ( n = 40) was divided randomly into two subsets, namely the training and testing subsets. Specifically, the ratio of training examples to testing examples was 30:10. Regarding the multiple linear regression, the training and testing subsets were established identically. The ratio of 30:10 was deployed in each training procedure with respect to all the models. Furthermore, a cross-validation strategy was not utilised, and the estimation errors could vary depending on the data subset.
The inputs and targets were normalised in order to have zero means and unity standard deviations. Moreover, the outputs were trained to produce outputs with zero means and unity standard deviations. Each network converged after reaching the maximum number of epochs. The training error was 0.0129 for MLP and 0.0409 for RBF. Then the network outputs were restored to their original values (raw data) in order to calculate the R 2 between estimated and observed values of the training and testing subsets. | Results
Table 1 shows the coefficients of determination (R 2 ) and root-mean-square errors that were obtained from the data subsets utilised both in training and in testing procedures in every model, including the multiple linear regression. As a whole, the linear regression exhibited the lowest accuracy (the lowest R 2 and highest root-mean-square error values).
Regarding the neural networks, the R 2 derived from the RBF (0.715) was the lowest in the testing subset, even though its R 2 in the training subset exhibited virtually a maximum value (0.999). All the models had lower R 2 values (less accuracy) in the testing subset compared with the training subset.
The ANFIS model permitted the achievement of the best testing performance (the highest R 2 and the lowest root-mean-square error). Hence, this model was deemed to be more effective than the MLP and RBF networks in predicting the number of survivors. The output values predicted by the ANFIS model fitted the actual data (original values) in a satisfactory way ( Figure 6 ). | Discussion
The sample size utilised in the present work ( n = 40) may be sufficient to conduct most ecological and biological approaches concerning the nutritional ecology of blowflies. Nevertheless it is important to note that larval density and amount of food (two of the input variables) provided the multiple regression model with few distinct values ( Figure 2 ), because only 11 densities (75, 150, 300, 450, 600, 900, 1200, 1800, 2400, 2700 and 3600 larvae) and four distinct amounts of available food were utilised (15, 30, 60 and 90g), making up the five proportions of larvae per gram of food used in this work (5, 10, 20, 30 and 40). Therefore, the larger amount of distinct values measured for the output variable (resultant adults), using the same combination of values of those input variables, caused a lack of fit that decreased the prediction capability of the models.
Nonetheless, the amount of distinct values of larval densities deployed in the present study was considerably larger than those widely used in experimental designs on nutritional ecology of blowflies ( Mackerras 1933 ; Slansky and Rodriguez 1987 ; Goodbrod and Goff 1990 ; Von Zuben et al. 2000 ). Furthermore, owing to operational limitations, such studies would be impracticable if numerous levels of larval density were used ( Von Zuben et al. 1993 , 2001 ). Thus, the use of artificial neural networks would be justified in this case, since the neural algorithms coped with the lack of fit better than the multiple regression method did ( Table 1 ).
The RBF network exhibited a less accurate performance (lowest R 2 and highest rootmean-square error in the testing subset) in the present study, considering only the three neural network models that were employed. On the other hand, in a study conducted by Zhang and Barrion ( 2006 ) based on invertebrate data sampled in an irrigated rice field, RBF was considered an effective approach to function approximation and documentation of sampling information. Such results may serve as a basis for further discussions about the feasibility of RBF networks in entomology in terms of their underlying attributes and robustness.
Howe et al. ( 2007 ) modelled the body temperature and activity of a widespread butterfly species ( Polyommatus icarus ) in relation to weather, with the aim of predicting how future climate may influence its activity. These authors utilised a multilayer feed-forward backpropagation network in order to accomplish their objectives, and this neural model was deemed to be superior to a generalised linear modelling approach to predicting body temperature. In the context of nutritional ecology, the MLP network performed better than two other models, namely, the linear regression and the RBF network ( Table 1 ).
Zhang et al. ( 2008b ) investigated the spatial distribution pattern of grassland insects by means of neural models. They concluded from their results that neural networks were more flexible than a conventional model. Additionally, these authors indicated that further research based on more complex distribution patterns should be conducted with the aim of obtaining more reliable conclusions. Similarly, neural networks were deemed to be superior to a conventional model (linear regression) in the current study. Nevertheless, the present work utilised simple experimental designs and only three explanatory variables (input). Therefore, more complex neural network studies should be implemented in order to explain the portion of the total variance that was not accounted for by the models. Furthermore, larger sample sizes are highly desirable because the number of examples (sample size) that is usually utilised in nutritional ecology may prevent the full utilisation of the potential of neural networks.
Owing to the relatively small number of distinct values, the input variables of larval density and amount of food did not permit the “best” possible fit, including with the neural models, between predicted and collected values. Nonetheless, their incorporation into the modelling was very important. If duration of immature stages were the only input variable, the modelling would have considerably lost its biological meaning ( Getz 1984 ; Arditi and Saïah 1992 ; Papandroulakis et al. 2000 ; Von Zuben et al. 2000 ). Moreover, it would not have been possible to establish the suitable larval density and amount of food that could determine a particular number of resultant adults ( Von Zuben et al. 2000 ).
The ANFIS network exhibited the most accurate performance in the testing subset, although further studies may be necessary for corroborating this superiority in other practical experiments. On the whole, the neural networks outperformed the multiple linear regression ( Table 1 ), indicating that neural models could be taken as feasible techniques for predicting bionomic variables concerning the nutritional ecology of blowflies. The present study constitutes only a first approach, albeit with promising applicability, in which the ecology of blowflies was effectively analysed by means of neural models.
The employment of a simple linear regression requires a nontrivial amount of statistical expertise. The use of a multiple non-linear regression model such as an MLP requires more knowledge and experience ( Sarle 1994 ). Therefore, it is important to highlight the usefulness of multidisciplinary studies with the aim of conducting effective investigations of bionomic parameters.
Future studies may consider other complex variables that were not assessed in the current work. Ambient temperature, for example, was utilised by Zhang et al. ( 2008a ). These authors deployed neural network algorithms (functional link artificial neural network) in order to model the food intake dynamics of larvae of S. litura (Lepidoptera). Six different temperatures were used for measuring the food intake, and the neural network approach was deemed to be accurate.
It is possible that unresolved problems concerning the bionomics of immature and adult individuals of blowflies could be disentangled and clarified by the use of neural models. The employment of such complex analytical tools may help entomologists to feasibly schematise the sort of practical situations in which the utilisation of artificial networks is able to provide new insights into the nutritional ecology of blowflies. | Associate Editor: David Morton was editor of this paper.
Bionomic features of blowflies may be clarified and detailed by the deployment of appropriate modelling techniques such as artificial neural networks, which are mathematical tools widely applied to the resolution of complex biological problems. The principal aim of this work was to use three well-known neural networks, namely Multi-Layer Perceptron (MLP), Radial Basis Function (RBF), and Adaptive Neural Network-Based Fuzzy Inference System (ANFIS), to ascertain whether these tools would be able to outperform a classical statistical method (multiple linear regression) in the prediction of the number of resultant adults (survivors) of experimental populations of Chrysomya megacephala (F.) (Diptera: Calliphoridae), based on initial larval density (number of larvae), amount of available food, and duration of immature stages. The coefficient of determination (R 2 ) derived from the RBF was the lowest in the testing subset in relation to the other neural networks, even though its R 2 in the training subset exhibited virtually a maximum value. The ANFIS model permitted the achievement of the best testing performance. Hence this model was deemed to be more effective in relation to MLP and RBF for predicting the number of survivors. All three networks outperformed the multiple linear regression, indicating that neural models could be taken as feasible techniques for predicting bionomic variables concerning the nutritional dynamics of blowflies.
Keywords | Acknowledgements
We wish to acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo — FAPESP (for the first author) and of the Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (for the second author).
Abbreviations
Adaptive Neural Network-Based Fuzzy Inference System;
Multi-Layer Perceptron;
Radial Basis Function | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Jun 9; 10:58 | oa_package/d0/d6/PMC3014792.tar.gz |
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PMC3014793 | 21209805 | 1. Introduction
Infertility affects approximately 10–15% of couples, and male factor infertility represents almost 50% of cases [ 1 ]. Among infertile men about 40–50% have azoospermia and severe oligozoospermia. Genetic abnormalities may cause infertility by affecting sperm production or sperm transport. These abnormalities are numerical (e.g., trisomy) and structural (e.g., inversions or translocations) chromosomal abnormalities, Y chromosome microdeletions, and gene mutations (e.g., cystic fibrosis). The frequencies of genetic abnormalities vary between 10–15% in oligozoospermic and azoospermic patients [ 2 ]. Because of this high prevalence, it is important to investigate the chromosomal abnormalities in these patients. | 3. Materials and Methods
Molecular cytogenetic and Y chromosome microdeletion analysis was performed according to standard methods on cultured cells from the patient's peripheral blood. Genomic DNA was isolated in accordance with kit protocol (High pure PCR template preparation kit, Roche). In the laboratory, kit which is used for determination of Y chromosome microdeletion is certified with IVD (in vitro diagnostic). 13 STS (sequence tagged site) regions on Y chromosome (ZFX/ZFY, SRY, AZFa: sY84, sY86, DFFRY, DBY, AZFb: sY117, sY125, sY127, sY134, AZFc: DAZ gene sY254, sY255) were analysed by using 3 different polymerase chain reaction (PCR) mixes (Genequality AZF MX, AB-Analitica). Multiplex PCR reaction was performed with isolated genomic DNA samples in conformity with kit protocols. PCR products were subjected to 85 V for electrophoresis during 2 hours and 30 minutes in 3% mucinous agarose gel. | 4. Results
When the results of electrophoresis were investigated with UV transilluminator, band patterns were detected only in ZFX/ZFY wells of all 3 PCR mixes. There was no band pattern in wells of other 12 STS regions ( Figure 1 ). This situation was not compatible with phenotype of patient and could occur due to allogenic BMT from his sister. Therefore, chimerism analysis was done with FISH using CepX/Y DNA probe (Vysis) in order to indicate chimerism rate of sex chromosomes in cells of the peripheric circulatory system of the patient. The CEP X/Y DNA probe is a mixture of a SpectrumOrange labeled CEP X DNA probe (Xp11.1-q11.1 Alpha Satellite DNA) and a SpectrumGreen labeled CEP Y DNA probe specific for the alpha satellite centromeric region of chromosome X and the satellite III (Yq12) region of chromosome Y. Two orange signals of Xp11.1-q11.1 Alpha Satellite DNA were found on the interphase chromosome in 200 cells of peripheric circulatory system of patient. It was determined that chimerism rate was 100% XX ( Figure 2 ). Towards this result, genomic DNA was isolated from buccal mucosa smear in order to obtain the patient's own cells. Isolated genomic DNA was evaluated with NanoDrop spectrophotometer (NanoDrop, Thermo Scientific, USA) to detect the density of this sample. 1 μ L DNA of patient was defined as 11.06 ng and rate of A260/A280 was defined as 1.42 which means that this amount was enough for PCR. Then, PCR was performed in accordance with kit protocol that was used previously. PCR products were subjected to 85 V for electrophoresis during 2 hours and 30 minutes in 3% mucinous agarose gel. Bands in wells of 3 different mixes including 13 STS regions were detected with UV transilluminator. There were band patterns in all wells, and Y chromosome microdeletion was not detected in patient ( Figure 3 ). Sex chromosomes of patient were evaluated in terms of aneuploidy with multiplex PCR in conformity with kit protocol (Aneufast-QF PCR) used with Quantitative Fluorescent PCR (QF-PCR) technique. The QF-PCR Kit contains six multiplex marker sets of short tandem repeats (STRs) that can be used for amplification of selected microsatellites and the Amelogenin-SRY. PCR products were subjected to capillary electrophoresis in automatized device of DNA strand analysis (ABI-3100 Avant). Fragment analysis showed that the patient had XY chromosomal constitution ( Figure 4 ). After PCR, AZF a, b, and c loci that were localized on Y chromosome long arm and did not generate recombination with X chromosome in meiosis were amplified (in the agarose gel, band pattern was positive in wells of 10 STS regions special to AZF loci). This also excludes the situation that patient could have XX sex chromosome in the beginning. | 5. Discussion
The incidence of Y chromosome microdeletions was reported 10–15% and 5–10% in azoospermic and oligozoospermic patients, respectively [ 3 ].
In the literature, there is only one report defining a male with the diagnosis of CML and 46,XX chromosomal constitution [ 4 ]. That patient, like the present case, had bilateral small testes, azoospermia, and hypergonadotropic hypogonadism. Cytogenetic and molecular analysis has demonstrated that male XX is caused by at least three mechanisms, abnormal Y-X interchange (90%), genes other than testes-determining factor (TDF), and mosaicism [ 5 ]. It is anticipated that malignant disease itself and adjuvant or neoadjuvant regimens used for the treatment may result in oligozoospermia and azoospermia [ 6 ]. Thachil et al. reported that cancer itself has negative effects on fertility before application of any treatment modality [ 7 ]. It is also reported that chemotherapeutic agents used in the treatment of CML, like cyclosphosphamide, hydroxyurea, busulphan, chlorambucil, and radiotherapy, have deleterious effects on germ cells which may result in azoospermia [ 8 , 9 ].
The reason of azoospermia in the present case in unclear whether it may be due to CML itself or the treatment regimens used to cure the disease.
In another study, it is reported that the rate of recovery of spermatogenesis was 17% in patients conditioned with cyclophosphamide combined with total body irradiation (TBI) before BMT. Recovery of spermatogenesis never occurred before the fourth year after transplantation and was observed up to 9 years. The incidence of azoospermia in those patients was 70.3% [ 10 ]. The rate of gonadal dysfunction in patients treated with cyclophosphamide with a cumulative dose of less than 400 mg/kg was less than 10% whereas this rate was increased up to 30% in prepubertal and 68–95% in adult patients treated with a cumulative dose of more than 400 mg/kg cyclophosphamide [ 9 , 10 ].
During TBI, the diffused testicular dose is estimated to be 1-2% of the total dose applied. Direct irradiation (0.15–0.35 Gy) causes oligozoospermia; doses between 0.35 Gy and 0.5 Gy cause reversible azoospermia. Doses of 1.2 Gy are associated with a reduced risk of recovery of spermatogenesis. Time to recovery is also likely to depend on the dose [ 8 ]. Cumulative doses of fractionated radiotherapy of >2.5 Gy generally result in prolonged and likely permanent azoospermia. Radiation doses to the germinal epithelium of the testis given in 3- to 7-week fractionated courses cause more gonadal damage than single doses. An irreversible damage of the spermatogenesis will begin at a cumulative dose of >2.5 Gy when the radiation is fractionated [ 11 ]. The present case was conditioned with cyclophosphamide 60 mg/kg for two days and TBI with a total dose of 12 Gy in 3 fractionated doses before BMT. Recovery of spermatogenesis never occurred during the 8-year followup after BMT.
Pretreatment sperm cryopreservation, although not applied in this case, should be offered to young, male cancer patients to prevent infertility due to the therapeutic regimens which causes oligozoospermia or azoospermia.
To the best of our knowledge, the study does not provide a new information methodologically but from the point of clinical view this is the first NOA case with the diagnosis of CML and 46,XX chromosomal constitution due to allogeneic BMT from his sister. In conclusion, genomic DNA investigation in patients undergoing BMT should be done with patients' own cells (buccal mucosa, tissue biopsy, etc.) to obtain accurate results about the chromosomal constitutions of these patients. | Academic Editor: Mamede de Carvalho
Genomic DNA of a patient diagnosed with nonobstructive azoospermia and with the history of allogenic bone marrow transplantation from his sister due to chronic myeloid leukemia was isolated from peripheral blood in order to screen Y chromosome microdeletions. 13 short tagged sites belonging to AZF a, b, and c loci were detected with multiplex polymerase chain reaction technique. Bands were determined in ZFX/ZFY wells, whereas no bands were determined in wells of other STS regions. DNA isolation was done from buccal mucosa smear to obtain genomic DNA from patient's own cells and multiplex polymerase chain reaction technique was performed again. Bands were seen in all wells of 13 STS regions. Y chromosome microdeletion was not detected in the patient. In conclusion, genomic DNA isolation in patients undergoing BMT should be done from patients' own cells. | 2. Case Report
A healthy 27-year-old man with normal phenotype was referred for the evaluation of infertility. In his physical examination, right testis was 10 cc, left testis was 8 cc with grade 1 varicocele. Vasa deferentia were palpable on both sides. Consecutive two semen analyses of the patient revealed azoospermia with normal ejaculate volume. His serum FSH was 24.29 mIU/mL (1.6–12.4 mIU/mL) and total testosterone was 495 ng/dL (260–1600 ng/dL). The patient with the diagnosis of nonobstructive azoospermia (NOA) had a history of allogeneic bone marrow transplantation (BMT) from his sister due to chronic myeloid leukemia (CML). Karyotyping and Y chromosome microdeletion analysis, which are necessary for the evaluation of NOA, were done with peripheral blood leucocytes and buccal mucosa samples because of the probable change in peripheral blood leucocytes after allogeneic bone marrow transplantation. There was no family history of infertility.
The patient, who had the diagnosis of CML with positive Philadelphia (Ph) translocation in February 1999, was treated with Hydroxyurea 500 mg/day for two years. The patient was conditioned with cyclophosphamide 60 mg/kg for two days and TBI with a total dose of 12 Gy in 3 fractionated doses before BMT. In 2001, allogeneic BMT was performed. After allogeneic BMT, the patient was treated with 26 mg methotrexate and 30 gr IVIG. In cytogenetic analysis performed on cultured cells from bone marrow material before allogeneic BMT, no metaphases were examined. After BMT, cytogenetic analysis revealed 6 metaphases examined by trypsin GTG banding, and 4 of them had 46,XX chromosomal constitution. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 16; 2010:541061 | oa_package/1a/44/PMC3014793.tar.gz |
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PMC3014794 | 20572786 | Introduction
In the twilight zone of caves, within recessed areas of walls and ceiling depressions the cave orb weaver spider, Meta ovalis Gertsch (Araneae: Tetragnathidae), is frequently observed coinciding with the presence of large roosting aggregations of the cave cricket, Hadenoecus cumberlandicus Hubble & Norton (Orthoptera: Rhaphidophoridae). In addition, H. cumberlandicus and M. ovalis can be found out in the open, at wall/ceiling junctures. This cave cricket-spider cooccurrence is so regular, in fact, that the presence of one typically is used to indicate the presence of the other in survey work; the large, easily noticed M. ovalis , in particular, is especially useful in this regard as a landmark for pointing out large aggregations of H. cumberlandicus (mixed stages) that would otherwise be concealed in expanded ceiling fractures or joints overhead. This codistribution between H. cumberlandicus and M. ovalis is not only observed in many of the caves in northeastern Kentucky (Carter County), USA, but also throughout the majority of cave systems in North America where cave crickets ( Hadenoecus spp.) and M. ovalis are found ( Hill 2003 ; Lavoie et al. 2007 ; HH Hobbs III, unpublished data). The large orb weaver spider, M. ovalis , although not present in all of these caves, is recognized as an H. cumberlandicus predator ( Lavoie et al. 2007 ). On the other hand, the cave cricket, Hadenoecus spp., is more ubiquitous and is a dominant member of the cave community as a keystone species, providing a predictable source of guano that supplies nutrients (both directly and indirectly) to an array of springtails, beetles, flies, and millipedes that thrive below the Hadenoecus roost on the cave floor ( Poulson et al. 1995 ). Thus, M. ovalis are commonly found at sites where H. cumberlandicus aggregate.
The size of the aggregation usually consists of 5–10 individuals per cluster but may reach densities greater than 200 individuals ( Hobbs 1992 ; Hill 2003 ). Relative humidity is high, approximately 97% RH, temperature is moderate, averaging 15° C, and dry air currents are reduced in kettles (or bells) in the upper walls and ceiling of the cave where these aggregations are formed ( Hobbs 1992 ). Selection of these stable, humid, unventilated sites by H. cumberlandicus is highly favorable for maintaining water balance ( Studier et al. 1987 ; Studier and Lavoie 1990 ) and also for protecting against predation by certain ground-dwelling beetles by keeping H. cumberlandicus high above the ground ( Peck 1976 ). The formation of the aggregation itself has the benefit of enhancing water conservation behaviorally via a group effect that further protects the H. cumberlandicus from desiccation ( Yoder et al. 2002 ). Lavoie et al. ( 2007 ) noted that H. cumberlandicus repeatedly use the same sites within the cave for aggregation, presumably one generation after the next. Poulson et al. ( 1995 ) and Hill ( 2003 ) reported that H. cumberlandicus return from the outside after nightly foraging bouts to previously occupied sites within the cave where they, in turn, defecate copiously. As such, these highly specific sites used by H. cumberlandicus within the cave are localized regions of heavy guano accumulation.
This study tests the hypothesis that H. cumberlandicus is responding to an aggregation pheromone, because of the apparent derivation of this pheromone from feces (i.e. guano), supported by previous work on crickets ( Nagel and Cade 1983 ; McFarlane et al. 1983 ) and locusts ( Byers 1991 ), site persistence, and resultant expression of aggregation behavior. This study also seeks to examine whether this H. cumberlandicus aggregation pheromone, if present, is attractive for M. ovalis , which may explain the co-occurrence of this predator in proximity to the H. cumberlandicus roost by acting as a host cue (kairomone). It is not uncommon for a predator to use excretory products of prey as a potential signal of healthy prey and their abundance ( Weiss 2006 ) or for predator excreta to be associated with reduced movement by prey to go undetected as a novel form of antipredator behavior ( Persons and Rypstra 2001 ; Kortet and Hedrick 2004 ; Wilder and Rypstra 2004 ). As such, uric acid (varying concentrations) is evaluated as one of possible active components mediating the aggregation response in H. cumberlandicus because of its high occurrence as a component of excreta in most insects ( Nation 2001 ). Additionally, a combination of laboratory and field experiments were used because of the sensitivity of trogloxenes, especially when taken out of the cave environment, so that it can be determined to what extent laboratory observations reflect how H. cumberlandicus may behave in the deep cave environment. | Materials and Methods
Crickets, spiders, and test conditions
The study site was the dry upper level of Laurel Cave (N 38° 22′ 30.8′′ W 83° 06′ 55.4′′) in Carter Cave State Resort Park (Carter County, Kentucky, USA) from August to October, 2003–2007. The study site was in a dry, phreatic, 280 m long passage that was situated above a lower active vadose stream level. Laurel Cave is a multi-entrance cave system developed in Mississippian limestone (total cave length 1091 m). This area is a horizontal, tubular paleo-trunk conduit ranging in height from 1–4 m that intersects the lower stream passage approximately 4.5 m above the stream and about 200 m northwest of a 1 m high × 4 m wide entrance ( Pfeffer et al. 1981 ). There are no active streams, only epikarstic drip input in the study area that has an extensive twilight zone (30+ m), a dark zone, and is easily accessible by H. cumberlandicus and M. ovalis through cave entrances or crevices leading to the surface. During winter, cold, dry air blows into this level via the entrance, causing H. cumberlandicus to move further into the cave; whereas in summer, air blows out of the entrance and H. cumberlandicus are not restricted to the deeper confines of the upper level. The area of the cave that was used for these experiments involved a total of five separate study sites within an expanded area of the twilight zone approximately 20 m from the entrance where H. cumberlandicus have been shown to be abundant ( Hill 2003 ).
A parthenogenic population of H. cumberlandicus exists in Laurel Cave ( Hubbell and Norton 1978 ). Adult H. cumberlandicus are distinguished by a large, darkly sclerotized ovipositor and are of larger size than nymphs. Male and female M. ovalis are easy to differentiate because of the male pedipalps ( Ubick et al. 2005 ). None of their ages were known, but experiments were conducted during the same time of year, from August to October. Transport back to the laboratory was done in plastic coolers (ca. 8,000 cc, l × w × h) containing moist leaf litter, and H. cumberlandicus and M. ovalis were used for experiments within 24 h after collection. All handling of specimens was done with an aspirator and forceps. Conditions for the laboratory experiments were 15° C ± 1° C, 97% RH (saturated K 2 SO 4 solution; Winston and Bates 1960 ), in total darkness (0:24 L:D), maintained in an environmental room. Conditions at the study site in Laurel Cave, where experiments were conducted, were 12 ± 2° C and 98 ± 3% RH (thermo/hygrometer, Fisher Scientific, www.fishersci.com ) and were consistent with measurements recorded in this region of the cave as reported by Hill ( 2003 ). Observations were made in red light.
Uric acid, natural pheromone collection and target preparation
Uric acid was purchased commercially (Sigma Chemical Co., www.sigmaaldrich.com ) and was diluted with HPLC grade acetone (Sigma); acetone also served as a control. All preparation and application of solutions were done using glass (miscellaneous glassware and 50 μ l pipettes, accuracy ± 0.25%, precision < 0.6%; Fisher). Filter paper was No. 3 Whatman ( www.whatman.com ) with an internal diameter of 9 cm. These filter paper discs served as targets in the attraction bioassays. All materials were handled using gloves and forceps to avoid contact with human skin oils. Any labeling on the filter paper disc was done with a graphite pencil (No. 2, Pentel, www.pentel.com ).
Natural aggregation pheromone was collected by placing H. cumberlandicus , 10 at a time, in a plastic cooler (ca. 2,000 cc, l × w × h) containing a filter paper disc for 48 h, modified from aggregation pheromone collection by Nagel and Cade ( 1983 ) and McFarlane et al. ( 1983 ). Collection of natural pheromone was made from female adults as well as nymphs (mixed stages) and a total of 10 filter paper discs were prepared each from a separate population of H. cumberlandicus (10 from adults and 10 from nymphs).
To prepare the filter paper discs of uric acid, each dilution, [0.1M], [0.01M] and [0.001M] uric acid, was tested separately and applied (four applications of 50 μ l each, with air drying in between applications) at the center of the 9 cm filter paper disc and then allowed to air dry, making a spot application that did not exceed 3 cm in diameter. Three different sets of uric acid dilutions were prepared, and a fresh filter paper disc was used for each experiment. Control filter paper discs were those treated with 200 μl acetone, as well as untreated filter paper discs. Only one dilution of uric acid was tested at a time.
Description of the bioassay
Experiments focused on a two-choice bioassay set up, providing a choice between two control filter paper discs (untreated or acetone) and two test filter paper discs (natural pheromone or dilution of uric acid) in a statistically valid four-quadrant block design (enlarged version of design from Arlian and Vyszenski-Moher ( 1995 ). The study area measured 1 m × 1 m and was divided into four equal sized quadrants (50 cm on each side of the midpoint); this was constructed using cotton thread (Anecot T-30, American and Efird, www.amefird.com ) on the walls of the cave (securing the ends of the thread with poster putty) and on the outside of a clear plexiglass chamber (described below) in the laboratory tests. Each 50 cm × 50 cm quadrant was subdivided into four quadrants and the filter paper disc was placed at the center of the point of intersection (25 cm from the sides of each quadrant). The filter paper discs were placed in the study arena such that they alternated between quadrants: control (quadrant 1), test (quadrant 2), control (quadrant 3), test (quadrant 4). Therefore, a test quadrant was in between two control quadrants.
H. cumberlandicus were placed, ten at time, at the center of the arena. In the laboratory, the experiments were conducted in a clear plexiglass chamber (491,400 cc; 140 cm 1 × 130 cm w × 27 cm h) with the filter paper discs secured on the floor of the chamber that was then inverted so that the crickets were in an upside down position like they are in the cave; discs were at least 15 cm away from the walls of the chamber. Counts of H. cumberlandicus in the various quadrants (two control, two test/ study area) were made at 1 h and again at 6 h in both cave and laboratory settings. Filter paper discs were secured using nontoxic, unscented poster putty (Duck, Dial Co., St. Louis, MO).
Sample size and data analysis
Each experiment represents observations on a total of 40 H. cumberlandicus each; thus 10 H. cumberlandicus per replicate, n = 4. In the experiments where M. ovalis was used, total sample size was 15 for each observation; thus, 3 M. ovalis per replicate, n = 5. Data were expressed as percentage of H. cumberlandicus or M. ovalis that were counted in the test quadrants. Untreated filter paper discs were used for comparison and to rule out potential directional left/right bias. Percentage data were arcsin transformed and analyzed using chi-square (χ 2 ) statistics using 50% (20 H. cumberlandicus ) or 47% (7 M. ovalis ) as the expected (E) value, a = 0.05, d.f. = 1 ( Sokal and Rohlf 1995 ). | Results
H. cumberlandicus responses
Within 1 h, 80% of female adult H. cumberlandicus in the laboratory bioassay crawled to filter paper discs that had been previously exposed and defecated upon by adult H. cumberlandicus , with the majority of them, 73%, remaining around these discs up to 6 h, all exhibiting significant attraction ( Table 1 ). Nymphs reacted similarly at 1 h and 6 h with 70%) (28/40 H. cumberlandicus ) and 78%) (31/40 H. cumberlandicus ), respectively (c 2 = 1.17; p > 0.05). Similar results were observed if adults were introduced to filter paper discs that had been exposed to nymphs and nymphs responded similarly to discs that had been exposed to adults, yielding high percentage attraction values ranging from 73% (29/40) - 83% (33/40). In all cases, H. cumberlandicus crawled directly to odor sources and stayed there. There was little movement between quadrants during the 6 h test period; in most cases, H. cumberlandicus made direct contact with the filter paper disc, with other individuals clustering around. The results to discs treated with uric acid produced significant attraction at [0.1M] uric acid at 1 h, but this was not significant at 6 h; and there was a significant response after 6 h at [0.001M] uric acid that was not significant at 1 h. Response to [0.01M] uric acid did not produce any significant stimulatory effect ( Table 1 ). No dose reaction to increasing uric acid concentration was evident, and the response to uric acid was lower than to natural aggregation pheromone that was collected from H. cumberlandicus ( Table 1 ). Over the range of uric acid concentrations, the combined effects of uric acid concentration compared favorably to controls (acetone-only and untreated filter paper) when averaged at 1 h (52% average of [0.1M], [0.01M] and [0.001M] uric acid responses) and 6 h (56% average of [0.1M], [0.01M] and [0.001M] uric acid responses) observations.
Similar results were obtained in the cave, with 68%) and 88% of H. cumberlandicus being attracted to filter paper discs that had been previously defecated upon and variable responses (low, if any, level attraction at [0.1M] and [0.01M] uric acid and even repellency at [0.001M] uric acid) to filter paper discs treated with uric acid. Filter paper discs that had been exposed to female adults were detected by nymphs, and filter paper discs that had been exposed to nymphs were attractive to female adults, with 78% (31/40) and 73% (29/40) attraction and arrestment, respectively, after 6 h (χ 2 = 0.94; p > 0.05). There seemed to be stronger recruitment at 6 h than at 1 h in the cave with the natural H. cumberlandicus aggregation pheromone ( Table 1 ). In both laboratory and cave settings, uric acid treatment failed to cause a retention effect, because there appeared to be more H. cumberlandicus movement to and from discs. Percentage attraction values for H. cumberlandicus in laboratory bioassays to acetone-only controls were 43% after 1 h and 55% after 6 h and were not significantly different from response to untreated filter paper, 48% and 40% attraction, respectively, (c 2 = 1.31; p > 0.05; Table 1 ) with fairly even distribution, implying that there was little left/right bias in the experiment. Similar results to these controls in the laboratory bioassays were obtained in the cave (χ 2 = 1.67; p > 0.05; Table 1 ), and these experimental values approximate the 50% expected (E) value that was used in the chisquare calculation. In conclusion, H. cumberlandicus respond by attraction and arrestment to filter paper discs that had been exposed to H. cumberlandicus but not to filter paper discs treated with uric acid.
Spider responses
In laboratory bioassays, filter paper discs that had been defecated upon by female adult H. ventured into the test quadrant. There was no dose effect to increasing uric acid concentration, and the number of M. ovalis in control quadrants (untreated and acetoneonly) was similar to values for M. ovalis that were counted in quadrants containing test attractants ( Table 2 ). Percentage attraction to uric acid by M. ovalis across all concentrations ([0.1M], [0.01M], [0.001M] uric acid) averaged 31% at 1 h and 47% at 6 h, and this was similar to control results .
In the cave, female adult H. cumberlandicus- contacted filter paper discs elicited no significant attraction activity to M. ovalis at 1 h or at 6 h ( Table 2 ). At no time did M. ovalis change their course of direction in response to the test materials after they had been released in the bioassay arena. Recruitment to discs that had been exposed to nymphal H. cumberlandicus was similar to that toward filter paper discs made by exposure to adult H. cumberlandicus by not prompting significant activity by M. ovalis (data not presented). Uric acid-treated discs produced significant attraction by M. ovalis at [0.1M] uric acid at 1 h and at [0.001M] uric acid at 6 h, and repellency at [0.001M] uric acid at 6 h ( Table 2 ). Results with increasing uric acid showed that it was not dose-dependent. When averaged across uric acid concentrations ([0.1M], [0.01M], [0.001M] uric acid), 55% of M. ovalis were in uric-acid treated areas at 1 h and 45% were present in uric acid-treated areas at 6 h, and these values were highly reminiscent to acetone-only and untreated controls ( Table 2 ). In both laboratory and cave settings, the reaction by M.ovalis to controls were variable, ranging from 20% to 67%, and this reflects the movement and rather constant change in position in the bioassay arena by M. ovalis within the test arena which characterized the spider's reactions to the various H. cumberlandicus -associated test materials not occurring with any regular frequency. Collectively, counts of M. ovalis in treatment quadrants overlapped with counts of M. ovalis in control quadrants. The conclusion is that female adults of the cave orb weaver spider, M. ovalis , displayed no preference for filter paper discs that had been exposed to H. cumberlandicus or to uric acid. | Discussion
In the cave cricket, H. cumberlandicus , the aggregation pheromone that operates is characterized by several key features, most of which are reminiscent of aggregation pheromone described in other orthopterans. The response involves attraction, indicating that there is long-range detection and thus a volatile active ingredient. There is a retention effect, where H. cumberlandicus exhibits reduced ambulatory activity (arrestment) on treated areas, leading to collections of numerous individuals, occasionally in direct contact with each other (little, if any, movement up to 6 h to adjacent quadrants after attraction). The response is not stage-specific, because nymphs react to excreta from female adults and female adults react to excreta from nymphs, which rules out classification of this chemical cue as a sex pheromone (furthermore, this test population of H. cumberlandicus is parthenogenic, where a sex pheromone has no purpose). Although speculative, the apparent derivation of aggregation pheromone from feces agrees with the abdominal origin of aggregation pheromone in the house cricket, Acheta domesticus ( McFarlane et al. 1983 ) and the camel cricket, Ceuthophilus secretus ( Nagel and Cade 1983 ). Although active components of H. cumberlandicus aggregation pheromone have not yet been identified (inconsistent results with uric acid rules it out experimentally), based upon related studies, it is conceivable that the active components are phenolic-based or organic acids derived from resident gut bacteria ( McFarlane et al. 1983 ; Dillon et al. 2002 ).
Cave orb weaver spiders, M. ovalis , displayed no attraction responses, and at no time did they stop crawling and remain immobile when coming into contact with filter paper discs that previously had been exposed to H. cumberlandicus ; i.e. this is earmarked by the number of M. ovalis in test quadrants that were similar to the number in control quadrants. Thus, there is no evidence that the aggregation pheromone of H. cumberlandicus contains an attractant or a retainer as a kairomone (host cue) to M. ovalis ; therefore, H. cumberlandicus excreta (i.e. guano accumulation) apparently fails to be detected by M. ovalis as a signal of H. cumberlandicus prey quality (healthy, well fed, undiseased individuals), presence, or abundance. There is also no evidence that H. cumberlandicus aggregation pheromone contains any kind of repellent to M. ovalis , because no blatant avoidance behavior was displayed by M. ovalis when introduced to the H. cumberlandicus aggregation pheromone. Often when spiders are predators, the spider's excreta is detected and modifies the behavior of potential prey by stillness and reduced movements to go undetected, as though proceeding cautiously, described as a novel form of antipredator behavior ( Persons and Rypstra 2001 ; Wilder and Rypstra 2004 ; Eiben and Persons 2007 ); prey (cricket) detects predators (spider) usually based upon the predator's diet ( Kortet and Hedrick 2004 ). In fact, reduced movement in response to spider excreta is precisely how field crickets, Gryllus integer , react to the presence of the spider, Hololena nedra ( Kortet and Hedrick 2004 ), and preliminary data testing spider excreta from M. ovalis suggest that this is also occurring in H. cumberlandicus (JB Benoit, The Ohio State University, Columbus, OH, unpublished data). In the present study, however, nymphs and female adults of H. cumberlandicus were shown to exhibit reduced activity (arrestment-retaining effect of aggregation pheromone) in response to their own excreta, thus it seems reasonable to suggest that the H. cumberlandicus aggregation pheromone similarly adds a useful defense function against predation by M. ovalis residing close to the H. cumberlandicus roost.
Cave crickets and cave spiders are characterized by losing water rapidly and drying out quickly ( Studier et al. 1987 ; Hadley et al. 1981 ), and, as such, they have a high moisture requirement (hydrophilic water balance classification; Yoder et al. 2002 ). Indeed, localized sites selected within the cave where H. cumberlandicus aggregations are formed are where air is nearly water-saturated, temperature is stable, and air currents are reduced; thus, based upon a similar ecologic hydrophilic water balance profile, it is not surprising that M. ovalis shares the same habitat site in in that they have the same water loss problem. This study demonstrated that selection of these preferred sites by H. cumberlandicus is guided by an aggregation pheromone. This aggregation pheromone acts to recruit H. cumberlandicus to these sites and to retain them at these sites. As a product of cluster formation, the net transpiration (water loss) rates of individuals are greatly suppressed by nearly two-fold, leading to a group effect that facilitates water conservation ( Yoder et al. 2002 ). Taken together, these considerations indicate that a few noteworthy attributes of H. cumberlandicus behavior are chemically-mediated by use of its aggregation pheromone: (1) habitat site selection within the cave, satisfying an absolute moisture requirement, presumably with access to outside food; (2) regulation of water balance via a resultant group effect; and, (3) perhaps defense against the predatory M. ovalis by antipredator behavior featuring reduced movement. | Associate Editor: Michael Breed was editor of this paper
Food input by the cave cricket, Hadenoecus cumberlandicus Hubble & Norton (Orthoptera: Rhaphidophoridae), is vital to the cave community, making this cricket a true keystone species. Bioassays conducted on cave walls and in the laboratory show that clustering in H. cumberlandicus is guided by a pheromone, presumably excreta. This aggregation pheromone was demonstrated by using filter paper discs that had previous adult H. cumberlandicus exposure, resulting in > 70% response by either nymphs or adults, prompting attraction (thus, active component is a volatile), followed by reduced mobility (arrestment) on treated surfaces. Adults were similarly responsive to pheromone from nymphs, agreeing with mixed stage composition of clusters in the cave. Effects of [0.001M – 0.1M] uric acid (insect excreta's principle component) on H. cumberlandicus behavior were inconsistent. This pheromone is not a host cue (kairomone) and is not used as a repellent (allomone) as noted through lack of responses to natural H. cumberlandicus pheromone and uric acid concentrations by a co-occurring predatory cave orb weaver spider, Meta ovalis Gertsch (Araneae: Tetragnathidae). This pheromone is not serving as a sex pheromone because nymphs were affected by it and because this population of H. cumberlandicus is parthenogenic. The conclusion of this study is that the biological value of the aggregation pheromone is to concentrate H. cumberlandicus in sheltered sites in the cave conducive for minimizing water stress. Rather than signaling H. cumberlandicus presence and quality, the reduced mobility expressed as a result of contacting this pheromone conceivably may act as a defense tactic (antipredator behavior) against M. ovalis , which shares this favored habitat site.
Keywords | Acknowledgments
Supported in part by a competitive research grant from Wittenberg University to JLT. | CC BY | no | 2022-01-12 16:13:46 | J Insect Sci. 2010 May 13; 10:47 | oa_package/9e/77/PMC3014794.tar.gz |
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PMC3014795 | 21209806 | 1. Background
Granulocytic sarcoma (GS) is a rare localized tumor composed of immature granulocytic precursors. These neoplasms are known as monocytic sarcoma and chloroma which refers to the green coloration of some of these tumors on gross examination [ 1 ]. It is usually associated with leukemia or other myeloproliferative disorders [ 2 ]. However, it occurs very rarely without hematologic diseases [ 3 ], and the diagnosis is difficult in such cases.
The most common sites of involvement are the skin, lymph nodes, soft tissues, and bone, but the tumor can occur virtually anywhere [ 2 – 6 ]. Although nonleukemic GS may be found in any location, primary occurrence in the knee is exceptional. Here we report a very rare case of GS of the knee but no bone marrow involvement at presentation which was accurately diagnosed and has been followed up for a long period. | 3. Discussion
GS is a rare extramedullary tumor that consists of immature granulocytic cells. this tumor was first described in 1811 and originally called chloroma by King in 1853 [ 7 ]. The term “chloroma” is derived from the Greek word chloros (green). The estimated incidence is about 0,7 per million in children and 2 per million in adults [ 8 ]. There is no sex predilection, with a mean age of 48 years (range: 2–81) [ 1 ]. GS occurs most commonly in bone, periosteum, soft tissue, lymph nodes, and skin, although it can occur anywhere throughout the body [ 9 ].
GS usually presents concomitantly with or after the onset of acute myelogenous leukemia, blastic phase of chronic myelogenous leukemia, or myelodysplastic syndrome [ 9 ]. However, it is seen very rarely without bone marrow disease as in our case and symptoms are secondary to the mass effect of the tumor. To the best of our knowledge, there has been only one case report of nonleukemic GS of the knee in the medical literature [ 10 ].
Macroscopically, GS is usually green in appearance. The green color, which is due to the presence of myeloperoxidase (MPO) in the leukemia cells, is not present in all tumors of this type [ 11 ]. According to the World Health Organization classification, the tumor is histologically classified into 3 types: well-differentiated, poorly differentiated, and blastic [ 12 , 13 ]. The diagnosis is often difficult in GS cases when the myeloblastic cells are poorly differentiated and the tumor lacks the characteristic green color. A large proportion (75%–86%) of GS in nonleukemic patients is initially misdiagnosed and frequently mistaken for non-Hodgkin lymphoma, small round cell tumor (neuroblastoma, rhabdomyosarcoma, Ewing sarcoma/PNET, and medulloblastoma), and undifferentiated carcinoma [ 8 ]. In such isolated presentation, early diagnosis is based on immunohistochemical analysis, especially staining of MPO and CD43. Traweek et al. suggested that an immunohistochemical panel including CD20, CD43, CD68, and MPO can successfully identify 96% of extramedullary myeloid cell tumors via paraffin sections [ 13 ].
The rarity of cases of nonleukemic GS has limited the availability of prospective studies on the effectiveness of various treatment strategies. Although no standard treatment protocol has been established, several previous reports suggested that local treatments such as surgical excision and/or radiation therapy alone would not be sufficient for the treatment of nonleukemic GS [ 1 , 2 , 14 ] and early aggressive chemotherapy may represent the best chance for remission [ 14 – 17 ]. Our patient received 6 cycles of CHOP-regimen because he refused bone marrow transplantation.
The long-term prognosis of nonleukemic GS remains poor [ 15 ], and the majority of these patients die of leukemia within an average of 16.5 months after diagnosis [ 9 ]. Uncommonly, however, cases of no progression have also been reported [ 18 , 19 ]. In our case, the patient has remained free of disease for 40 months.
GS is rare, and clinical diagnosis is especially difficult without bone marrow involvement. Immunohistochemical studies using an appropriate panel of markers are extremely helpful to make a correct diagnosis. The strategy for the management of nonleukemic GS should be individualized. Aggressive chemotherapy is beneficial and may induce complete remission. Early diagnosis and strict followup of these patients are essential for a better outcome in GS. | Academic Editor: Werner Rabitsch
Granulocytic sarcoma (GS) is a rare extramedullary tumor composed of immature myeloid cells. It is usually associated with leukemia or other myeloproliferative disorders. It occurs very rarely without overt hematologic diseases. A 19-year-old man presented with left knee mass. Biopsy with pathological analysis showed lymphoma aspect. Immunostains yielded the diagnosis of GS with myeloperoxidase and CD43 positivity. There was no systemic manifestation of leukemia, and bone marrow biopsiy was negative for neoplastic infiltration. Chemotherapy by CHOP was efficient, and the patient remaind alive and healthy 40 months after the end of treatment. The case is discussed in the framework of the existing literature about the diagnosis, treatment, and prognosis of this very rare condition. | 2. Case Report
A 19-year-old Berber Moroccan man began experiencing pain in his left knee. His medical history was otherwise unremarkable. Physical examination revealed no abnormality except for an 8 cm, mobile mass of left knee with no involvement of overlying skin. CTscan revealed a large mass of anterior left knee involving bone and soft tissue suspicious of sarcoma ( Figure 1 ). A radionuclide bone scan showed a single area of uptake of radiotracer in the left knee.
Biopsy showed a green colour of the tumour, and pathological analysis revealed diffuse proliferation of round cells with medium-sized or large nuclei lymphoma aspect. Immunostains showed myeloperoxidase and CD43 positivity and leukocyte common antigen (LCA) and CD 3, 5, 56 negativity indicating a diagnosis of a well-differentiated granulocytic sarcoma. There was no systemic manifestation of leukemia. The blood cell count, thoracoabdominal CTscan, and bone marrow histology were normal.
The patient received 6 cycles of chemotherapy by CHOP (consisting of 750 mg/m2 cyclophosphamide intravenously injected on day 1, 50 mg/m2 doxorubicin injected intravenously, on day 1; 1.4 mg/m2 vincristine intravenously injected on day 1, and 100 mg prednisolone orally administrated on days 1–5 repeated every three weeks). The patient responded well to the treatment, and complete remission was achieved. He remained alive and healthy 40 months after the end of treatment.
Consent
Written informed consent was obtained from the patient's father for publication of this case report. A copy of the written consent is available for review with the Editor-in-Chief of this journal.
Conflict of Interests
The authors declare that they have no conflict of interests.
Authors' Contributions
I. Elghissassi conceived the case report, analyzed and interpreted the patient data regarding the haematological disease H. Innrhaoun analyzed and interpreted the patient data and was a major contributor in the literature review H. Mrabti analyzed and interpreted the patient data and was a major contributor in the literature review H. Errihani contributed to revision, supervision and approval of the work. All authors read and approved the final manuscript. | Abbreviations
Granulocytic sarcoma
Cluster of differentiation
Cyclophosphamide, doxorubicin, vincristine and prednisolone
Leukocyte common antigen
Myeloperoxidase
Primitive neuroectodermal tumor. | CC BY | no | 2022-01-13 01:48:12 | Case Rep Med. 2010 Dec 14; 2010:235295 | oa_package/e1/56/PMC3014795.tar.gz |
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PMC3014796 | 20569136 | Introduction
The acoustic system plays an important role in communication and behaviour of orthopteran insects. This auditory communication system can be divided into the sound production organs and the sound perceiving organs, as well as their neuronal processing systems. Acoustic signals are used for intraspecific communications but also for interspecific interactions ( Alexander 1967 ; Ewing 1989 ; Bailey 1990 ; Gerhardt and Huber 2002 ; Greenfield 2002 ). Most species of Tettigoniidae use acoustic communication, and consequently, it is relatively well studied ( Bailey and Rentz 1990 ). However, the taxon Hetrodinae has received little attention in this respect, despite its potential importance for biological control of pest species.
One main function of the intraspecific auditory communication between females and males is to assist pair formation ( Robinson 1990 ). Therefore these acoustic signals are stereotypical with a distinct structure for a given species. The temporal pattern and frequency components of these songs are species specific and are widely used for taxonomy and ecological analysis ( Heller 1988 ; Ragge and Reynolds 1998 ; Walker et al. 2003 ; Elliott and Hershberger 2007 ).
Another type of acoustic signal that is used in many insect taxa (e.g. Coleoptera ( Lewis and Cane 1990 ; Schilman et al. 2001 ) and Homoptera ( Stölting et al. 2004 )), is the disturbance sound. These alarm signals are made by insects disturbed in different manners e.g. by touching. In contrast to the calling song, the disturbance sound has a simple and irregular temporal pattern ( Masters 1980 ). Alexander ( 1967 ) reported that arthropods use the sound production for a defensive mechanism more often than for any other acoustical communication.
The ear of Tettigoniidae is located in the proximal area of the foreleg tibia ( Graber 1876 ; Schumacher 1979 ). The scolopidial cells, specialized for detecting mechanical forces, show a typical arrangement in the proximal tibia of Tettigoniidae ( Schumacher 1973 ; Lakes and Schikorski 1990 ). These cells form a complex tibial organ, consisting of the subgenual organ, the intermediate organ, and the crista acustica; the latter perceives airborne sound ( Stumpner 1996 ). The auditory fibres run from the tibial organ through nerve 5B1 into the prothoracic ganglion where they terminate in the auditory neuropile ( Römer et al. 1988 ).
The Hetrodinae are distributed all over Africa and neighbouring areas ( Grzeschik 1969 ; Irish 1992 ) and are called armoured ground (or bush) crickets because of spikes on their pronotum and legs. These bush crickets are flightless with rudimentary wings that are covered under the pronotum ( Weidner 1955 ). Acanthoplus longipes (Orthoptera: Tettigoniidae: Hetrodinae) is a dark brown and ventrally green bushcricket with spines only on the pronotum. They are sexually dimorphic, and males use an elytroelytral stridulatory mechanism, as is the case with most bushcrickets. A. longipes lives in the low grassland of Southwest Africa (Namibia, Angola, and Congo) where it can have plague status in field crops when its population climaxes between March and May ( Weidner 1955 ; Mbata 1992 ). The importance for agricultural ecosystems leads to investigations about the reproductive system of Acanthoplus spp. ( Mbata 1992 ; Bateman and Ferguson 2004 ). The acoustic system of Tettigoniidae is an important part of the reproductive system. In respect to the auditory system it has been shown that Acanthoplus spp. have a pulsed calling song ( Conti and Viglianisi 2005 ), but the sensory organs have not been investigated. Therefore, the acoustic signals, as well as the anatomy and physiology of the sound receiver, are described. | Materials and Methods
Bushcrickets
A. longipes ( Figure 1 ) were collected as nymphs on roads near Keetmanshoop (26° 32′ S, 18° 6′ E), Namibia in March 2008 and transferred to the University of Giessen. The species was identified based on the key from Irish ( 1992 ). Four female and seven male A. longipes were used for the experiments. The animals were sorted by sex and kept between 22° C and 30° C with a 12: 12 lightdark cycle. They were fed with wheat seedlings, dog and fish food, and water ad libitum .
Sound recordings and analysis
For the sound recordings, the bushcrickets were placed within a cage of fly-screen in an anechoic chamber (50 × 50 × 50 cm). Each of six males was recorded once.
The recordings of the calling song were made in the dark, while the recordings of the disturbance stridulation were made under light conditions. To evoke a disturbance sound, the resting insects ( n = 2) were briefly touched with a stick. The songs were recorded at a temperature between 23° C and 27° C. An ultrasound microphone (Ultra Sound Gate CPVS, Avisoft Bioacoustics, www.avisoft.com ) with a frequency range of 10 to 95 kHz connected to a digital recorder (Tascam HD-P2) with a sampling rate of 192 kHz was used. The microphone was placed 15 to 40 cm away from the bushcrickets. Sound pressure level was measured with a Voltcraft meter (DT-8820). Both temporal structure and frequency range of the recordings were analyzed on a computer with the AviSoft program. For statistical analysis, Prism 4.03 (GraphPad Software, Inc., www.graphpad.com ) was used. The following terminology was used for describing the insect sounds:
Impulse: A single impulse probably caused by movement of one tooth of the stridulatory file.
Pulse: A train of impulses which are produced by opening or closing the wings. Verse: A group of impulses, which can contain one or two pulses.
For the analysis of the courtship behaviour, four virgin female A. longipes were tested. For each test, one female and one male were put together into a terrarium.
Hearing threshold
For electrophysiological investigations, A. longipes ( n = 5) were waxed on a metal holder with the ventral side up, and the forelegs were fixed approximately in their natural position. The hindlegs were removed and the midlegs were fixed with wax. The prothorax was opened ventrally and the prothoracic ganglion, the leg nerve, and the tympanal nerve were exposed. The recordings were made extracellularly from the tympanal nerve close to the bifurcation from the leg nerve. The tympanal nerve was put on a silver wire electrode, and the indifferent electrode was inserted contralaterally in the thorax. The signals from the nerve were amplified 1.000x by a preamplifier (T122, Tektronix, Inc., www.tek.com ), visualized on an oscilloscope, and connected to earphones. The sound signals were computer generated and amplified. They were made audible by a loudspeaker (SEAS 11 F-GX), which was positioned laterally 38 cm from the insect. The tested frequencies ranged from 3 to 40 kHz and were played back with sound pressure levels from 30 to 80 dB. Each sound intensity was tested five times. The lowest acoustic stimulus which elicited neuronal responses was defined as the auditory threshold.
Neuroanatomy
For the anatomical studies of the periphery, all legs (of 7 A. longipes ) were removed and placed into Petri dishes filled with saline solution. The legs were opened proximally at the femur-tibia joint, and the tympanal nerve (N5B1) was cut and placed in a glass capillary filled with 5% cobalt chloride solution in distilled water. Preparations were placed in a moist chamber for two days at 4° C. The staining was visualized with a 1% solution of ammonium sulphide in phosphate buffer. The legs were fixed in 4% of paraformaldehyde, dehydrated in a graded ethanol series, and cleared in methylsalicylate. As it was not possible to see the scolopidial cells through the dark cuticle, the tibia was opened dorsally by careful dissection.
For the anatomical studies of the central nervous system, the prothoracic ganglion was removed from the animal and placed in a Petri dish. The tympanal nerve (N5B1) was placed in a glass capillary, which was filled with a 5% neurobiotin solution in distilled water. The preparation was incubated at 4° C in a moist chamber for 48 hours. Thereafter, the ganglion was fixed in 4% paraformaldehyde. Then it was dehydrated, cleared in xylene for 5 minutes, and rehydrated. The next step was incubation in collagenase and hyaluronidase solution (1 mg each, Sigma Chemicals, www.sigmaaldrich.com ) in 1 ml phosphate buffer for one hour at 37° C. The ganglion was placed in an Avidin-Biotin-Complex (Vectastain ABC Kit PK-6100 Vector Laboratories, www.vectorlabs.com ) over night. After washing with phosphate buffer, the marking was visualized with DAB and H 2 O 2 (Vector Peroxidase Substrate Kit DAB SK-4100, Vector Laboratories) under visual control. The ganglion was dehydrated and cleared in methylsalicylate.
All preparations were documented by drawings (Leitz Dialux microscope with a drawing tube) and photographs (Olympus BH-2 microscope, www.olympus.com , with a Leica DCF-320 camera, www.leicamicrosystems.com ). | Results
Sound of A. longipes
The calling song of A. longipes males ( Figure 2A ) was produced in the late evening. The males were persistent singers, often singing for several minutes without any interruptions. Most stayed in one place, usually elevated, while singing, but some walked around without stopping to sing. The sound pressure level reached about 87 dB SPL in a distance of 10 cm caudal ( n = 4).
The calling song consisted of a sequence of verses that were separated into two pulses by a pause of about 16 ms ( Figures 2A, 2C ). These two pulses consisted of 2 to 7 impulses, which differed between the tested males ( Figure 3 ), but all males had fewer impulses in the first pulse than in the second pulse. In one of the six males (M2 in Figure 3 ) the second pulse more than doubled the number of impulses in the first pulse. The impulse interval (3.5 ms, n = 2811; SD = 0.68) was similar in the first and the second pulses ( Figure 2C ), which were separated by an interpuise interval of about 16 ms. The verse interval was about 50 ms. The mean verse duration was 40 ms ( Figure 4A ), and the mean number of impulses per verse was 8.53 pulses ( Figure 4B ).
Disturbance stridulation ( Figure 2B ) could be more easily elicited during the day than during the night and from resting insects than from walking insects. For two males, recordings from both types of sounds were compared ( Figure 4 ). The disturbance sound showed three characteristic differences to the calling song. First, the disturbance stridulation lasted only a few seconds. Second, the disturbance stridulation consisted of verses with only one pulse. Third, the pulses consisted of about 13 or 14 impulses per verse in contrast to the maximum number of 10 impulses per verse in the calling song ( Figure 4A ). The mean number of impulses per verse between the calling song and the disturbance stridulation was significantly different ( Figure 4 ; unpaired t-test; p < 0.0001; t = 45.45; df = 1495; calling song: n = 1262; disturbance sound: n = 235) in both males. However, the duration of the verses of both sounds was not different ( Figure 4B ). The sound pattern resulted in two groups of interval durations ( Figure 2D ). The verse interval was rather variable (mean = 98 ms; n = 220; SD = 62.50), but the impulse interval (2.9 ms; n = 2028; SD = 0.78) was invariant and significantly different from that of the calling song (p < 0.0001, unpaired T-test, dft = 26.13, df= 4837).
Both types of songs had similar frequency spectra within the investigated range with a peak around 15 kHz and a steady decrease in the ultrasonic range ( Figure 5 ).
Defense behaviour
Disturbance stridulation can be regarded as one mechanism of defense. While producing the sound, A. longipes always started to run away. As an additional defense mechanism, both sexes used reflex bleeding. They extruded hemolymph liquid from the coxa-trochanter joint. The squirt intensity and the bleeding coxa-trochanter joints could vary. The bleeding could not be elicited by a brief touch, but by handling the insects, e.g. during preparation for experiments. Otherwise, no complex defense mechanisms were observed.
Courtship
Females performed positive phonotaxis toward singing males. Whereas 3 of 4 females paused during phonotaxis, 1 female approached the male very quickly. When females reached the males, they touched them with their long antennae, and the males stopped singing. All observed pairs met each other under the top of the cage, and the male climbed underneath the female from a lateral position. Mating only started in the late evening and took at least 2 hours. On the next morning, 3 of 4 females still carried the spermatophore. One spermatophore was removed and weighted: 0.46 g, 5.4% of the respective male's weight. Females were heavier (mean 11.6 g, n = 3) than the males (mean 8.5 g, n = 2). During the day, the females fed on the spermatophore. For egg laying, the female, with its abdomen, made a small hole in the sand and placed a cluster of eggs into it.
Electrophysiology
The hearing threshold showed the highest sensitivity from 4 and 10 kHz with a threshold between 40 and 45 dB SPL ( Figure 6 ). The threshold rose to about 60 dB SPL in the ultrasonic range (20 – 40 kHz). No differences between males and females were found.
Neuroanatomy
The anterograde backfills of the tympanal nerve into the prothoracic ganglion showed that the nerve, 5B1, projects through the leg nerve. The axonal fibres of the auditory receptors continued in a posterior curve to the midline of the ganglion and terminated ipsilaterally in a dense neuropile ( Figure 7 ).
Peripheral backfills into the tibia showed the typical tripartite organization of the sensory complex for Tettigoniidae: subgenualorgan, intermediate organ, and crista acustica ( Figure 8 ). A. longipes had about 27 neurons in the crista acustica of the foreleg, 18 cells in the midleg, and 14 crista acustica neurons in the hindleg ( Table 1 ), with no sexual dimorphism. | Discussion
Calling song and courtship of A. longipes
The calling song of A. longipes is a sequence of two pulse verses, which can last several minutes. Each verse consists of two pulses, which consist of a few impulses. The impulse numbers in the pulses vary among individuals (see also Conti and Viglianisi 2005 ). The songs often show some variations within a basic pattern ( Schul 1998 ), which could be important for sexual selection. Larger variation might raise a problem when females need an exact pattern of the calling song to recognize the species-specific song ( Klappert and Reinhold 2003 ), which is the case with females from areas of sympatry ( Gwynne 2001 ). Variable song pattern could lead to heterospecific mating in closely related species, as has been shown for Acrididae ( von Helversen and von Helversen 1975 ).
The results on the frequency spectrum extend those of Conti and Viglianisi ( 2005 ) in the ultrasonic range and confirm a broad peak between 10 and 15 kHz. This frequency spectrum lies within the range of other Tettigoniidae ( Heller 1988 ; Römer et al. 1989 ; Schul and Patterson 2003 ). The fact of frequency attenuation of the vegetation, especially for the ultrasonic components of the calling song ( Keuper et al. 1986 ; Römer and Lewald 1991 ), might be the reason that A. longipes males seemed to prefer singing from a higher position. This has to be confirmed by field studies.
The auditory threshold shows the greatest sensitivity to between 4 and 10 kHz, which reflects a mismatch to the frequency spectrum of the calling song. In other Tettigoniidae, a species-specific tuning to the song spectrum is found, although the temporal pattern might be even more important ( Dobler et al. 1994 ; Römer and Bailey 1998 ; Schul and Patterson 2003 ; Lehmann et al. 2007 ). Phonotaxis experiments with song models could clarify how species recognition in A. longipes is influenced by song frequency or by song pattern. In the laboratory, no chorusing of A. longipes could be observed, as was observed in the Hetrodinae Acanthoplus speiseri ( Mbata 1992 ) and Eugaster spp. ( Grzeschik 1969 ). This shows a considerable variation of acoustic signalling in a genus similar to other Tettigoniidae ( Greenfield et al. 2004 ; Fertschai et al. 2007 ).
The phonotactic behaviour of the females, which is a common reaction to the conspecific calling song in tettigoniids, is also described for other Acanthoplus species ( Power 1958 ) and for Eugaster species ( Weidner 1955 ; Grzeschik 1969 ). However, no courtship song could be observed for A. longipes as in A. speiseri ( Mbata 1992 ) and Eugaster spp. ( Grzeschik 1969 ). The mating and egg laying behaviour is similar to those of other Hetrodinae species ( Weidner 1955 ; Power 1958 ; Grzeschik 1969 ; Mbata 1992 ) although the mating duration seems to be much longer.
Disturbance sound and defense
The disturbance sounds of orthopterans are less well studied ( Field 1993 ; Desutter-Grandcolas 1998 ). Some Tettigoniidae use their stridulation mechanism for both intraspecific communication and as a defense mechanism ( Kaltenbach 1990 ), and other species use different organs for disturbance stridulation ( Heller 1996 ). Additionally, other defensive behaviour with and without sound production evolved ( Belwood 1990 ). The disturbance stridulation of A. longipes could be evoked by disturbing resting animals. It was a brief sound that stopped shortly after the disturbance. The verses consist of more impulses that are not separated into pulses, compared to the calling song. The verse interval is variable; thus, the rather plain and variable pattern fits two of four characteristics of a disturbance sound (simple and irregular) proposed by Masters ( 1980 ). The two other characteristics (broad frequency band and a maximum energy at 1 kHz) could not be found in the disturbance stridulation of A. longipes . The frequency spectrum of the disturbance stridulation and the calling song are similar. It has been found for other orthopterans, as well, that the four characteristics do not always fit to disturbance sounds ( Desutter-Grandcolas 1998 ). The different impulse interval together with the different verse structure indicate that the disturbance stridulation does not simply reflect the neuronal and functional networks involved in calling song stridulation.
Many species who use disturbance sounds are large, flightless, slow-moving and night-singing bushcrickets, for example Pterophylla camellifolia, Liparoscelis nigrispina and Aglaothorax armiger ( Alexander 1960 ), which leads some authors to the assumption that this kind of sound production is a defense mechanism, especially against vertebrates ( Alexander 1967 ; Belwood 1990 ). The disturbance stridulation might increase the chance of survival of an insect after a predatory attack because it might startle the predator ( Robinson and Hall 2002 ). Or it might have a warning function for an additional defense mechanism, e.g. noxious signals ( Masters 1979 ). Furthermore, it is possible that a defense sound is mimicking an aposematic signal. While camouflage and mimicry are primary defense mechanisms ( Gwynne 2001 ), the disturbance stridulation is a secondary defense mechanism, which is used after the predator has made contact with the potential prey. There are also some arguments against the hypothesis of a defense mechanism: if this type of sound is an important defense mechanism both sexes should be able to produce it ( Heller 1996 ). Only in tettigoniid species, where the females also produce a sound for intraspecific communication, both sexes produce disturbance sounds ( Shaw and Galliart 1987 ). Furthermore, nymphs should also benefit from such a defense mechanism, as in some tettigoniid species ( Dadour and Bailey 1990 ).
A. longipes showed no complex behavioural pattern for defense, as other orthopterans do ( Sandow and Bailey 1978 ), but like other Hetrodinae ( Weidner 1955 ; Power 1958 ; Grzeschik 1969 ), both sexes use reflex bleeding as an additional, secondary defense mechanism. However, there is no evidence that the hemolymph of A. longipes is noxious. Additionally, A. longipes is well armed with spines, making it a difficult prey for small animals. The complement of different defense mechanisms might be necessary for day-active, ground-living flightless animals that otherwise might become an easy prey.
Neuroanatomy of the auditory system
Retrograde backfills of the legs show a complex of scolopidial cells in the proximal tibia, which can be divided into three parts. The most proximal group of cells is the subgenual organ, which detects substrate vibrations. The middle part is the intermediate organ, and the third part is the crista acustica, which perceives airborne sound ( Stumpner 1996 ). This complex tibial organ can be found in all legs, although tympana are only present in the foreleg. In the crista acustica, the cell number is species-specific and ranges between 20 and 50 cells in different species of the Tettigoniidae ( Schumacher 1979 ; Lakes and Schikorski 1990 ; Kalmring et al. 1993 ; Robinson and Hall 2002 ). The number of crista acustica receptor cells of A. longipes ( n = 27) fits well into this range. Like in other Tettigoniidae, the number of crista acustica cells decreases in the midleg and the hindleg ( Houtermans and Schumacher 1974 ). The central projection of auditory fibres has a typical arrangement in the prothoracic ganglion. The fibers project into the auditory neuropile and terminate at the midline. It can be presumed that the crista acustica cells have a tonotopic projection like in other Tettigoniidae ( Oldfield 1982 ). Thus, the neuroanatomy of this first-described Hetrodinae is in accordance with that of other Tettigoniidae ( Lakes and Schikorski 1990 ). | Associate Editor: David Morton was editor of this paper.
The auditory sensory system of the taxon Hetrodinae has not been studied previously. Males of the African armoured ground cricket, Acanthoplus longipes (Orthoptera: Tettigoniidae: Hetrodinae) produce a calling song that lasts for minutes and consists of verses with two pulses. About three impulses are in the first pulse and about five impulses are in the second pulse. In contrast, the disturbance stridulation consists of verses with about 14 impulses that are not separated in pulses. Furthermore, the inter-impulse intervals of both types of sounds are different, whereas verses have similar durations. This indicates that the neuronal networks for sound generation are not identical. The frequency spectrum peaks at about 15 kHz in both types of sounds, whereas the hearing threshold has the greatest sensitivity between 4 and 10 kHz. The auditory afferents project into the prothoracic ganglion. The foreleg contains about 27 sensory neurons in the crista acustica; the midleg has 18 sensory neurons, and the hindleg has 14. The auditory system is similar to those of other Tettigoniidae.
Keywords | Acknowledgments
We thank Dr. John Irish for sharing his knowledge about these animals with us. | CC BY | no | 2022-01-12 16:13:47 | J Insect Sci. 2010 Jun 10; 10:59 | oa_package/d2/43/PMC3014796.tar.gz |
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