Identification of a viral gene essential for the genome replication of a domesticated endogenous virus in ichneumonid parasitoid wasps.

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor "domesticated endogenous viruses" (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposoter didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis.

Massive Somatic and Germline Chromosomal Integrations of Polydnaviruses in Lepidopterans.

Increasing numbers of horizontal transfer (HT) of genes and transposable elements are reported in insects. Yet the mechanisms underlying these transfers remain unknown. Here we first quantify and characterize the patterns of chromosomal integration of the polydnavirus (PDV) encoded by the Campopleginae Hyposoter didymator parasitoid wasp (HdIV) in somatic cells of parasitized fall armyworm (Spodoptera frugiperda). PDVs are domesticated viruses injected by wasps together with their eggs into their hosts in order to facilitate the development of wasp larvae. We found that six HdIV DNA circles integrate into the genome of host somatic cells. Each host haploid genome suffers between 23 and 40 integration events (IEs) on average 72 h post-parasitism. Almost all IEs are mediated by DNA double-strand breaks occurring in the host integration motif (HIM) of HdIV circles. We show that despite their independent evolutionary origins, PDV from both Campopleginae and Braconidae wasps use remarkably similar mechanisms for chromosomal integration. Next, our similarity search performed on 775 genomes reveals that PDVs of both Campopleginae and Braconidae wasps have recurrently colonized the germline of dozens of lepidopteran species through the same mechanisms they use to integrate into somatic host chromosomes during parasitism. We found evidence of HIM-mediated HT of PDV DNA circles in no less than 124 species belonging to 15 lepidopteran families. Thus, this mechanism underlies a major route of HT of genetic material from wasps to lepidopterans with likely important consequences on lepidopterans.

Functional characterization of Microplitis demolitor bracovirus genes that encode nucleocapsid components.

IMPORTANCE: Understanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of the Microplitis demolitor bracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding protein p6.9-1 are required for DNA processing and packaging into nucleocapsids.

Bracoviruses recruit host integrases for their integration into caterpillar's genome.

Some DNA viruses infect host animals usually by integrating their DNAs into the host genome. However, the mechanisms for integration remain largely unknown. Here, we find that Cotesia vestalis bracovirus (CvBV), a polydnavirus of the parasitic wasp C. vestalis (Haliday), integrates its DNA circles into host Plutella xylostella (L.) genome by two distinct strategies, conservatively and randomly, through high-throughput sequencing analysis. We confirmed that the conservatively integrating circles contain an essential "8+5" nucleotides motif which is required for integration. Then we find CvBV circles are integrated into the caterpillar's genome in three temporal patterns, the early, mid and late stage-integration. We further identify that three CvBV-encoded integrases are responsible for some, but not all of the virus circle integrations, indeed they mainly participate in the processes of early stage-integration. Strikingly, we find two P. xylostella retroviral integrases (PxIN1 and PxIN2) are highly induced upon wasp parasitism, and PxIN1 is crucial for integration of some other early-integrated CvBV circles, such as CvBV_04, CvBV_12 and CvBV_24, while PxIN2 is important for integration of a late-integrated CvBV circle, CvBV_21. Our data uncover a novel mechanism in which CvBV integrates into the infected host genome, not only by utilizing its own integrases, but also by recruiting host enzymes. These findings will strongly deepen our understanding of how bracoviruses regulate and integrate into their hosts.

Genome-wide identification of lncRNAs associated with viral infection in Spodoptera frugiperda.

The role of lncRNAs in immune defence has been demonstrated in many multicellular and unicellular organisms. However, investigation of the identification and characterization of long non-coding RNAs (lncRNAs) involved in the insect immune response is still limited. In this study, we used RNA sequencing (RNA-seq) to investigate the expression profiles of lncRNAs and mRNAs in the fall armyworm Spodoptera frugiperda in response to virus infection. To assess the tissue- and virus-specificity of lncRNAs, we analysed and compared their expression profiles in haemocytes and fat body of larvae infected with two entomopathogenic viruses with different lifestyles, i.e. the polydnavirus HdIV (Hyposoter didymator IchnoVirus) and the densovirus JcDV (Junonia coenia densovirus). We identified 1883 candidate lncRNAs, of which 529 showed differential expression following viral infection. Expression profiles differed considerably between samples, indicating that many differentially expressed (DE) lncRNAs showed virus- and tissue-specific expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and target prediction analyses indicated that DE-LncRNAs were mainly enriched in metabolic process, DNA replication and repair, immune response, metabolism of insect hormone and cell adhesion. In addition, we identified three DE-lncRNAs potentially acting as microRNA host genes, suggesting that they participate in gene regulation by producing miRNAs in response to virus infection. This study provides a catalogue of lncRNAs expressed in two important immune tissues and potential insight into their roles in the antiviral defence in S. frugiperda. The results may help future in-depth functional studies to better understand the biological function of lncRNAs in interaction between viruses and the fall armyworm.

The Complete Genome of Chelonus insularis Reveals Dynamic Arrangement of Genome Components in Parasitoid Wasps That Produce Bracoviruses.

Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae (Microplitis demolitor and Cotesia congregata) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis, which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized compared to M. demolitor and C. congregata. Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. IMPORTANCE Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This work characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis, which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function.

Symbiotic bracovirus of a parasite manipulates host lipid metabolism via tachykinin signaling.

Parasites alter host energy homeostasis for their own development, but the mechanisms underlying this phenomenon remain largely unknown. Here, we show that Cotesia vestalis, an endoparasitic wasp of Plutella xylostella larvae, stimulates a reduction of host lipid levels. This process requires excess secretion of P. xylostella tachykinin (PxTK) peptides from enteroendocrine cells (EEs) in the midgut of the parasitized host larvae. We found that parasitization upregulates PxTK signaling to suppress lipogenesis in midgut enterocytes (ECs) in a non-cell-autonomous manner, and the reduced host lipid level benefits the development of wasp offspring and their subsequent parasitic ability. We further found that a C. vestalis bracovirus (CvBV) gene, CvBV 9-2, is responsible for PxTK induction, which in turn reduces the systemic lipid level of the host. Taken together, these findings illustrate a novel mechanism for parasite manipulation of host energy homeostasis by a symbiotic bracovirus gene to promote the development and increase the parasitic efficiency of an agriculturally important wasp species.

Chelonus inanitus bracovirus encodes lineage-specific proteins and truncated immune IκB-like factors.

Bracoviruses and ichnoviruses are endogenous viruses of parasitic wasps that produce particles containing virulence genes expressed in host tissues and necessary for parasitism success. In the case of bracoviruses the particles are produced by conserved genes of nudiviral origin integrated permanently in the wasp genome, whereas the virulence genes can strikingly differ depending on the wasp lineage. To date most data obtained on bracoviruses concerned species from the braconid subfamily of Microgastrinae. To gain a broader view on the diversity of virulence genes we sequenced the genome packaged in the particles of Chelonus inanitus bracovirus (CiBV) produced by a wasp belonging to a different subfamily: the Cheloninae. These are egg-larval parasitoids, which means that they oviposit into the host egg and the wasp larvae then develop within the larval stages of the host. We found that most of CiBV virulence genes belong to families that are specific to Cheloninae. As other bracoviruses and ichnoviruses however, CiBV encode v-ank genes encoding truncated versions of the immune cactus/IκB factor, which suggests these proteins might play a key role in host-parasite interactions involving domesticated endogenous viruses. We found that the structures of CiBV V-ANKs are different from those previously reported. Phylogenetic analysis supports the hypothesis that they may originate from a cactus/IκB immune gene from the wasp genome acquired by the bracovirus. However, their evolutionary history is different from that shared by other V-ANKs, whose common origin probably reflects horizontal gene transfer events of virus sequences between braconid and ichneumonid wasps.

Genome-Wide Patterns of Bracovirus Chromosomal Integration into Multiple Host Tissues during Parasitism.

Bracoviruses are domesticated viruses found in parasitic wasp genomes. They are composed of genes of nudiviral origin that are involved in particle production and proviral segments containing virulence genes that are necessary for parasitism success. During particle production, proviral segments are amplified and individually packaged as DNA circles in nucleocapsids. These particles are injected by parasitic wasps into host larvae together with their eggs. Bracovirus circles of two wasp species were reported to undergo chromosomal integration in parasitized host hemocytes, through a conserved sequence named the host integration motif (HIM). Here, we used bulk Illumina sequencing to survey integrations of Cotesia typhae bracovirus circles in the DNA of its host, the maize corn borer (Sesamia nonagrioides), 7 days after parasitism. First, assembly and annotation of a high-quality genome for C. typhae enabled us to characterize 27 proviral segments clustered in proviral loci. Using these data, we characterized large numbers of chromosomal integrations (from 12 to 85 events per host haploid genome) for all 16 bracovirus circles containing a HIM. Integrations were found in four S. nonagrioides tissues and in the body of a caterpillar in which parasitism had failed. The 12 remaining circles do not integrate but are maintained at high levels in host tissues. Surprisingly, we found that HIM-mediated chromosomal integration in the wasp germ line has occurred accidentally at least six times during evolution. Overall, our study furthers our understanding of wasp-host genome interactions and supports HIM-mediated chromosomal integration as a possible mechanism of horizontal transfer from wasps to their hosts. IMPORTANCE Bracoviruses are endogenous domesticated viruses of parasitoid wasps that are injected together with wasp eggs into wasp host larvae during parasitism. Several studies have shown that some DNA circles packaged into bracovirus particles become integrated into host somatic genomes during parasitism, but the phenomenon has never been studied using nontargeted approaches. Here, we use bulk Illumina sequencing to systematically characterize and quantify bracovirus circle integrations that occur in four tissues of the Mediterranean corn borer (Sesamia nonagrioides) during parasitism by the Cotesia typhae wasp. Our analysis reveals that all circles containing a HIM integrate at substantial levels (from 12 to 85 integrations per host cell, in total) in all tissues, while other circles do not integrate. In addition to shedding new light on wasp-bracovirus-host interactions, our study supports HIM-mediated chromosomal integration of bracovirus as a possible source of wasp-to-host horizontal transfer, with long-term evolutionary consequences.

Investigating bracovirus chromosomal integration and inheritance in lepidopteran host and nontarget species.

Bracoviruses (BVs) are domesticated viruses found in braconid parasitoid wasp genomes. They are composed of domesticated genes from a nudivrius, coding viral particles in which wasp DNA circles are packaged. BVs are viewed as possible vectors of horizontal transfer of genetic material (HT) from wasp to their hosts because they are injected, together with wasp eggs, by female wasps into their host larvae, and because they undergo massive chromosomal integration in multiple host tissues. Here, we show that chromosomal integrations of the Cotesia typhae BV (CtBV) persist up to the adult stage in individuals of its natural host, Sesamia nonagrioides, that survived parasitism. However, while reproducing host adults can bear an average of nearly two CtBV integrations per haploid genome, we were unable to retrieve any of these integrations in 500 of their offspring using Illumina sequencing. This suggests either that host gametes are less targeted by CtBVs than somatic cells or that gametes bearing BV integrations are nonfunctional. We further show that CtBV can massively integrate into the chromosomes of other lepidopteran species that are not normally targeted by the wasp in the wild, including one which is divergent by at least 100 million years from the natural host. Cell entry and chromosomal integration of BVs are thus unlikely to be major factors shaping wasp host range. Together, our results shed new light on the conditions under which BV-mediated wasp-to-host HT may occur and provide information that may be helpful to evaluate the potential risks of uncontrolled HT associated with the use of parasitoid wasps as biocontrol agents.

The naked truth: An updated review on nudiviruses and their relationship to bracoviruses and baculoviruses.

Nudiviruses (Nudiviridae) are double-stranded DNA viruses with enveloped and rod-shaped virions. Several insect orders (e.g., Diptera, Lepidoptera, Coleoptera, Orthoptera) and aquatic crustaceans are susceptible to nudivirus infections, which can result in varied degrees of disease in all developmental host stages. Their pathogenicity endangers insect rearing and crustacean aquacultures, but has also proven effective in biocontrol against Oryctes rhinoceros infestations. This literature review aims to present all known nudivirus species and provide a comprehensive Nudiviridae phylogeny by including recently described nudiviral isolates, and discuss this phylogeny in comparison to current opinions and taxonomical propositions. Moreover, we aim to clarify biological, pathological and genomic differences or similarities between nudiviruses and related entomopathogenic viruses, including baculoviruses (Baculoviridae) and bracoviruses (Polydnaviridae). A phylogenetic analysis using 17 concatenated nudivirus core genes resulted in the expected structure with the genera Alphanudivirus and Betanudivirus, as well as the most recently recognized genera Gammanudivirus and Deltanudivirus. The hymenopteran Osmia cornuta nudivirus (OcNV) groups closest with the hymenopteran Fopius arisanus endogenous nudivirus (FaENV) and does not share a most common ancestor with the hymenopteran bracoviruses. Except for one node, all clades are highly supported. The proposition of a recent study to assign subgroups to the alphanudiviruses might be legitimate, but more hymenopteran and orthopteran nudiviruses, especially in bees and cricket, need to be identified to resolve this proposal. In addition, freshwater and marine nudiviruses might form taxonomic subgroups among gammanudiviruses as well, but more aquatic nudiviruses need to be identified and sequenced for better resolution. Furthermore, the search for nudiviruses in insects with (semi)aquatic life stages may aid in finding the missing link that led to the manifestation of aquatic nudiviruses.

MdBVe46 is an envelope protein that is required for virion formation by Microplitis demolitor bracovirus.

Bracoviruses (BVs) are endogenized nudiviruses that braconid parasitoid wasps have coopted for functions in parasitizing hosts. Microplitis demolitor is a braconid wasp that produces Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of the moth Chrysodeixis includens. Some BV core genes are homologs of genes also present in baculoviruses while others are only known from nudiviruses or other BVs. In this study, we had two main goals. The first was to separate MdBV virions into envelope and nucleocapsid fractions before proteomic analysis to identify core gene products that were preferentially associated with one fraction or the other. Results indicated that nearly all MdBV baculovirus-like gene products that were detected by our proteomic analysis had similar distributions to homologs in the occlusion-derived form of baculoviruses. Several core gene products unknown from baculoviruses were also identified as envelope or nucleocapsid components. Our second goal was to functionally characterize a core gene unknown from baculoviruses that was originally named HzNVorf64-like. Immunoblotting assays supported our proteomic data that identified HzNVorf64-like as an envelope protein. We thus renamed HzNVorf64-like as MdBVe46, which we further hypothesized was important for infection of C. includens. Knockdown of MdBVe46 by RNA interference (RNAi) greatly reduced transcript and protein abundance. Knockdown of MdBVe46 also altered virion morphogenesis, near-fully inhibited infection of C. includens, and significantly reduced the proportion of hosts that were successfully parasitized by M. demolitor.

RNA interference identifies domesticated viral genes involved in assembly and trafficking of virus-derived particles in ichneumonid wasps.

There are many documented examples of viral genes retained in the genomes of multicellular organisms that may in some cases bring new beneficial functions to the receivers. The ability of certain ichneumonid parasitic wasps to produce virus-derived particles, the so-called ichnoviruses (IVs), not only results from the capture and domestication of single viral genes but of almost entire ancestral virus genome(s). Indeed, following integration into wasp chromosomal DNA, the putative and still undetermined IV ancestor(s) evolved into encoding a 'virulence gene delivery vehicle' that is now required for successful infestation of wasp hosts. Several putative viral genes, which are clustered in distinct regions of wasp genomes referred to as IVSPERs (Ichnovirus Structural Protein Encoding Regions), have been assumed to be involved in virus-derived particles morphogenesis, but this question has not been previously functionally addressed. In the present study, we have successfully combined RNA interference and transmission electron microscopy to specifically identify IVSPER genes that are responsible for the morphogenesis and trafficking of the virus-derived particles in ovarian cells of the ichneumonid wasp Hyposoter didymator. We suggest that ancestral viral genes retained within the genomes of certain ichneumonid parasitoids possess conserved functions which were domesticated for the purpose of assembling viral vectors for the delivery of virulence genes to parasitized host animals.

Genomic architecture of endogenous ichnoviruses reveals distinct evolutionary pathways leading to virus domestication in parasitic wasps.

BACKGROUND: Polydnaviruses (PDVs) are mutualistic endogenous viruses inoculated by some lineages of parasitoid wasps into their hosts, where they facilitate successful wasp development. PDVs include the ichnoviruses and bracoviruses that originate from independent viral acquisitions in ichneumonid and braconid wasps respectively. PDV genomes are fully incorporated into the wasp genomes and consist of (1) genes involved in viral particle production, which derive from the viral ancestor and are not encapsidated, and (2) proviral segments harboring virulence genes, which are packaged into the viral particle. To help elucidating the mechanisms that have facilitated viral domestication in ichneumonid wasps, we analyzed the structure of the viral insertions by sequencing the whole genome of two ichnovirus-carrying wasp species, Hyposoter didymator and Campoletis sonorensis. RESULTS: Assemblies with long scaffold sizes allowed us to unravel the organization of the endogenous ichnovirus and revealed considerable dispersion of the viral loci within the wasp genomes. Proviral segments contained species-specific sets of genes and occupied distinct genomic locations in the two ichneumonid wasps. In contrast, viral machinery genes were organized in clusters showing highly conserved gene content and order, with some loci located in collinear wasp genomic regions. This genomic architecture clearly differs from the organization of PDVs in braconid wasps, in which proviral segments are clustered and viral machinery elements are more dispersed. CONCLUSIONS: The contrasting structures of the two types of ichnovirus genomic elements are consistent with their different functions: proviral segments are vehicles for virulence proteins expected to adapt according to different host defense systems, whereas the genes involved in virus particle production in the wasp are likely more stable and may reflect ancestral viral architecture. The distinct genomic architectures seen in ichnoviruses versus bracoviruses reveal different evolutionary trajectories that have led to virus domestication in the two wasp lineages.

Quantitative trait loci involved in the reproductive success of a parasitoid wasp.

Dissecting the genetic basis of intraspecific variations in life history traits is essential to understand their evolution, notably for potential biocontrol agents. Such variations are observed in the endoparasitoid Cotesia typhae (Hymenoptera: Braconidae), specialized on the pest Sesamia nonagrioides (Lepidoptera: Noctuidae). Previously, we identified two strains of C. typhae that differed significantly for life history traits on an allopatric host population. To investigate the genetic basis underlying these phenotypic differences, we used a quantitative trait locus (QTL) approach based on restriction site-associated DNA markers. The characteristic of C. typhae reproduction allowed us generating sisters sharing almost the same genetic content, named clonal sibship. Crosses between individuals from the two strains were performed to generate F2 and F8 recombinant CSS. The genotypes of 181 clonal sibships were determined as well as the phenotypes of the corresponding 4,000 females. Informative markers were then used to build a high-quality genetic map. These 465 markers spanned a total length of 1,300 cM and were organized in 10 linkage groups which corresponded to the number of C. typhae chromosomes. Three QTLs were detected for parasitism success and two for offspring number, while none were identified for sex ratio. The QTLs explained, respectively, 27.7% and 24.5% of the phenotypic variation observed. The gene content of the genomic intervals was investigated based on the genome of C. congregata and revealed 67 interesting candidates, as potentially involved in the studied traits, including components of the venom and of the symbiotic virus (bracovirus) shown to be necessary for parasitism success in related wasps.

Intracellular dynamics of polydnavirus innexin homologues.

Polydnaviruses associated with ichneumonid parasitoid wasps (Ichnoviruses) encode large numbers of genes, often in multigene families. The Ichnovirus Vinnexin gene family, which is expressed in parasitized lepidopteran larvae, encodes homologues of Innexins, the structural components of insect gap junctions. Here, we have examined intracellular behaviours of the Campoletis sonorensis Ichnovirus (CsIV) Vinnexins, alone and in combination with a host Innexin orthologue, Innexin2 (Inx2). QRT-PCR verified that transcription of CsIV vinnexins occurs contemporaneously with inx2, implying co-occurrence of Vinnexin and Inx2 proteins. Confocal microscopy demonstrated that epitope-tagged VinnexinG (VnxG) and VinnexinQ2 (VnxQ2) exhibit similar subcellular localization as Spodoptera frugiperda Inx2 (Sf-Inx2). Surface biotinylation assays verified that all three proteins localize to the cell surface, and cytochalasin B and nocodazole that they rely on actin and microtubule cytoskeletal networks for localization. Immunomicroscopy following co-transfection of constructs indicates extensive co-localization of Vinnexins with each other and Sf-Inx2, and live-cell imaging of mCherry-labelled Inx2 supports that Vinnexins may affect Sf-Inx2 distribution in a Vinnexin-specific fashion. Our findings support that the Vinnexins may disrupt host cell physiology in a protein-specific manner through altering gap junctional intercellular channel communication, as well as indirectly by affecting multicellular junction characteristics.

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.

The recurrent domestication of viruses: major evolutionary transitions in parasitic wasps.

Several lineages of endoparasitoid wasps, which develop inside the body of other insects, have domesticated viruses, used as delivery tools of essential virulence factors for the successful development of their progeny. Virus domestications are major evolutionary transitions in highly diverse parasitoid wasps. Much progress has recently been made to characterize the nature of these ancestrally captured endogenous viruses that have evolved within the wasp genomes. Virus domestication from different viral families occurred at least three times in parasitoid wasps. This evolutionary convergence led to different strategies. Polydnaviruses (PDVs) are viral gene transfer agents and virus-like particles of the wasp Venturia canescens deliver proteins. Here, we take the standpoint of parasitoid wasps to review current knowledge on virus domestications by different parasitoid lineages. Then, based on genomic data from parasitoid wasps, PDVs and exogenous viruses, we discuss the different evolutionary steps required to transform viruses into vehicles for the delivery of the virulence molecules that we observe today. Finally, we discuss how endoparasitoid wasps manipulate host physiology and ensure parasitism success, to highlight the possible advantages of viral domestication as compared with other virulence strategies.

Recurrent Domestication by Lepidoptera of Genes from Their Parasites Mediated by Bracoviruses.

Bracoviruses are symbiotic viruses associated with tens of thousands of species of parasitic wasps that develop within the body of lepidopteran hosts and that collectively parasitize caterpillars of virtually every lepidopteran species. Viral particles are produced in the wasp ovaries and injected into host larvae with the wasp eggs. Once in the host body, the viral DNA circles enclosed in the particles integrate into lepidopteran host cell DNA. Here we show that bracovirus DNA sequences have been inserted repeatedly into lepidopteran genomes, indicating this viral DNA can also enter germline cells. The original mode of Horizontal Gene Transfer (HGT) unveiled here is based on the integrative properties of an endogenous virus that has evolved as a gene transfer agent within parasitic wasp genomes for ≈100 million years. Among the bracovirus genes thus transferred, a phylogenetic analysis indicated that those encoding C-type-lectins most likely originated from the wasp gene set, showing that a bracovirus-mediated gene flux exists between the 2 insect orders Hymenoptera and Lepidoptera. Furthermore, the acquisition of bracovirus sequences that can be expressed by Lepidoptera has resulted in the domestication of several genes that could result in adaptive advantages for the host. Indeed, functional analyses suggest that two of the acquired genes could have a protective role against a common pathogen in the field, baculovirus. From these results, we hypothesize that bracovirus-mediated HGT has played an important role in the evolutionary arms race between Lepidoptera and their pathogens.

Relationship between oviposition, virulence gene expression and parasitism success in Cotesia typhae nov. sp. parasitoid strains.

Studying mechanisms that drive host adaptation in parasitoids is crucial for the efficient use of parasitoids in biocontrol programs. Cotesia typhae nov. sp. (Fernández-Triana) (Hymenoptera: Braconidae) is a newly described parasitoid of the Mediterranean corn borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Braconidae are known for their domesticated bracovirus, which is injected with eggs in the host larva to overcome its resistance. In this context, we compared reproductive success traits of four Kenyan strains of C. typhae on a French and a Kenyan populations of its host. Differences were found between the four strains and the two most contrasted ones were studied more thoroughly on the French host population. Parasitoid offspring size was correlated with parasitism success and the expression of bracovirus virulence genes (CrV1 and Cystatin) in the host larva after parasitism. Hybrids between these two parasitoid strains showed phenotype and gene expression profiles similar to the most successful parental strain, suggesting the involvement of dominant alleles in the reproductive traits. Ovary dissections revealed that the most successful strain injected more eggs in a single host larva than the less successful one, despite an equal initial ovocyte number in ovaries. It can be expected that the amount of viral particles increase with the number of eggs injected. The ability to bypass the resistance of the allopatric host may in consequence be related to the oviposition behaviour (eggs allocation). The influence of the number of injected eggs on parasitism success and on virulence gene expression was evaluated by oviposition interruption experiments.