Evaluation of Fopius arisanus as a biological control agent for the olive fruit fly in California

1 The egg‐prepupal parasitoid Fopius arisanus (Hymenoptera: Braconidae) was evaluated in quarantine facilities as a potential biological control agent for the olive fruit fly Bactrocera oleae (Diptera: Tephritidae) in California, U.S.A. 2 Nontarget testing of two weed biological control agents confirmed that F. arisanus will not attack Tephritidae that feed in inflorescences or galls. It may, however, pose risks to native Tephritidae that feed in fruit. 3 Females preferentially oviposited in eggs, although first‐instar B. oleae were also attacked. Low lifetime reproductive potential and high rates of direct mortality inflicted on host eggs indicate that rearing on B. oleae may prove difficult. 4 In multiparasitized B. oleae, F. arisanus prevailed in competition against two species of larval–pupal parasitoids, Diachasmimorpha kraussii and Psyttalia concolor (both Hymenoptera: Braconidae). 5 The broad host‐range of F. arisanus with respect to fruit‐feeding Tephritidae may preclude its introduction to California, as may its low fecundity and its intrinsic competitive superiority over larva l–pupal parasitoids, which include specialists on B. oleae that are currently being introduced to California. High rates of direct mortality, however, point to potential uses in augmentative biological control. Whether or not F. arisanus is released in California, its biology as a parasitoid of B. oleae has been little studied to date and the results herein may be applied in other regions worldwide where B. oleae is a problem.     

Foraging Behavior and Patch Time Allocation by Fopius arisanus (Hymenoptera: Braconidae), an Egg-Larval Parasitoid of Tephritid Fruit Flies

This study quantitatively describes the host-searching behavior of Fopius arisanus (Sonan) (Hymenoptera: Braconidae), an important egg-larval parasitoid of tephritid fruit fly pests, on coffee berries infested with host eggs of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). We also investigate the parasitoid's response to local variation in host patch quality. The temporal pattern of behavioral organisation was examined by constructing an ethogram. The parasitoid spent over 90% of its foraging time in detecting and locating hosts after arriving on a host-infested fruit, and displayed a relatively fixed behavioral pattern leading to oviposition. Patch residence time increased in the presence of host-associated cues, following successful ovipositions, and with increasing size of host clutches per fruit, but decreased with each successive visit to the same host patch and with increasing availability of alternative host patches. The parasitoid females discriminated against previously parasitized hosts and spent significantly less time and searching effort on patches previously exploited by herself or by conspecific females. The effective host-searching behavior, perfect host discrimination ability, and success-motivated searching strategy shown by F. arisanus ensured a thorough exploitation of host resources by this parasitoid.     

Description of a New Species of Microsporidia from Muscidifurax raptor (Hymenoptera: Pteromalidae), a Pupal Parasitoid of Muscoid Flies

ABSTRACT. A microsporidian parasite, Nosema muscidifuracis n. sp., has been found in Muscidifurax raptor , a parasitoid of muscoid flies. Stages of the parasite developed in direct contact with the host cell cytoplasm and were detected in midgut epithelium, Malpighian tubules, ovaries (including oocytes) and fat body of larvae and adults. Spores were also detected within eggs deposited on the host. Light and electron microscopy revealed a developmental cycle with diplokaryotic stages dividing by binary fission and disporous sporulation sequences producing diplokaryotic spores of three morphological classes, differing significantly only in length of the polar filament. Two of the classes were found in larvae, pupae and adults. One of these, with about five turns in the coiled polar filament, is presumed to be responsible for transmission from cell to cell within the host (autoinfection) and the other, with about 10 turns, responsible for transmission from host to host. A third class, with about 15 turns in the polar filament, was found in eggs of M. raptor . It is, presumably, either involved in initiation and spread of the infection at eclosion or is responsible for horizontal transmission to a new host individual when eggs are cannibalized.     

Condition-Dependent Mating Success in Male Fruit Flies: Ingestion of a Pheromone Precursor Compensates for a Low-Quality Diet

Previous studies revealed that males of the oriental fruit fly, Bactrocera dorsalis, require protein in the adult diet to obtain matings and that ingestion of methyl eugenol, which acts as a pheromone precursor, increases male attractiveness and mating competitiveness. The goal of this study was to investigate the interaction between diet quality and methyl eugenol consumption in affecting the mating frequency of B. dorsalis males. In one set of experiments, mature males were deprived of protein for 1, 3, or 7 days and were either given or denied access to methyl eugenol (ME). These males competed against control males (continuously protein-fed, no feeding on ME) for copulations in field cages. Without ME, males held without protein for 3 or 7 days obtained significantly fewer matings than control males. With ME, however, males held for even 7 days without protein achieved higher mating success than control males. In a second set of experiments, mature males were held without protein for 7 days and then given a protein-rich diet for 1, 3, or 7 days before testing and were either given or denied access to ME. Without ME, males were competitively inferior to control males when tested 1 or 3 days after resumption of protein feeding and equivalent to control males only after 7 days of protein feeding. With ME, however, males obtained significantly more matings than control males when tested 3 or 7 days after resumed protein feeding and had similar mating success as control males after 1 day of access to the protein-rich diet. Results show that mating success in this species is condition-dependent, with both nutritional state and ME consumption influencing male mating success. Under the test conditions, feeding on ME counteracted a low quality diet and enhanced male mating success.     

Effect of Two Opiine Parasitoids (Hymenoptera: Braconidae) Introduced for Fruit Fly Control on a Native Hawaiian Tephritid,Trupanea dubautiae(Diptera: Tephritidae)

Diachasmimorpha longicaudata(Ashmead) andPsyttalia fletcheri(Silvestri) are opiine parasitoids introduced into Hawaii for control of the Oriental fruit fly,Bactrocera dorsalis(Hendel) and the melon fly,Bactrocera cucurbitae(Coquillett), respectively. Both species have recently been mass-reared and released for research in augmentative biocontrol programs. Laboratory and field sleeve cage experiments were conducted to investigate the potential impact of mass-producedD. longicaudataandP. fletcherion a native Hawaiian tephritid,Trupanea dubautiae(Bryan), infesting the flowerheads of the native composite shrubDubautia raillardioidesHillebrand. Gravid females ofD. longicaudataandP. fletcheriwere confined with bloomingD. raillardioidesflowerheads infested with late instarT. dubautiae.BothD. longicaudataandP. fletcherilacked positive oviposition responses toT. dubautiaelarvae in infested flowerheads and caused neither parasitism nor mortality to the flies. However, when larvae were removed from the flowerheads and presented in screened dishes containing artificial diet of the parasitoids' normal rearing hosts (B. dorsalisandB. cucurbitae), bothD. longicaudataandP. fletcherireadily oviposited in the test larvae. Oviposition byD. longicaudatadid not significantly affect the percentage pupation ofT. dubautiae,but did reduce the emergence of adult flies. Oviposition byP. fletcherisignificantly reduced both pupation and adult fly emergence. All progeny of both parasitoid species died as eggs or first-instar larvae. Results from our experiments demonstrate that biological control programs targeted against frugivorous tephritid pests byD. longicaudataandP. fletcherihave no harmful impact on flowerhead-infestingT. dubautiae.     

Selection and evaluation of RT-qPCR reference genes for expression analysis in the tiny egg parasitoid wasp,Trichogramma dendrolimi matsumura (Hymenoptera: Trichogrammatidae)

The egg parasitoid, Trichogramma spp., is an important biological control agent used against a broad range of Lepidopteran pests in agriculture and forestry. The biology of Trichogramma has been studied in details. Further studies should focus on the molecular mechanisms of Trichogramma by qualifying the expression of related genes It is critical to select appropriate reference genes for normalizing RT-qPCR results and establishing a robust method for quantifying target gene expression. This study aimed to identify and validate appropriate reference genes for use in RT-qPCR analysis of Trichogramma dendrolimi. Ten candidate housekeeping genes, namely beta-actin (ACTIN), forkhead box O (FOXO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 90 (HSP90), ribosomal protein L10a (RPL10a), L18 (RPL18), L28 (RPL28), S13 (RPS13), S15 (RPS15), and superoxide dismutase (SOD), were tested for their suitability as reference genes for developmental stage (3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th day after parasitization), tissue (head, thorax, and abdomen of adults), sex of adults (male and female), and temperature (17℃, 25℃, and 32℃). According to the GeNorm analysis, a robust analysis should involve using an appropriate combination of reference genes, namely, at least three genes for different development stages, two genes for different tissues, two genes for different sex, and two genes for different temperatures, respectively. According to the RelFinder method by the integrated results of GeNorm, NormFinder, BestKeeper, and the ΔCt method, we identified the developmental stage-specific reference genes SOD, GAPDH, and ACTIN; tissue-specific reference genes RPL18 and RPS15; sex-specific reference genes RPL18 and SOD; and temperature-specific reference genes RPL18 and RPL10a. This study provides a standardized procedure for the quantification of gene expression in T. dendrolimi and will be helpful for future biological control programs using Trichogramma wasps.     

Transfection,Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.     

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.Anna Karlgren and Jenny Carlsson contributed equally to this study.Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se     

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.     

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.     

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures,Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability ({"type":"clinical-trial","attrs":{"text":"NCT01151124","term_id":"NCT01151124"}}NCT01151124 and {"type":"clinical-trial","attrs":{"text":"NCT02117635","term_id":"NCT02117635"}}NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER^TAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.     

Release and establishment of a biological control agent, Psyllaephagus pilosus for eucalyptus psyllid (Ctenarytaina eucalypti) in Ireland

An Australian parasitoid wasp, Psyllaephagus pilosus, was collected from a previous release site in France and introduced into a commercial eucalyptus foliage plantation in Co. Kerry in the south west of Ireland to control the eucalyptus (blue gum) psyllid Ctenarytaina eucalypti. The first parasitised psyllid nymphs were observed 26 days after the release was made in late May 1998, and 49 days elapsed before a new generation of adult parasitoids was seen. Visually assessed indices of psyllid parasitism and adult wasp incidence were used to quantify the pattern of adult dispersion and establishment. A second generation of adult wasps emerged in late August, initiating a rapid spread of parasitism throughout the release site during September that culminated in a peak rate of almost 100% parasitism by late October. Subsequently by the end of 1998, an apparently random process of dispersion and successful natural colonisation occurred at eight out of ten monitored plantations at distances up to 70 km from the release point. The first colonisers of the other two sites arrived very late in 1998, and consequently failed to establish viable populations by the following summer. Probably this failure was because the small numbers of colonisers had arrived too late in the growing season to effect population establishment before the onset of winter. Assisted introductions were, therefore, made at these sites in June and August 1999, respectively. Once fully established, the parasitoid had no difficulty in surviving winter conditions at all monitored sites and demonstrated excellent potential as a biological control agent.     

Response of Three Hymenopteran Parasitoids Introduced for Fruit Fly Control to a Gall-Forming Tephritid,Procecidochares alani(Diptera: Tephritidae)

The response of three larval–pupal parasitoids,Diachasmimorpha longicaudata(Ashmead),Diachasmimorpha tryoni(Cameron), andTetrastichus giffardianusSilvestri, to the Hamakua pamakani gall fly,Procecidochares alaniL., was determined in the laboratory. We also observed responses of these parasitoids to their normal rearing hosts,Bactrocera dorsalis(Hendel) andCeratitis capitata(Weidemann).D. tryonilanded on pamakani galls or on dishes containingP. alanilarvae as frequently as on dishes containingC. capitata.In contrast,D. longicaudataonly rarely landed on the galls.D. tryoniandD. longicaudataoviposited in galls in fewer than 1% of our observations.D. longicaudataprobedP. alanilarvae as frequently asB. dorsalislarvae, but no parasitoid offspring were observed.D. tryonioviposited more frequently inC. capitatathanP. alani.NoD. tryonideveloped inP. alanilarvae.T. giffardianuslanded on pamakani galls andP. alanilarvae more frequently than any other host substrate. In contrast,T. giffardianusentered galls with artificially opened windows one time in 10 observations. We observed 12% parasitism ofP. alanilarvae dissected from the galls and 20% of parasitismP. alaniin the windowed galls. We discuss the implications of our results for future augmentative or classical biological control studies.     

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling,Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available¹. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment². Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice³. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown⁴, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)⁵ and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate^5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for ¹⁵N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein⁷ (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state⁸. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1⁹.     

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine¹ and TH is also present in a subset of adult pelvic ganglia neurons^2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc¹. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine^2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.     

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.     

Development of Aerial Releases of Diachasmimorpha tryoni (Cameron) (Hymenoptera: Braconidae), a Parasitoid that Attacks the Mediterranean Fruit Fly, Ceratitis capitata (Weidemann) (Diptera: Tephritidae), in the Guatemalan Highlands

A braconid parasitoid, Diachasmimorpha tryoni (Cameron), was released from the air into Guatemalan coffee plantations that contained Mediterranean fruit flies, Ceratitis capitata (Weidemann). Parasitoid adults were chilled, placed in paper bags, and dropped from an altitude of 100 m and at an airspeed of ~ 130 km/h. Releases were made at four different densities over a period of two years. At the higher release rates parasitism levels reached as high as 84%. The feasibility of using a more technically sophisticated aerial release technique, the auger sterile-insect release machine utilized in C. capitata sterile-fly aerial eradication projects in California and Florida, was also examined. Chilled D. tryoni either alone or in combination with chilled, sterile C. capitata , were dropped over target areas and the released parasitoids examined for mortality and damage. Samples of released parasitoids were taken and tested for 'flight ability'; i.e. flight response after an opportunity to recover from chilling. There was no evidence of significant mortality due to aerial release, and the flight-ability of insects released at various rates and altitudes did not significantly differ from chilled controls that were not released from an airplane.     

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules ¹. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes ² and restrict their environment to a more favorable low oxygen pressure ³. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria ^4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor ⁶. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)^7,8 has also been associated with severe disease ⁹.One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps ¹⁰. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM ¹¹. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian ¹² and Mozambican patients ¹³, (although not in Malian ¹⁴).With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay ¹⁵. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.     

Engineering Platform and Experimental Protocol for Design and Evaluation of a Neurally-controlled Powered Transfemoral Prosthesis

To enable intuitive operation of powered artificial legs, an interface between user and prosthesis that can recognize the user''s movement intent is desired. A novel neural-machine interface (NMI) based on neuromuscular-mechanical fusion developed in our previous study has demonstrated a great potential to accurately identify the intended movement of transfemoral amputees. However, this interface has not yet been integrated with a powered prosthetic leg for true neural control. This study aimed to report (1) a flexible platform to implement and optimize neural control of powered lower limb prosthesis and (2) an experimental setup and protocol to evaluate neural prosthesis control on patients with lower limb amputations. First a platform based on a PC and a visual programming environment were developed to implement the prosthesis control algorithms, including NMI training algorithm, NMI online testing algorithm, and intrinsic control algorithm. To demonstrate the function of this platform, in this study the NMI based on neuromuscular-mechanical fusion was hierarchically integrated with intrinsic control of a prototypical transfemoral prosthesis. One patient with a unilateral transfemoral amputation was recruited to evaluate our implemented neural controller when performing activities, such as standing, level-ground walking, ramp ascent, and ramp descent continuously in the laboratory. A novel experimental setup and protocol were developed in order to test the new prosthesis control safely and efficiently. The presented proof-of-concept platform and experimental setup and protocol could aid the future development and application of neurally-controlled powered artificial legs.