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 Hyposotor 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.

Conserved Viral Transcription Plays a Key Role in Virus-Like Particle Production of the Parasitoid Wasp Venturia canescens.

Nudiviruses are large double-stranded DNA viruses related to baculoviruses known to be endogenized in the genomes of certain parasitic wasp species. These wasp-virus associations allow the production of viral particles or virus-like particles that ensure wasp parasitism success within lepidopteran hosts. Venturia canescens is an ichneumonid wasp belonging to the Campopleginae subfamily that has endogenized nudivirus genes belonging to the Alphanudivirus genus to produce "virus-like particles" (Venturia canescens virus-like particles [VcVLPs]), which package proteic virulence factors. The main aim of this study was to determine whether alphanudivirus gene functions have been conserved following endogenization. The expression dynamics of alphanudivirus genes was monitored by a high throughput transcriptional approach, and the functional role of lef-4 and lef-8 genes predicted to encode viral RNA polymerase components was investigated by RNA interference. As described for baculovirus infections and for endogenized nudivirus genes in braconid wasp species producing bracoviruses, a transcriptional cascade involving early and late expressed alphanudivirus genes could be observed. The expression of lef-4 and lef-8 was also shown to be required for the expression of alphanudivirus late genes allowing correct particle formation. Together with previous literature, the results show that endogenization of nudiviruses in parasitoid wasps has repeatedly led to the conservation of the viral RNA polymerase function, allowing the production of viruses or viral-like particles that differ in composition but enable wasp parasitic success. IMPORTANCE This study shows that endogenization of a nudivirus genome in a Campopleginae parasitoid wasp has led to the conservation, as for endogenized nudiviruses in braconid parasitoid wasps, of the viral RNA polymerase function, required for the transcription of genes encoding viral particles involved in wasp parasitism success. We also showed for the first time that RNA interference (RNAi) can be successfully used to downregulate gene expression in this species, a model in behavioral ecology. This opens the opportunity to investigate the function of genes involved in other traits important for parasitism success, such as reproductive strategies and host choice. Fundamental data acquired on gene function in Venturia canescens are likely to be transferable to other parasitoid wasp species used in biological control programs. This study also renders possible the investigation of other nudivirus gene functions, for which little data are available.

Identifying bracovirus and ichnovirus genes involved in virion morphogenesis.

Bracoviruses (BVs) and ichnoviruses (IVs) evolved from different endogenized viruses but through convergence have been coopted by parasitoids in the families Braconidae and Ichneumonidae for similar functions in parasitizing hosts. Experimentally studying the role of endogenized viral genes in virion morphogenesis remains a key challenge in the study of BVs and IVs. Here we summarize how multiomics, electron microscopy, and RNA interference (RNAi) methods have provided new insights about BV and IV gene function.

Proteo-Trancriptomic Analyses Reveal a Large Expansion of Metalloprotease-Like Proteins in Atypical Venom Vesicles of the Wasp Meteorus pulchricornis (Braconidae).

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be "metalloproteinase bombs" that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified.

Plant-phenotypic changes induced by parasitoid ichnoviruses enhance the performance of both unparasitized and parasitized caterpillars.

There is increasing awareness that interactions between plants and insects can be mediated by microbial symbionts. Nonetheless, evidence showing that symbionts associated with organisms beyond the second trophic level affect plant-insect interactions are restricted to a few cases belonging to parasitoid-associated bracoviruses. Insect parasitoids harbour a wide array of symbionts which, like bracoviruses, can be injected into their herbivorous hosts to manipulate their physiology and behaviour. Yet, the function of these symbionts in plant-based trophic webs remains largely overlooked. Here, we provide the first evidence of a parasitoid-associated symbiont belonging to the group of ichnoviruses which affects the strength of plant-insect interactions. A comparative proteomic analysis shows that, upon parasitoid injection of calyx fluid containing ichnovirus particles, the composition of salivary glands of caterpillars changes both qualitatively (presence of two viral-encoded proteins) and quantitatively (abundance of several caterpillar-resident enzymes, including elicitors such as glucose oxidase). In turn, plant phenotypic changes triggered by the altered composition of caterpillar oral secretions affect the performance of herbivores. Ichnovirus manipulation of plant responses to herbivory leads to benefits for their parasitoid partners in terms of reduced developmental time within the parasitized caterpillar. Interestingly, plant-mediated ichnovirus-induced effects also enhance the performances of unparasitized herbivores which in natural conditions may feed alongside parasitized ones. We discuss these findings in the context of ecological costs imposed to the plant by the viral symbiont of the parasitoid. Our results provide intriguing novel findings about the role played by carnivore-associated symbionts on plant-insect-parasitoid systems and underline the importance of placing mutualistic associations in an ecological perspective.

Influence of parasitoid-associated viral symbionts on plant-insect interactions and biological control.

Insect parasitoids have evolved symbiotic interactions with several viruses and thousands of parasitoid species have established mutualistic associations with polydnaviruses (PDVs). While PDVs have often been described as virulence factors allowing development of immature parasitoids inside their herbivore hosts, there is increasing awareness that PDVs can affect plant-insect interactions. We review recent literature showing that PDVs alter not only host physiology, but also feeding patterns and composition of herbivore's oral secretions. In turn PDV-induced changes in herbivore phenotype affect plant responses to herbivory with consequences ranging from differential expression of plant defense-related genes to wider ecological effects across multiple trophic levels. In this opinion paper we also highlight important missing gaps to fully understand the role of PDVs and other parasitoid-associated viral symbionts in a plant-insect interaction perspective. Because PDVs negatively impact performance and survival of herbivore pests, we conclude arguing that PDV genomes offer potential opportunities for biological control.

Progeny fitness determines the performance of the parasitoid Therophilus javanus, a prospective biocontrol agent against the legume pod borer.

Therophilus javanus (Bhat & Gupta) is an exotic larval endoparasitoid newly imported from Asia into Africa as a classical biological control agent against the pod borer Maruca vitrata (Fabricius). The parasitoid preference for the five larval instars of M. vitrata and their influence on progeny sex ratio were assessed together with the impact of larval host age at the time of oviposition on development time, mother longevity and offspring production. In a choice situation, female parasitoids preferred to oviposit in the first three larval instars. The development of immature stages of the parasitoid was observed inside three-day-old hosts, whereby the first two larval instars of T. javanus completed their development as endoparasites and the third larval instar as ectoparasite. The development time was faster when first larval instars (two- and three-day-old) of the host caterpillars were parasitized compared to second larval instar (four-day-old). The highest proportion of daughters (0.51) was observed when females were provided with four-day-old hosts. The lowest intrinsic rate of increase (r) (0.21 ± 0.01), the lowest rate of increase (λ) (1.23 ± 0.01), and the lowest net reproductive rate (Ro) (35.93 ± 6.51) were recorded on four-day-old hosts. These results are discussed in the light of optimizing mass rearing and release strategies.

The Unconventional Viruses of Ichneumonid Parasitoid Wasps.

To ensure their own immature development as parasites, ichneumonid parasitoid wasps use endogenous viruses that they acquired through ancient events of viral genome integration. Thousands of species from the campoplegine and banchine wasp subfamilies rely, for their survival, on their association with these viruses, hijacked from a yet undetermined viral taxon. Here, we give an update of recent findings on the nature of the viral genes retained from the progenitor viruses and how they are organized in the wasp genome.

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.

[Polydnaviruses, a unique example of viral machinery domesticated by parasitoid wasps]. / Les Polydnavirus, un exemple unique de machinerie virale domestiquée par des insectes parasitoïdes.

Polydnaviruses are unique mutualistic viruses associated with thousands of parasitoid wasps. They are characterized by a segmented packaged DNA genome and are necessary for parasitic success. Virus particles are produced in the wasp ovaries from a set of "viral" sequences integrated into the wasp genome. The polydnavirus/wasp associations as observed today result from the integration of a viral genomes into the wasp genome during evolution. Recent years have been marked by the discovery of the viral ancestors of the two known types of polydnavirus, bracovirus and ichnovirus, which has helped to shed some light on the evolution of the symbiosis. Some of the viral genes have been conserved in the genome of the parasitoid, allowing the latter to produce non-replicative viral particles, that contain DNA molecules encoding essentially "virulence" genes, probably of insect origin. Thus polydnaviruses can be considered as endogenous viral elements (EVE) that have been domesticated by the wasp to become a "weapon" allowing its survival.

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.

Venom Atypical Extracellular Vesicles as Interspecies Vehicles of Virulence Factors Involved in Host Specificity: The Case of a Drosophila Parasitoid Wasp.

Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to protect their offspring from the host immune response. The hymenopteran species of the genus Leptopilina, parasites of Drosophila, rely on the injection of a venom which contains proteins and peculiar vesicles (hereafter venosomes). We show here that the injection of purified L. boulardi venosomes is sufficient to impair the function of the Drosophila melanogaster lamellocytes, a hemocyte type specialized in the defense against wasp eggs, and thus the parasitic success of the wasp. These venosomes seem to have a unique extracellular biogenesis in the wasp venom apparatus where they acquire specific secreted proteins/virulence factors and act as a transport system to deliver these compounds into host lamellocytes. The level of venosomes entry into lamellocytes of different Drosophila species was correlated with the rate of parasitism success of the wasp, suggesting that this venosome-cell interaction may represent a new evolutionary level of host-parasitoid specificity.

The dual life of ichnoviruses.

Ichnoviruses (IVs) are mutualistic, double-stranded DNA viruses playing a key role in the successful parasitism of thousands of endoparasitoid wasp species. IV particles are produced exclusively in the female wasp reproductive tract. They are co-injected along with the parasitoid egg into caterpillar hosts upon parasitization. The expression of viral genes by infected host cells leads to an immunosuppressive state and delayed development of the host, two pathologies that are critical to the successful development of the wasp egg and larva. Ichnovirus is one of the two recognized genera within the family Polydnaviridae (polydnaviruses or PDVs), the other genus being Bracovirus (BV), associated with braconid wasps. IVs are associated with ichneumonid wasps belonging to the subfamilies Campopleginae and Banchinae; attempts to identify IV particles in other ichneumonid subfamilies have so far been unsuccessful. Functional studies targeting IV genes expressed in parasitized hosts, along with investigations of the molecular mechanisms responsible for viral morphogenesis in the female wasp, have resulted in a better understanding of the biology of these atypical viruses.

Volatiles from Maruca vitrata (Lepidoptera, Crambidae) host plants influence olfactory responses of the parasitoid Therophilus javanus (Hymenoptera, Braconidae, Agathidinae).

Plants damaged by herbivores are known to release odors attracting parasitoids. However, there is currently no information how leguminous plants damaged by the pod borer Maruca vitrata attract the exotic larval parasitoid Therophilus javanus, which was imported into Benin from the putative area of origin of the pod borer in tropical Asia for assessing its potential as a biological control agent. In this study, we used Y-tube olfactometer bioassays to investigate T. javanus response towards odors emitted by four M. vitrata-damaged host plants: cowpea Vigna unguiculata, the most important cultivated host, and the naturally occurring legumes Lonchocarpus sericeus, Sesbania rostrata and Tephrosia platycarpa. Olfactory attraction of T. javanus was influenced by the species of plant damaged by the pod borer. Moreover, odors released from M. vitrata-infested host plant organs (flowers and pods) were discriminated over non-infested organs in cowpea and T. platycarpa, respectively. These results are discussed in the context of the possible impact of M. vitrata host plants on T. javanus foraging activity and subsequent establishment in natural environments following experimental releases.

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.

The Domestication of a Large DNA Virus by the Wasp Venturia canescens Involves Targeted Genome Reduction through Pseudogenization.

Polydnaviruses (PDVs) are compelling examples of viral domestication, in which wasps express a large set of genes originating from a chromosomally integrated virus to produce particles necessary for their reproductive success. Parasitoid wasps generally use PDVs as a virulence gene delivery system allowing the protection of their progeny in the body of parasitized host. However, in the wasp Venturia canescens an independent viral domestication process led to an alternative strategy as the wasp incorporates virulence proteins in viral liposomes named virus-like particles (VLPs), instead of DNA molecules. Proteomic analysis of purified VLPs and transcriptome sequencing revealed the loss of some viral functions. In particular, the genes coding for capsid components are no longer expressed, which explains why VLPs do not incorporate DNA. Here a thorough examination of V. canescens genome revealed the presence of the pseudogenes corresponding to most of the genes involved in lost functions. This strongly suggests that an accumulation of mutations that leads to gene specific pseudogenization precedes the loss of viral genes observed during virus domestication. No evidence was found for block loss of collinear genes, although extensive gene order reshuffling of the viral genome was identified from comparisons between endogenous and exogenous viruses. These results provide the first insights on the early stages of large DNA virus domestication implicating massive genome reduction through gene-specific pseudogenization, a process which differs from the large deletions described for bacterial endosymbionts.

Parasitic wasp-associated symbiont affects plant-mediated species interactions between herbivores.

Microbial mutualistic symbiosis is increasingly recognised as a hidden driving force in the ecology of plant-insect interactions. Although plant-associated and herbivore-associated symbionts clearly affect interactions between plants and herbivores, the effects of symbionts associated with higher trophic levels has been largely overlooked. At the third-trophic level, parasitic wasps are a common group of insects that can inject symbiotic viruses (polydnaviruses) and venom into their herbivorous hosts to support parasitoid offspring development. Here, we show that such third-trophic level symbionts act in combination with venom to affect plant-mediated interactions by reducing colonisation of subsequent herbivore species. This ecological effect correlated with changes induced by polydnaviruses and venom in caterpillar salivary glands and in plant defence responses to herbivory. Because thousands of parasitoid species are associated with mutualistic symbiotic viruses in an intimate, specific relationship, our findings may represent a novel and widespread ecological phenomenon in plant-insect interactions.

First extensive characterization of the venom gland from an egg parasitoid: structure, transcriptome and functional role.

The venom gland is a ubiquitous organ in Hymenoptera. In insect parasitoids, the venom gland has been shown to have multiple functions including regulation of host immune response, host paralysis, host castration and developmental alteration. However, the role played by the venom gland has been mainly studied in parasitoids developing in larval or pupal hosts while little is known for parasitoids developing in insect eggs. We conducted the first extensive characterization of the venom of the endoparasitoid Ooencyrtus telenomicida (Vassiliev), a species that develops in eggs of the stink bug Nezara viridula (L.). In particular we investigated the structure of the venom apparatus, its functional role and conducted a transcriptomic analysis of the venom gland. We found that injection of O. telenomicida venom induces: 1) a melanized-like process in N. viridula host eggs (host-parasitoid interaction), 2) impairment of the larval development of the competitor Trissolcus basalis (Wollaston) (parasitoid-parasitoid interaction). The O. telenomicida venom gland transcriptome reveals a majority of digestive enzymes (peptidases and glycosylases) and oxidoreductases (laccases) among the most expressed genes. The former enzymes are likely to be involved in degradation of the host resources for the specific benefit of the O. telenomicida offspring. In turn, alteration of host resources caused by these enzymes may negatively affect the larval development of the competitor T. basalis. We hypothesize that the melanization process induced by venom injection could be related to the presence of laccases, which are multicopper oxidases that belong to the phenoloxidases group. This work contributed to a better understanding of the venom in insect parasitoids and allowed to identify candidate genes whose functional role can be investigated in future studies.