The multispecies stinkbug iflavirus Halyomorpha halys virus detected in the multispecies stinkbug egg parasitoid microwasp, Telenomus podisi (Ashmead) (Hymenoptera: Platygastridae).

Wasps are important parasitoids of stinkbugs and frequently exposed to various types of microorganisms through environmental contact and fecal-oral transmission route. Many parasitize stinkbug eggs and are commercially used in the field to control insect population. The parasitoid T. podisi is known for its high parasitism capacity and ability to target multiple species of stinkbugs. In this study we asked whether T. podisi exposed to eggs infected by a multispecies asymptomatic stinkbug virus, the Halyomorpha halys virus (HhV) would get infected. HhV is a geographically distributed multispecies iflavirus previously found to infect four stinkbug hosts, including three Brazilian species, Chinavia ubica, Euschistus heros and Diceraeus melacanthus, and T. posidi can parasitize all of them. As results, RT-PCR screening revealed positive samples for the HhV genome in two out of four tested pools of T. podisi, whereas the antigenome, indicative of replicative activity, was not detected. The wasps were raised in E. heros eggs that presented both the genome and the antigenome forms of the HhV genome. Subsequent RNA-deep sequencing of HhV positive T. podisi RNA pools yielded a complete genome of HhV with high coverage. Phylogenetic analysis positioned the isolate HhV-Tp (isolate Telenomus podisi) alongside with the stinkbug HhV. Analysis of transcriptomes from several hymenopteran species revealed HhV-Tp reads in four species. However, the transmission mechanism and the ecological significance of HhV remain elusive, warranting further studies to illuminate both the transmission process and its capacity for environmental propagation using T. podisi as a potential vector.

Interactions of Vank proteins from Microplitis bicoloratus bracovirus with host Dip3 suppress eIF4E expression.

Microplitis bicoloratus bracovirus (MbBV) inhibits the immune response of the host Spodoptera litura by disrupting nuclear factor (NF)-κB signaling and downstream gene expression. However, the underlying molecular mechanisms are not well understood. Herein, we report that viral ankyrin (Vank) proteins interacted with host dorsal-interacting protein 3 (Dip3) to selectively inhibit the transcription of eukaryotic translation initiation factor 4 E (eIF4E). Dip3 and Vank proteins were co-expressed and colocalized in the nucleus. Furthermore, ectopic expression of Dip3 rescued the transcription of some NF-κB-dependent genes suppressed by Vank proteins, including eIF4E. Co-immunoprecipitation and pull-down assays confirmed that Vank proteins interacted with and bound to full-length Dip3, which including MADF, DNA-binding protein, BESS, and protein-protein interaction motifs as well as non-motif sequences. In vivo, RNAi-mediated dip3 silencing decreased eIF4E levels and was accompanied by an immunosuppressive phenotype in S. litura. Our results provided novel insights into the regulation of host transcription during immune suppression by viral proteins that modulate nuclear NF-κB signaling.

Phylogenomics of Ichneumonoidea (Hymenoptera) and implications for evolution of mode of parasitism and viral endogenization.

Ichneumonoidea is one of the most diverse lineages of animals on the planet with >48,000 described species and many more undescribed. Parasitoid wasps of this superfamily are mostly beneficial insects that attack and kill other arthropods and are important for understanding diversification and the evolution of life history strategies related to parasitoidism. Further, some lineages of parasitoids within Ichneumonoidea have acquired endogenous virus elements (EVEs) that are permanently a part of the wasp's genome and benefit the wasp through host immune disruption and behavioral control. Unfortunately, understanding the evolution of viral acquisition, parasitism strategies, diversification, and host immune disruption mechanisms, is deeply limited by the lack of a robust phylogenetic framework for Ichneumonoidea. Here we design probes targeting 541 genes across 91 taxa to test phylogenetic relationships, the evolution of parasitoid strategies, and the utility of probes to capture polydnavirus genes across a diverse array of taxa. Phylogenetic relationships among Ichneumonoidea were largely well resolved with most higher-level relationships maximally supported. We noted codon use biases between the outgroups, Braconidae, and Ichneumonidae and within Pimplinae, which were largely solved through analyses of amino acids rather than nucleotide data. These biases may impact phylogenetic reconstruction and caution for outgroup selection is recommended. Ancestral state reconstructions were variable for Braconidae across analyses, but consistent for reconstruction of idiobiosis/koinobiosis in Ichneumonidae. The data suggest many transitions between parasitoid life history traits across the whole superfamily. The two subfamilies within Ichneumonidae that have polydnaviruses are supported as distantly related, providing strong evidence for two independent acquisitions of ichnoviruses. Polydnavirus capture using our designed probes was only partially successful and suggests that more targeted approaches would be needed for this strategy to be effective for surveying taxa for these viral genes. In total, these data provide a robust framework for the evolution of Ichneumonoidea.

The First Complete Genome Sequence of a Novel Tetrastichus brontispae RNA Virus-1 (TbRV-1).

The complete sequence of a novel RNA virus isolated from Tetrastichus brontispae (TbRV-1) was determined to be 12,239 nucleotides in length with five non-overlapping, linearly arranged coding sequences (CDS), potentially encoding nucleoproteins, hypothetical proteins, matrix proteins, glycoproteins, and RNA-dependent RNA polymerases. Sequence analysis indicated that the RNA-dependent RNA polymerase of TbRV-1 shares a 65% nucleotide and 67% amino acid sequence identity with Hubei dimarhabdovirus 2, suggesting that TbRV-1 is a member of the dimarhabdovirus supergroup. This corresponded to the result of the phylogenetic analysis. The affiliation of TbRV-1 with members of the family Rhabdoviridae was further validated by similar transcription termination motifs (GGAACUUUUUUU) to the Drosophila sigmavirus. The prevalence of TbRV-1 in all tissues suggested that the virus was constitutive of, and not specific to, any wasp tissue. To our knowledge, this is the first report on the complete genome sequence of a dimarhabdovirus in parasitoids.

Evidence for an ichnovirus machinery in parasitoids of coleopteran larvae.

Bathyplectes spp. are ichneumonid solitary larval parasitoids of the alfalfa weevil which have been classified in the subfamily Campopleginae and which harbor atypical virus particles. Despite the morphological differences between Bathyplectes spp. particles and the polydnaviruses carried by a number of related campoplegine species, called ichnoviruses, the process by which they are produced is very similar to that of ichnoviruses. To address the question of the nature and origin of these atypical particles, the Bathyplectes anurus ovary transcriptome has been analyzed. We found a number of highly expressed transcripts displaying similarities with genes belonging to the machinery involved in the production of ichnovirus particles. In addition, transcripts with similarities with repeat-element genes, which are characteristic of the packaged campoplegine ichnovirus genome were identified. Altogether, our results provide evidence that Bathyplectes particles are related to ichnoviruses.

Genetic footprints of adaptive divergence in the bracovirus of Cotesia sesamiae identified by targeted resequencing.

The African parasitoid wasp Cotesia sesamiae is a generalist species structured in locally adapted populations showing differences in host range. The recent discovery of Cotesia typhae, a specialist, sister species to C. sesamiae, provides a good framework to study the genetic determinants of parasitoid host range. To investigate the genomic bases of divergence between these populations and species, we used a targeted sequencing approach on 24 samples. We targeted the bracovirus genomic region encoding virulence genes involved in the interaction with the lepidopteran hosts of the wasps. High sequencing coverage was obtained for all samples, allowing the study of genetic variation between wasp populations and species. By combining population genetic estimations, such as nucleotide diversity (π), relative differentiation (FST ) and absolute divergence (dxy ), with branch-site dN/dS measures, we identified six of 98 bracovirus genes showing significant divergence and evidence of positive selection. These genes, belonging to different gene families, are potentially involved in host adaptation and in the specialization process. Fine-scale analyses of genetic variation also revealed mutations and large deletions in certain genes inducing pseudogenization and loss of function. The image emerging from these results is that adaptation mediated by bracovirus genes happens through selection of particularly adaptive alleles and loss of nonadaptive genes. These results highlight the central role of the bracovirus in the molecular interactions between the wasps and their hosts and in the evolutionary processes of specialization.

A polydnavirus-encoded ANK protein has a negative impact on steroidogenesis and development.

Polydnaviruses (PDV) are viral symbionts associated with ichneumonid and braconid wasps parasitizing moth larvae, which are able to disrupt the host immune response and development, as well as a number of other physiological pathways. The immunosuppressive role of PDV has been more intensely investigated, while very little is known about the PDV-encoded factors disrupting host development. Here we address this research issue by further expanding the functional analysis of ankyrin genes encoded by the bracovirus associated with Toxoneuron nigriceps (Hymenoptera, Braconidae). In a previous study, using Drosophila melanogaster as experimental model system, we demonstrated the negative impact of TnBVank1 impairing the ecdysone biosynthesis by altering endocytic traffic in prothoracic gland cells. With a similar approach here we demonstrate that another member of the viral ank gene family, TnBVank3, does also contribute to the disruption of ecdysone biosynthesis, but with a completely different mechanism. We show that its expression in Drosophila prothoracic gland (PG) blocks the larval-pupal transition by impairing the expression of steroidogenic genes. Furthermore, we found that TnBVank3 affects the expression of genes involved in the insulin/TOR signaling and the constitutive activation of the insulin pathway in the PG rescues the pupariation impairment. Collectively, our data demonstrate that TnBVANK3 acts as a virulence factor by exerting a synergistic and non-overlapping function with TnBVANK1 to disrupt the ecdysone biosynthesis.

Evidence of Varroa-mediated deformed wing virus spillover in Hawaii.

Varroa destructor, a parasitic mite of honey bees, is also a vector for viral diseases. The mite displays high host specificity and requires access to colonies of Apis spp. to complete its lifecycle. In contrast, the Deformed Wing Virus (DWV), one of the many viruses transmitted by V. destructor, appears to have a much broader host range. Previous studies have detected DWV in a variety of insect groups that are not directly parasitized by the mite. In this study, we take advantage of the discrete distribution of the Varroa mite in the Hawaiian archipelago to compare DWV prevalence on non-Apis flower visitors, and test whether Varroa presence is linked to a "viral spillover". We selected two islands with different viral landscapes: Oahu, where V. destructor has been present since 2007, and Maui, where the mite is absent. We sampled individuals of Apis mellifera, Ceratina smaragdula, Polistes aurifer, and Polistes exclamens, to assess and compare the DWV prevalence in the Hymenoptera community of the two islands. The results indicated that, as expected, honey bee colonies on Oahu have much higher incidence of DWV compared to Maui. Correspondingly, DWV was detected on the Non-Apis Hymenoptera collected from Oahu, but was absent in the species examined on Maui. The study sites selected shared a similar geography, climate, and insect fauna, but differed in the presence of the Varroa mite, suggesting an indirect, but significant, increase on DWV prevalence in the Hymenoptera community on mite-infected islands.

A Horizontally Transferred Autonomous Helitron Became a Full Polydnavirus Segment in Cotesia vestalis.

Bracoviruses associate symbiotically with thousands of parasitoid wasp species in the family Braconidae, working as virulence gene vectors, and allowing the development of wasp larvae within hosts. These viruses are composed of multiple DNA circles that are packaged into infective particles, and injected together with wasp's eggs during parasitization. One of the viral segments of Cotesia vestalis bracovirus contains a gene that has been previously described as a helicase of unknown origin. Here, we demonstrate that this gene is a Rep/Helicase from an intact Helitron transposable element that covers the viral segment almost entirely. We also provide evidence that this element underwent at least two horizontal transfers, which appear to have occurred consecutively: first from a Drosophila host ancestor to the genome of the parasitoid wasp C. vestalis and its bracovirus, and then from C. vestalis to a lepidopteran host (Bombyx mori). Our results reinforce the idea of parasitoid wasps as frequent agents of horizontal transfers in eukaryotes. Additionally, this Helitron-bracovirus segment is the first example of a transposable element that effectively became a whole viral circle.

Selection of reference genes for expression analysis using RT-qPCR in the dissemination system of Heliothis virescens ascovirus 3 h (HvAV-3h).

Ascoviruses are double-stranded DNA viruses that mainly infect noctuid larvae, and are transmitted by the parasitoid wasp Microplitis similis Lyle. Ascovirus-parasitoids wasp-noctuid insects constitute the dissemination system. Selection of suitable reference genes for the dissemination system could play an important role in elucidating the pathogenic molecular mechanisms of ascovirus. Unfortunately, such studies on potential reference genes in the dissemination system of ascoviruses are lacking. In the present study, we evaluated 11 candidate reference genes: ß-actin1 (ACT1), ß-actin2 (ACT2), elongation factor 1 (EF1), elongation factor 2 (EF2), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), 28S ribosome (28S), Tubulin (TUB) and 18S ribosome (18S). The samples were originally from various virus concentrations and points-in-time of experimental treatments using RefFinder and four algorithms. The results showed that EF1 was the most stable internal gene in S. exigua and M. similis and that EF2 was the most stable in the IOZCAS-Spex-II-A cell line, and the stability of reference genes were confirmed via the expression levels of two inhibitor of apoptosis-like (iap-like) genes from Heliothis virescens ascovirus 3 h (HvAV-3h). This study provides a crucial basis for future research that explores the molecular mechanisms of the pathogenesis of ascoviruses.

A polydnaviral genome of Microplitis bicoloratus bracovirus and molecular interactions between the host and virus involved in NF-κB signaling.

Polydnaviruses (PDVs) play a critical role in altering host gene expression to induce immunosuppression. However, it remains largely unclear how PDV genes affect host genes. Here, the complete genome sequence of Microplitis bicoloratus bracovirus (MbBV), which is known to be an apoptosis inducer, was determined. The MbBV genome consisted of 17 putative double-stranded DNA circles and 179 fragments with a total size of 336,336 bp and contained 116 open reading frames (ORFs). Based on conserved domains, nine gene families were identified, of which the IκB-like viral ankyrin (vank) family included 28 members and was one of the largest families. Among the 116 ORFs, 13 MbBV genes were expressed in hemocytes undergoing MbBV-induced apoptosis and further analyzed. Three vank genes (vank86, vank92, vank101) were expressed in hemocytes collected from Spodoptera litura larvae parasitized by M. bicoloratus, in which host NF-κB/IκBs, including relish, dorsal, and cactus, were also persistently expressed. When Spli221 cells were infected with MbBV viral particles, mRNA levels of host and viral NF-κB/IκB genes were persistent and also varied in Spli221 cells undergoing virus-induced pre-apoptosis cell from 1 to 5 hours postinfection. Both were then expressed in a time-dependent expression in virus-induced apoptotic cells. These data show that viral IκB-like transcription does not inhibit host NF-κB/IκB expression, suggesting that transcription of these genes might be regulated by different mechanisms.

The high-throughput production of dsRNA against sacbrood virus for use in the honey bee Apis cerana (Hymenoptera: Apidae).

Sacbrood virus (SBV) is a serious threat to honey bees. Currently, there is no specific drug available for the treatment of SBV that does not affect the quality of the bee product. RNA interference (RNAi) is an important antiviral strategy for disease control. To effectively utilize this technology, the large-scale production and purification of double-stranded RNA (dsRNA) is necessary. Here, a dsRNA-expressing plasmid targeting the VP1 gene of Chinese sacbrood virus (CSBV) was constructed and expressed in Escherichia coli (E. coli) HT115 (DE3). After lysing and ethanol precipitation from E. coli, dsRNA VP1 was purified with an anion exchange chromatography column. Second instar larvae of Apis cerana were fed the purified dsRNA VP1. A significant decrease in larval mortality and the level of expression of the VP1 gene after CSBV infection was demonstrated after the ingestion of dsRNA VP1. This result provides a potential method for the large-scale production of dsRNA to protect A. cerana from CSBV infection.

Pervasiveness of parasites in pollinators.

Many pollinator populations are declining, with large economic and ecological implications. Parasites are known to be an important factor in the some of the population declines of honey bees and bumblebees, but little is known about the parasites afflicting most other pollinators, or the extent of interspecific transmission or vectoring of parasites. Here we carry out a preliminary screening of pollinators (honey bees, five species of bumblebee, three species of wasp, four species of hoverfly and three genera of other bees) in the UK for parasites. We used molecular methods to screen for six honey bee viruses, Ascosphaera fungi, Microsporidia, and Wolbachia intracellular bacteria. We aimed simply to detect the presence of the parasites, encompassing vectoring as well as actual infections. Many pollinators of all types were positive for Ascosphaera fungi, while Microsporidia were rarer, being most frequently found in bumblebees. We also detected that most pollinators were positive for Wolbachia, most probably indicating infection with this intracellular symbiont, and raising the possibility that it may be an important factor in influencing host sex ratios or fitness in a diversity of pollinators. Importantly, we found that about a third of bumblebees (Bombus pascuorum and Bombus terrestris) and a third of wasps (Vespula vulgaris), as well as all honey bees, were positive for deformed wing virus, but that this virus was not present in other pollinators. Deformed wing virus therefore does not appear to be a general parasite of pollinators, but does interact significantly with at least three species of bumblebee and wasp. Further work is needed to establish the identity of some of the parasites, their spatiotemporal variation, and whether they are infecting the various pollinator species or being vectored. However, these results provide a first insight into the diversity, and potential exchange, of parasites in pollinator communities.

Biology and genomics of viruses within the genus Gammabaculovirus.

Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens.

Polydnaviruses (PDVs) are symbionts of parasitoid wasps that function as gene delivery vehicles in the insects (hosts) that the wasps parasitize. PDVs persist in wasps as integrated proviruses but are packaged as circularized and segmented double-stranded DNAs into the virions that wasps inject into hosts. In contrast, little is known about how PDV genomic DNAs persist in host cells. Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the host Pseudoplusia includens. MdBV infects primarily host hemocytes and also infects a hemocyte-derived cell line from P. includens called CiE1 cells. Here we report that all 15 genomic segments of the MdBV encapsidated genome exhibited long-term persistence in CiE1 cells. Most MdBV genes expressed in hemocytes were persistently expressed in CiE1 cells, including members of the glc gene family whose products transformed CiE1 cells into a suspension culture. PCR-based integration assays combined with cloning and sequencing of host-virus junctions confirmed that genomic segments J and C persisted in CiE1 cells by integration. These genomic DNAs also rapidly integrated into parasitized P. includens. Sequence analysis of wasp-viral junction clones showed that the integration of proviral segments in M. demolitor was associated with a wasp excision/integration motif (WIM) known from other bracoviruses. However, integration into host cells occurred in association with a previously unknown domain that we named the host integration motif (HIM). The presence of HIMs in most MdBV genomic DNAs suggests that the integration of each genomic segment into host cells occurs through a shared mechanism.

VLPs of Leptopilina boulardi share biogenesis and overall stellate morphology with VLPs of the heterotoma clade.

Viruses and virus-like particles (VLPs) of insect parasitoids modify host-parasite interactions. The Drosophila wasp, Leptopilina heterotoma, produce 300 nm spiked VLPs that bind to the host's blood cells via surface projections. L. heterotoma is a generalist wasp that attacks over a dozen Drosophila species. Oviposition introduces VLPs into the hemolymph of Drosophila larvae. VLPs lyse hemocytes and obliterate immune signaling in infected larval hosts. L. boulardi, a member of a distinct Leptopilina clade, is a specialist, whose host range is limited to the melanogaster group. As a step toward understanding a potential relationship between venom contents and host range in these wasps, we used electron microscopy to characterize VLPs from the virulent L. boulardi-17 (Lb-17) strain. While the Lb-17 VLPs can neither lyse blood cells nor suppress host defense, their biogenesis is surprisingly similar to that of L. heterotoma. Like L. heterotoma VLPs, L. boulardi VLPs are stellate; but they have fewer spikes, each spike being significantly longer than the spikes in L. heterotoma VLPs. The Lb-17 VLPs possess a dimple, making them clearly distinct from L. heterotoma VLPs. We discuss the significance of these cross-clade differences in VLP morphologies in relation to their biological activities and the host range of the wasp.

Transcriptomic profiling of Microplitis demolitor bracovirus reveals host, tissue and stage-specific patterns of activity.

The polydnaviruses (PDVs) are a family of DNA viruses that are symbiotically associated with parasitoid wasps. The transcription of particular genes or gene-family members have been reported for several PDVs, but no studies have characterized the spatio-temporal patterns of expression for the entire complement of predicted genes in the encapsidated genome of any PDV isolate. The braconid wasp Microplitis demolitor carries the PDV Microplitis demolitor bracovirus (MdBV) and parasitizes larval stage Pseudoplusia (Chrysodeixis) includens. The encapsidated genome consists of 15 genomic segments with 51 predicted ORFs encoding proteins ≥100 aa. A majority of these ORFs form four multimember gene families (ptp, ank, glc and egf) while the remaining ORFs consist of single copy (orph) genes. Here we used RT-PCR and quantitative real-time PCR methods to profile the encapsidated transcriptome of MdBV in P. includens and M. demolitor. Our results indicate that most predicted genes are expressed in P. includens. Spatial patterns of expression in P. includens differed among genes, but temporal patterns of expression were generally similar, with transcript abundance progressively declining between 24 and 120 h. A subset of ptp, ank and orph genes were also expressed in adult female but not male M. demolitor. Only one encapsidated gene (ank-H4) was expressed in all life stages of M. demolitor, albeit at much lower levels than in P. includens. However, another encapsidated gene (orph-B1) was expressed in adult M. demolitor at similar levels to those detected in P. includens.

Combined effect of nucleopolyhedrovirus and Microplitis pallidipes for the control of the beet armyworm, Spodoptera exigua.

BACKGROUND: Nucleopolyhedrovirus (SeNPV) and Microplitis pallidipes are important biological control agents of Spodoptera exigua populations. The interactions between these agents and their combined effect on pest control were investigated in the laboratory and in commercial greenhouses. RESULTS: Microplitis pallidipes searched for and deposited eggs in more healthy larvae than virus-infected larvae 3 days after viral infection. Each female parasitoid that developed in a virus-infected host oviposited in a virus-infected host, or emerged from a cocoon carrying virus transmitted to 4.0, 7.6 or 2.4 healthy larvae respectively. Each female parasitoid exposed to a mixture of virus and 10% honey water solution transmitted the virus to 2.2 healthy larvae. In an experiment with cabbage growing in commercial greenhouses, the pest population reduction was greater by M. pallidipes carrying SeNPV (82.3-89.7% reduction) than by parasitoids without virus (59.5-62.4% reduction). CONCLUSIONS: Control of S. exigua was greater with M. pallidipes plus SeNPV than with M. pallidipes alone. Microplitis pallidipes preferred healthy hosts to infected hosts. Parasitoids were able to complete their development in virus-infected hosts before the hosts died from the virus infection. The parasitoid ovipositors contaminated with the virus could carry and transmit SeNPV.

Decoupling of host-symbiont-phage coadaptations following transfer between insect species.

Transferring endosymbiotic bacteria between different host species can perturb the coordinated regulation of the host and bacterial genomes. Here we use the most common maternally transmitted bacteria, Wolbachia pipientis, to test the consequences of host genetic background on infection densities and the processes underlying those changes in the parasitoid wasp genus Nasonia. Introgressing the genome of Nasonia giraulti into the infected cytoplasm of N. vitripennis causes a two-order-of-magnitude increase in bacterial loads in adults and a proliferation of the infection to somatic tissues. The host effect on W. pipientis distribution and densities is associated with a twofold decrease in densities of the temperate phage WO-B. Returning the bacteria from the new host species back to the resident host species restores the bacteria and phage to their native densities. To our knowledge, this is the first study to report a host-microbe genetic interaction that affects the densities of both W. pipientis and bacteriophage WO-B. The consequences of the increased bacterial density include a reduction in fecundity, an increase in levels of cytoplasmic incompatibility (CI), and unexpectedly, male-to-female transfer of the bacteria to uninfected females and an increased acceptance of densely infected females to interspecific mates. While paternal inheritance of the W. pipientis was not observed, the high incidence of male-to-female transfer in the introgressed background raises the possibility that paternal transmission could be more likely in hybrids where paternal leakage of other cytoplasmic elements is also known to occur. Taken together, these results establish a major change in W. pipientis densities and tissue tropism between closely related species and support a model in which phage WO, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis.

Identification of bracovirus particle proteins and analysis of their transcript levels at the stage of virion formation.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitic wasps; they replicate only in the calyx cells of a wasp's ovaries and are transferred at oviposition along with the parasitoid egg into the lepidopteran host. The DNA packaged in the viral particles encodes factors that manipulate the host's immune defences and development to benefit the parasitoid. PDVs are found in two subfamilies of ichneumonids (ichnoviruses) and in braconids of the microgastroid complex (bracoviruses). We recently showed that the latter derive from an ancestral nudivirus, as 24 nudivirus-related genes were identified in ovaries of two distantly related braconids at the stage of virion formation. Here, we present a comprehensive analysis of the viral particle proteins of the Chelonus inanitus bracovirus (CiBV). Proteins of purified CiBV particles were analysed by mass spectrometry and amino acid sequences matched to the existing ovarian-cDNA database. In addition, transcript quantities of identified genes were measured by quantitative real-time PCR in female pupae at the onset and peak of virion formation and at corresponding stages in male pupae. This combined approach allowed the identification of 44 CiBV particle proteins: 16 were nudivirus-related, three had similarity to ovarian proteins of another braconid, 11 had similarity to cellular proteins and 14 had no similarity to known proteins. The transcripts of all of them increased in female, but not male, pupae. These data confirm the important contribution of nudivirus genes but also indicate the presence of many lineage- or species-specific proteins possibly involved in the parasitoid-host interaction.