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Abstract Background The GMC oxidoreductases comprise a large family of diverse FAD enzymes that share a homologous backbone. The relationship and origin of the GMC oxidoreductase genes, however, was unknown. Recent sequencing of entire genomes has allowed for the evolutionary analysis of the GMC oxidoreductase family. Results Although genes that encode enzyme families are rarely linked in higher eukaryotes, we discovered that the majority of the GMC oxidoreductase genes in the fruit fly (D. melanogaster), mosquito (A. gambiae), honeybee (A. mellifera), and flour beetle (T. castaneum) are located in a highly conserved cluster contained within a large intron of the flotillin-2 (Flo-2) gene. In contrast, the genomes of vertebrates and the nematode C. elegans contain few GMC genes and lack a GMC cluster, suggesting that the GMC cluster and the function of its resident genes are unique to insects or arthropods. We found that the development patterns of expression of the GMC cluster genes are highly complex. Among the GMC oxidoreductases located outside of the GMC gene cluster, the identities of two related enzymes, glucose dehydrogenase (GLD) and glucose oxidase (GOX), are known, and they play major roles in development and immunity. We have discovered that several additional GLD and GOX homologues exist in insects but are remotely similar to fungal GOX. Conclusion We speculate that the GMC oxidoreductase cluster has been conserved to coordinately regulate these genes for a common developmental or physiological function related to ecdysteroid metabolism. Furthermore, we propose that the GMC gene cluster may be the birthplace of the insect GMC oxidoreductase genes. Through tandem duplication and divergence within the cluster, new GMC genes evolved. Some of the GMC genes have been retained in the cluster for hundreds of millions of years while others might have transposed to other regions of the genome. Consistent with this hypothesis, our analysis indicates that insect GOX and GLD arose from a different ancestral GMC gene than that of fungal GOX.

DAS-ELISA, immunohistochemistry and electron microscopy were used toinvestigate the association of the causal agent of bacterial wilt, Erwinia tracheiphila (Smith), within the beetle Acalymma vittatum (F.). After a 24-h acquisition period, a high percentage of individuals tested positive for E. tracheiphila antigen using both immunohistochemistry (100%) and DAS-ELISA (70- 60%). Both assays showed that the antigen remained in beetles long after the initial acquisition, with thepercentage declining during incubation. Using ELISA, the percentage decreased to 4.7% within 3 d after acquistion, then increased to 10% within 10 d and remained at 10% for 30 d. Immunoperoxidase assays of paraffin embedded gut sections were more sensitive, and showed that 95% of the beetles harbored the pathogen after 10 d and 20% after 30 d. E. tracheiphila antigen was present throughout the digestive tract soon after acquisition, but only small clusters of E. tracheiphila were observed along the alimentary canal 3 d after transfer onto clean plants. After 10 and 30 d oil clean plants, E. tracheiphila antigen reaction was stronger and clusters of bacteria were more numerous, primarily in the posterior midgut and ariterior portion of the hindgut. Scanning electron microscopy and TEM photomicrographs confirmed the presence of bacterial cells resembling E'. tracheiphila associated with the intima of the hindgut I and 30 d after acquisition. This demonstrated the sensitivity of immunohistochemisty for detecting E. tracheiphila within its vector, and suggests a long-term extracellular endosymbiotic association of E. tracheiphila with the alimentary canal ofA. vittatum. [ABSTRACT FROM AUTHOR]

Osmia lignaria Say is used in combination with Apis mellifera L. to pollinate U.S. orchard crops. The deployment of O. lignaria requires artificial warming to synchronize adult bee emergence with crop bloom. However, current methods for emerging bees are time consuming and inefficient; the Hivetop Incubator (HTI) invention creates a space atop an A. mellifera hive whose heat flows through the screened bottom of the HTI to incubate cocooned O. lignaria adults therein. In response to HTI heat, O. lignaria adults chew out of cocoons, find an exit hole, and fly away to nest in provided nesting sites and, thereby, pollinate a crop. Objectives for studies performed in Utah and Washington were to: 1) determine whether HTIs inhibit A. mellifera thermoregulation or colony growth, 2) compare O. lignaria emergence duration from an HTI with and without hive heat, and 3) assess whether O. lignaria females leave HTIs located at the orchard edge to nest throughout the orchard. We found no significant differences between the internal temperatures of A. mellifera colonies with and without HTIs and no impact on A. mellifera food storage or brood production. Osmia lignaria in hive-heated HTIs emerged 3× faster than bees in unheated HTIs. Heated HTIs were significantly cooler than hive temperatures but significantly warmer than HTIs atop empty hive boxes. Osmia lignaria nest distribution was not correlated to the location of HTIs at the orchard edge. Overall, HTIs were effective for timely, on-site emergence of O. lignaria for orchard pollination without negatively impacting A. mellifera colonies., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Cuckoo bumble bees (Psithyrus) (Lepeletier, 1832) (Hymenoptera: Apidae) are a unique lineage of bees that depend exclusively on a host bumble bee species to provide nesting material, nutritional resources, and labor to rear offspring. In this study, we document usurpation incidence and population genetic data of Bombus insularis (Smith, 1861) (Hymenoptera: Apidae), a bumble bee species in the Psithyrus subgenus, on field-deployed B. huntii colonies in northern Utah, United States. Within 12 d of deploying B. huntii Greene, 1860 (Hymenoptera: Apidae) colonies at two field sites, 13 of the 16 colonies contained at least one established B. insularis female. Although our results demonstrate that field-deployed bumble bee colonies are highly susceptible to B. insularis usurpation, applying a fabricated excluder to prevent the inquiline from invading a colony was 100% effective. Sibship analysis using microsatellite genotype data of 59 B. insularis females estimates that they originated from at least 49 unique colonies. Furthermore, sibship analysis found siblings distributed between the field sites that were 7.04 km apart. Our result suggests that B. insularis females have the capacity to disperse across the landscape in search of host colonies at distances of at least 3.52 km and up to 7.04 km. Our study underscores the detrimental impact B. insularis usurpation has on the host bumble bee colony. As B. insularis significantly impacts the success of bumble bee colonies, we briefly discuss how the utilization of excluders may be useful for commercial bumble bee colonies that are used to pollinate open field crops., (Published by Oxford University Press on behalf of Entomological Society of America 2021.)

Bumble bees provide valuable pollination services to many wild and agricultural plants. Populations of some bumble bee species are in decline, prompting the need to better understand bumble bee biology and to develop methodologies for assessing the effects of environmental stressors on these bees. Use of bumble bee microcolonies as an experimental tool is steadily increasing. This review closely examines the microcolony model using peer-reviewed published literature identified by searching three databases through November 2018. Microcolonies have been successfully used for investigating a range of endpoints including behavior, the gut microbiome, nutrition, development, pathogens, chemical biology, and pesticides/xenobiotics. Methods for the initiation and monitoring of microcolonies, as well as the recorded variables were catalogued and described. From this information, we identified a series of recommendations for standardizing core elements of microcolony studies. Standardization is critical to establishing the foundation needed to support use of this model for biological response investigations and particularly for supporting use in pesticide risk assessment., (Published by Oxford University Press on behalf of Entomological Society of America 2019.)

Managed pollinators such as the alfalfa leafcutting bee, Megachile rotundata, are essential to the production of a wide variety of agricultural crops. These pollinators encounter a diverse array of microbes when foraging for food and nest-building materials on various plants. To test the hypothesis that food and nest-building source affects the composition of the bee-nest microbiome, we exposed M. rotundata adults to treatments that varied both floral and foliar source in a 2 × 2 factorial design. We used 16S rRNA gene and internal transcribed spacer (ITS) sequencing to capture the bacterial and fungal diversity of the bee nests. We found that nest microbial communities were significantly different between treatments, indicating that bee nests become inoculated with environmentally derived microbes. We did not find evidence of interactions between the fungi and bacteria within our samples. Furthermore, both the bacterial and fungal communities were quite diverse and contained numerous exact sequence variants (ESVs) of known plant and bee pathogens that differed based on treatment. Our research indicates that bees deposit plant-associated microbes into their nests, including multiple plant pathogens such as smut fungi and bacteria that cause blight and wilt. The presence of plant pathogens in larval pollen provisions highlights the potential for bee nests to act as disease reservoirs across seasons. We therefore suggest that future research should investigate the ability of bees to transmit pathogens from nest to host plant.

Hymenoptera is the second-most sequenced arthropod order, with 52 publically archived genomes (71 with ants, reviewed elsewhere), however these genomes do not capture the breadth of this very diverse order (Figure 1, Table 1). These sequenced genomes represent only 15 of the 97 extant families. Although at least 55 other genomes are in progress in an additional 11 families (see Table 2), stinging wasps represent 35 (67%) of the available and 42 (76%) of the in progress genomes. A more comprehensive catalog of hymenopteran genomes is needed for research into the evolutionary processes underlying the expansive diversity in terms of ecology, behavior, and physiological traits within this group. Additional sequencing is needed to generate an assembly for even 0.05% of the estimated 1 million hymenopteran species, and we recommend premier level assemblies for at least 0.1% of the >150,000 named species dispersed across the order. Given the haplodiploid sex determination in Hymenoptera, haploid male sequencing will help minimize genome assembly issues to enable higher quality genome assemblies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent ecological research has revealed that environmental factors can strongly affect insect immunity and influence the outcome of host-parasite interactions. To date, however, most studies examining immune function in mosquitoes have ignored environmental variability. We argue that one such environmental variable, temperature, influences both vector immunity and the parasite itself. As temperatures in the field can vary greatly from the ambient temperature in the laboratory, it will be essential to take temperature into account when studying vector immunology.

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its host L. dispar. L. dispar exhibits midgut-based and systemic, age-dependent resistance to LdMNPV within the fourth instar; the LD(50) in newly molted larvae is approximately 18-fold lower than in mid-instar larvae (48-72h post-molt). We examined the role of the immune system in systemic resistance by measuring differences in hemocyte immunoresponsiveness to foreign targets, hemolymph phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD) activities, and melanization of infected tissue culture cells. Mid-instar larvae showed a higher degree of hemocyte immunoresponsiveness, greater potential PO activity (pro-PO) at the time the virus is escaping the midgut to enter the hemocoel (72h post-inoculation), greater GLD activity, and more targeted melanization of infected tissue, which correlate with reduced viral success in the host. These findings support the hypothesis that innate immune responses can play an important role in anti-viral defenses against baculoviruses and that the success of these defenses can be age-dependent.

Background: Over the last two winters, there have been large-scale, unexplained losses of managed honey bee (Apis mellifera L.) colonies in the United States. In the absence of a known cause, this syndrome was named Colony Collapse Disorder (CCD) because the main trait was a rapid loss of adult worker bees. We initiated a descriptive epizootiological study in order to better characterize CCD and compare risk factor exposure between populations afflicted by and not afflicted by CCD., Methods and Principal Findings: Of 61 quantified variables (including adult bee physiology, pathogen loads, and pesticide levels), no single measure emerged as a most-likely cause of CCD. Bees in CCD colonies had higher pathogen loads and were co-infected with a greater number of pathogens than control populations, suggesting either an increased exposure to pathogens or a reduced resistance of bees toward pathogens. Levels of the synthetic acaricide coumaphos (used by beekeepers to control the parasitic mite Varroa destructor) were higher in control colonies than CCD-affected colonies., Conclusions/significance: This is the first comprehensive survey of CCD-affected bee populations that suggests CCD involves an interaction between pathogens and other stress factors. We present evidence that this condition is contagious or the result of exposure to a common risk factor. Potentially important areas for future hypothesis-driven research, including the possible legacy effect of mite parasitism and the role of honey bee resistance to pesticides, are highlighted.

Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92+/-5% in the untreated check to 1+/-3% when protected by injection of 230ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3+/-4% in the untreated group to 57+/-15% with an SCPs dose of 940ng. At 2h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300ng/microl SCPs, 70% and 96% were stained, respectively. At 6h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38kDa (PI=4.6) and 56kDa (PI=3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi.

The peritrophic matrix (PM) lines the midgut of most insects, providing protection to the midgut epithelial cells while permitting passage of nutrients and water. Herein, we provide evidence that plant-mediated alteration of the PM contributes to the well-documented inhibition of fatal infection by Autographa californica multiple nucleopolyhedrovirus (AcMNPV) of Heliothis virescens F. larvae fed cotton foliage. We examined the impact of the PM on pathogenesis using a viral construct expressing a reporter gene (AcMNPV-hsp70/lacZ) orally inoculated into larvae with either intact PMs or PMs disrupted by Trichoplusia ni granulovirus occlusion bodies containing enhancin, known to degrade insect intestinal mucin. Larvae possessing disrupted PMs displayed infection foci (lacZ signaling) earlier than those with intact PMs. We then examined PMs from larvae fed artificial diet or plant foliage using electron microscopy; foliage-fed larvae had significantly thicker PMs than diet-fed larvae. Moreover, mean PM width was inversely related to both the proportion of larvae with lacZ signaling at 18h post-inoculation and the final percentage mortality from virus. Thus, feeding on foliage altered PM structure, and these foliage-mediated changes reduced baculoviral efficacy. These data indicate that the PM is an important factor determining the success of an ingested pathogen in foliage-fed lepidopteran larvae.

Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F(2) progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F(2) genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

Reproduction of entomopathogenic nematodes requires that they escape recognition by a host's immune system or that they have mechanisms to escape encapsulation and melanization. We investigated the immune responses of larvae for the greater wax moth (Galleria mellonella), tobacco hornworm (Manduca sexta), Japanese beetle (Popillia japonica), northern masked chafer (Cyclocephala borealis), oriental beetle (Exomala orientalis) and adult house crickets (Acheta domesticus), challenged with infective juveniles from different species and strains of entomopathogenic nematodes. The in vivo immune responses of hosts were correlated with nematode specificity and survival found by infection assays. In P. japonica, 45% of injected infective juveniles from Steinernema glaseri NC strain survived; whereas the hemocytes from the beetle strongly encapsulated and melanized the Heterorhabditis bacteriophora HP88 strain, S. glaseri FL strain, Steinernema scarabaei and Steinernema feltiae. Overall, H. bacteriophora was intensively melanized in resistant insect species (E. orientalis, P. japonica and C. borealis) and had the least ability to escape the host immune response. Steinernema glaseri NC strain suppressed the immune responses in susceptible hosts (M. sexta, E. orientalis and P. japonica), whereas S. glaseri FL strain was less successful. Using an in vitro assay, we found that hemocytes from G. mellonella, P. japonica, M. sexta and A. domestica recognized both nematode species quickly. However, many S. glaseri in M. sexta and H. bacteriophora in G. mellonella escaped from hemocyte encapsulation by 24h. These data indicate that, while host recognition underlies some of the differences between resistant and susceptible host species, escape from encapsulation following recognition can also allow successful infection. Co-injected surface-coat proteins from S. glaseri did not protect H. bacteriophora in M. sexta but did protect H. bacteriophora in E. orientalis larva; therefore, surface coat proteins do not universally convey host susceptibility. Comparisons of surface coat proteins by native and SDS-PAGE demonstrated different protein compositions between H. bacteriophora and S. glaseri and between the two strains of S. glaseri.

To determine the roles of varroa mites in activating or vectoring viral infections, we performed quantitative comparison of viral infections between bees with and without mites by dot blot analysis and enzyme-linked immunosorbent assay (ELISA). Under natural and artificial mite infestations, bee pupae contained significantly higher levels of Kashmir bee virus (KBV) and deformed wing virus (DWV) RNAs and KBV structural proteins than mite-free pupae. Moreover, in mite-infested bee pupae, DWV had amplified to extremely high titers with viral genomic RNA being clearly visible after separation of total bee RNA in agarose gels. Linear regression analysis has shown a positive correlation between the number of mites introduced and the levels of viral RNAs. The detection of viral RNAs in the nymph and adult mites underline the possible role of varroa in virus transmission. However, most groups of virus-free adult mites (9/12) were associated with bee pupae heavily infected by viruses, suggesting that the elevated viral titers in mite-infested pupae more likely resulted from activated viral replication. Based on these observations and our concurrent research demonstrating suppressed immune responses in bees infested with mites, we propose that parasitization by varroa suppresses the immunity of honey bees, leading to activation of persistent, latent viral infection.

Viral diseases of honeybees are a major problem in apiculture, causing serious economic losses worldwide, especially in combination with varroa mites. To increase understanding of the relationship among viruses, mites and colony decline, the tripartite relationships among bees, viruses [Kashmir bee virus (KBV) and sacbrood virus (SBV)] and varroa mites have been investigated systematically. To develop an antibody-based test for KBV, two structural recombinant proteins were purified for polyclonal-antibody production. By using ELISA and RT-PCR, the presence of KBV and SBV was studied comparatively in different developmental stages and castes of bees. The results demonstrated that KBV may persist as a viral genome with extremely low levels of viral-capsid proteins and that KBV and SBV can co-infect honeybees. This study indicated the presence of KBV and SBV RNAs in both queens and eggs by RT-PCR, suggesting a route of transovarial transmission. Horizontal transmission is also very likely among adult bees and from adult workers to larvae through contaminated food resources, because both viruses have been detected in all developmental stages and food sources (brood food, honey, pollen and royal jelly). Furthermore, it was demonstrated that mites were another possible route of horizontal transmission, as both viruses were detected in mites and their saliva. This study, for the first time, detected co-occurrence of viruses in varroa, further underlining the importance of the mites in vectoring different bee viruses. Therefore, these results indicated that multiple infection routes exist for honeybee viral diseases.

The cDNA sequence of a salivary lysozyme in Helicoverpa zea (Lepidoptera: Noctuidae) was determined. The full-length cDNA is 1,032 bp, and it encodes a protein of 142 amino acids. This lysozyme has 90% identity with Heliothis virescens lysozyme and 76% identity with Manduca sexta lysozyme. There is a signal peptide of 20 amino acids at the N-terminus. The mature protein is about 14.4 kDa without the signal peptide. The pI value is greater than 9.5 as determined by isoelectric focusing. From genomic DNA, two introns and three exons were within the open reading frame (ORF). Southern blot analysis indicated that it is a single-copy gene. A time-course study revealed that the H. zea lysozyme gene was differentially expressed in the labial glands during the development of fifth-instar larvae, with the peak level of lysozyme mRNA being detected on day 1. Dot blot analysis showed different levels of H. zea lysozyme expression when the caterpillars fed on different plants. Further, the H. zea lysozyme could be detected with antibodies raised against the M. sexta lysozyme, and it was one of the most abundant secreted proteins in saliva collected directly from the caterpillar's spinneret. The potential role of the lysozyme on host plants in mediating susceptibility to bacterial disease is discussed in the context of tritrophic interactions.

Polydnaviruses (PDV) are obligate mutualistic symbionts found in association with some groups of parasitic Hymenoptera. In these groups, they suppress the immune response of the parasitoid's host and are required for successful parasitoid reproduction. Several PDV effects have been described in different experimental systems, but no clear picture of PDV mode of immunosuppression has emerged. No study to date has directly tested if PDV modes of action are evolutionarily conserved or divergent among parasitoid taxa within the Ichneumonoidea. We hypothesize the divergence in PDV mode of immunosuppression can be detected by identifying points of divergence in the immune response of different host species to PDV from one parasitoid species. This study tests the effects of purified PDV from Cotesia congregata on the immune response of three larval lepidopteran species that naturally are hosts of parasitoid species that differ in taxonomic relatedness to C. congregata. Here we demonstrate that despite associations with distantly related parasitoids (Ichneumonidae and Braconidae), Manduca sexta and Heliothis virescens showed similar patterns of increased glucose dehydrogenase (GLD) activity, suppressed cellular encapsulation in vitro, and increased time to pupation. In contrast, Lymantria dispar showed no response to C. congregata PDV across any of the parameters measured, even though it has an evolutionary association with several parasitoids closely related to C. congregata and within the Microgastrinae. The PDV immunosuppression in H. virescens and M. sexta does not correlate with host molecular phylogeny either. The suborganismal effects shown in M. sexta and H. virescens translated into significantly reduced pupation success in M. sexta only. Results demonstrate that while some PDV modes of immunosuppression in hosts may be divergent, others may be conserved across broad host groups.

Two proteins (SaM35 and SaM50) isolated from head tissues of the aphid vector, Sitobion avenae, were identified as potential receptors for barley yellow dwarf virus MAV isolate (Luteoviridae) based on MAV virus overlay assays and immunoblots of urea SDS 2-D gels. An anti-idiotypic antibody (MAV4 anti-ID) that mimics an epitope on MAV virions and competes with MAV in antibody binding assays also bound to SaM50 and SaM35 and to six additional proteins including a GroEL homolog. No MAV-binding proteins were detected from the nonvector aphid, Rhopalosiphum maidis, although MAV4 anti-ID did react with four proteins from R. maidis. It is hypothesized that SaM35 and SaM50 may be MAV receptors involved in MAV transmission based on their high affinity for MAV and their unique association with the vector, S. avenae. The additional aphid proteins binding the MAV4 anti-ID may represent less specific virus-binding proteins facilitating transmission through different aphid tissues., (Copyright 2001 Academic Press.)

Cotesia congregata and Manduca sexta were used as a model system to study the mechanism and effect of a polydnavirus (PDV). We hypothesized that (1) FAD-glucose dehydrogenase (GLD) (EC 1.1.99.10) hemolymph titer would increase in response to parasitism, (2) hemocyte targeting behavior would be altered by parasitism, and (3) changes observed in GLD activity and hemocyte behavior immediately post-parasitization would be due to the presence of PDV. GLD specific activity was measured at four time points early during parasitism using a spectrophotometric enzyme assay. Hemocyte behavior was measured using direct observations of hemocyte response to a foreign target in vitro. Results demonstrate that GLD increases immediately post-oviposition and post-injection of purified PDV, indicating that virions elicit nonself recognition. This increase relative to unparasitized controls also occurs in response to trioxsalen-UV inactivated virus, indicating that the initial disruption of the host immune response is not dependent upon viral transcription. Further, we demonstrate that plasmatocytes are actively spreading and aggregating but are not targeting nonself material in both parasitized and polydnavirus treatments. These results indicate that purified PDV is recognized as nonself and is triggering an immediate cellular immune response prior to viral transcription.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.