Bracovirus gene products are highly divergent from insect proteins

Recently, several polydnavirus (PDV) genomes have been completely sequenced. The dsDNA circles enclosed in virus particles and injected by wasps into caterpillars appear to mainly encode virulence factors potentially involved in altering host immunity and/or development, thereby allowing the survival of the parasitoid larvae within the host tissues. Parasitoid wasps generally inject virulence factors produced in the venom gland. As PDV genomes are inherited vertically by wasps through a proviral form, wasp virulence genes may have been transferred to this chromosomal form, leading to their incorporation into virus particles. Indeed, many gene products from Cotesia congregata bracovirus (CcBV), such as PTPs, IkappaB-like, and cystatins, contain protein domains conserved in metazoans. Surprisingly however, CcBV virulence gene products are not more closely related to insect proteins than to human proteins. To determine whether the distance between CcBV and insect proteins is a specific feature of BV proteins or simply reflects a general high divergence of parasitoid wasp products, which might be due to parasitic lifestyle, we have analyzed the sequences of wasp genes obtained from a cDNA library. Wasp sequences having a high similarity with Apis mellifera genes involved in a variety of biological functions could be identified indicating that the high level of divergence observed for BV products is a hallmark of these viral proteins. We discuss how this divergence might be explained in the context of the current hypotheses on the origin and evolution of wasp-bracovirus associations.     

The few virus-like genes of Cotesia congregata bracovirus

The origin of the symbiotic association between parasitoid wasps and bracoviruses is still unknown. From phylogenetic analyses, bracovirus-associated wasp species constitute a monophyletic group, the microgastroid complex. Thus all wasp-bracovirus associations could have originated from the integration of an ancestral virus in the genome of the ancestor of the microgastroids. In an effort to identify a set of virus genes that would give clues on the nature of the ancestral virus, we have recently performed the complete sequencing of the genome of CcBV, the bracovirus of the wasp Cotesia congregata. We describe here the putative proteins encoded by CcBV genome having significant similarities with sequences from known viruses and mobile elements. The analysis of CcBV gene content does not lend support to the hypothesis that bracoviruses originated from a baculovirus. Moreover, no consistent homology was found between CcBV genes and any set of genes constituting the core genome of a known free-living virus. We discuss the significance of the scarce homology found between proteins from CcBV and other viruses or mobile elements.     

Comparative genomics of mutualistic viruses of Glyptapanteles parasitic wasps

Background

Polydnaviruses, double-stranded DNA viruses with segmented genomes, have evolved as obligate endosymbionts of parasitoid wasps. Virus particles are replication deficient and produced by female wasps from proviral sequences integrated into the wasp genome. These particles are co-injected with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and, thereby, survival of proviral DNA. Here we characterize and compare the encapsidated viral genome sequences of bracoviruses in the family Polydnaviridae associated with Glyptapanteles gypsy moth parasitoids, along with near complete proviral sequences from which both viral genomes are derived.

Results

The encapsidated Glyptapanteles indiensis and Glyptapanteles flavicoxis bracoviral genomes, each composed of 29 different size segments, total approximately 517 and 594 kbp, respectively. They are generated from a minimum of seven distinct loci in the wasp genome. Annotation of these sequences revealed numerous novel features for polydnaviruses, including insect-like sugar transporter genes and transposable elements. Evolutionary analyses suggest that positive selection is widespread among bracoviral genes.

Conclusions

The structure and organization of G. indiensis and G. flavicoxis bracovirus proviral segments as multiple loci containing one to many viral segments, flanked and separated by wasp gene-encoding DNA, is confirmed. Rapid evolution of bracovirus genes supports the hypothesis of bracovirus genes in an 'arms race' between bracovirus and caterpillar. Phylogenetic analyses of the bracoviral genes encoding sugar transporters provides the first robust evidence of a wasp origin for some polydnavirus genes. We hypothesize transposable elements, such as those described here, could facilitate transfer of genes between proviral segments and host DNA.     

Characterization of a venom gland-associated rhabdovirus in the parasitoid wasp Diachasmimorpha longicaudata

Parasitoid wasps reproduce by laying their eggs on or inside of a host insect, which triggers a defense response in the host insect that kills the developing wasp. To counteract the host’s lethal response, some parasitoid wasps are associated with symbiotic viruses that alter host metabolism and development to promote successful development of the wasp embryo. These symbiotic viruses display a number of characteristics that differ from those of pathogenic viruses, but are poorly understood with the exception of one group, the polydnaviruses. Here, we characterize the genome of a non-polydnavirus associated with parasitoid wasps, Diachasmimorpha longicaudata rhabdovirus (DlRhV), and assess its role as a potential mutualistic virus. Our results show that the DlRhV genome contains six open reading frames (ORFs). Three ORFs show sequence homology to known viral genes and one ORF encodes a previously identified protein, called parasitism-specific protein 24 (PSP24), that has been hypothesized to play a role in promoting successful parasitism by D. longicaudata. We constructed a phylogeny that shows that DlRhV is most closely related to other insect-infecting rhabdoviruses. Finally, we report that DlRhV infection does not occur in all populations of D. longicaudata, and is not required for successful parasitism.     

Viral cystatin evolution and three-dimensional structure modelling: A case of directional selection acting on a viral protein involved in a host-parasitoid interaction

Background  

In pathogens, certain genes encoding proteins that directly interact with host defences coevolve with their host and are subject to positive selection. In the lepidopteran host-wasp parasitoid system, one of the most original strategies developed by the wasps to defeat host defences is the injection of a symbiotic polydnavirus at the same time as the wasp eggs. The virus is essential for wasp parasitism success since viral gene expression alters the immune system and development of the host. As a wasp mutualist symbiont, the virus is expected to exhibit a reduction in genome complexity and evolve under wasp phyletic constraints. However, as a lepidopteran host pathogenic symbiont, the virus is likely undergoing strong selective pressures for the acquisition of new functions by gene acquisition or duplication. To understand the constraints imposed by this particular system on virus evolution, we studied a polydnavirus gene family encoding cyteine protease inhibitors of the cystatin superfamily.     

The Bracovirus Genome of the Parasitoid Wasp Cotesia congregata Is Amplified within 13 Replication Units,Including Sequences Not Packaged in the Particles

The relationship between parasitoid wasps and polydnaviruses constitutes one of the few known mutualisms between viruses and eukaryotes. Viral particles are injected with the wasp eggs into parasitized larvae, and the viral genes thus introduced are used to manipulate lepidopteran host physiology. The genome packaged in the particles is composed of 35 double-stranded DNA (dsDNA) circles produced in wasp ovaries by amplification of viral sequences from proviral segments integrated in tandem arrays in the wasp genome. These segments and their flanking regions within the genome of the wasp Cotesia congregata were recently isolated, allowing extensive mapping of amplified sequences. The bracovirus DNAs packaged in the particles were found to be amplified within more than 12 replication units. Strikingly, the nudiviral cluster, the genes of which encode particle structural components, was also amplified, although not encapsidated. Amplification of bracoviral sequences was shown to involve successive head-to-head and tail-to-tail concatemers, which was not expected given the nudiviral origin of bracoviruses.     

Transfer of a chromosomal <Emphasis Type="Italic">Maverick</Emphasis> to endogenous bracovirus in a parasitoid wasp

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.     

Bracoviruses contain a large multigene family coding for protein tyrosine phosphatases

The relationship between parasitic wasps and bracoviruses constitutes one of the few known mutualisms between viruses and eukaryotes. The virions produced in the wasp ovaries are injected into host lepidopteran larvae, where virus genes are expressed, allowing successful development of the parasite by inducing host immune suppression and developmental arrest. Bracovirus-bearing wasps have a common phylogenetic origin, and contemporary bracoviruses are hypothesized to have been inherited by chromosomal transmission from a virus that originally integrated into the genome of the common ancestor wasp living 73.7 +/- 10 million years ago. However, so far no conserved genes have been described among different braconid wasp subfamilies. Here we show that a gene family is present in bracoviruses of different braconid wasp subfamilies (Cotesia congregata, Microgastrinae, and Toxoneuron nigriceps, Cardiochilinae) which likely corresponds to an ancient component of the bracovirus genome that might have been present in the ancestral virus. The genes encode proteins belonging to the protein tyrosine phosphatase family, known to play a key role in the control of signal transduction pathways. Bracovirus protein tyrosine phosphatase genes were shown to be expressed in different tissues of parasitized hosts, and two protein tyrosine phosphatases were produced with recombinant baculoviruses and tested for their biochemical activity. One protein tyrosine phosphatase is a functional phosphatase. These results strengthen the hypothesis that protein tyrosine phosphatases are involved in virally induced alterations of host physiology during parasitism.     

Polydnaviruses: potent mediators of host insect immune dysfunction

Endoparasitic insects are used as biological control agents to kill many species of insect pest. One key to the success of parasitoids that develop in the hemocoel of their host is their ability to knock out the host's immune system, inducing a decline in the responsiveness of a variety of cellular and humoral components so that parasitoid eggs are not encapsulated. In many species parasitized by braconid and ichneumonid wasps, host immunosuppression appears to be mediated by polydnaviruses (PDVs) injected by the female parasitoid into the host hemocoel. The viruses exhibit a complex and intimate genetic relationship with the wasp, since viral sequences are integrated within the wasp's chromosomal DNA. Here Mark Lavine and Nancy Beckage summarize the current evidence for mechanisms of virally induced host immunosuppression in parasitized insects, as well as the roles of other factors including wasp ovarian proteins and venom components, in suppressing hemocyte-mediated and humoral immune responses. Interestingly, in some species, the PDV-induced host immunosuppression appears transitory, with older parasitoid larvae probably exploiting other mechanisms to protect themselves from the host's immune system during the final stages of parasitism. During the final stages of parasitism, the parasitoids likely exploit other mechanisms of immunoevasion via antigen masking, antigen mimicry, or production of active inhibitors of the hemocyte-mediated encapsulation response as well as inhibiting melanization.     

Effects of Intraspecific Competition and Host-Parasitoid Developmental Timing on Foraging Behaviour of a Parasitoid Wasp

In a context where hosts are distributed in patches and susceptible to parasitism for a limited time, female parasitoids foraging for hosts might experience intraspecific competition. We investigated the effects of host and parasitoid developmental stage and intraspecific competition among foraging females on host-searching behaviour in the parasitoid wasp Hyposoter horticola. We found that H. horticola females have a pre-reproductive adult stage during which their eggs are not mature yet and they forage very little for hosts. The wasps foraged for hosts more once they were mature. Behavioural experiments showed that wasps’ foraging activity also increased as host eggs aged and became susceptible to parasitism, and as competition among foraging wasps increased.     

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.     

Genomic analysis reveals an exogenous viral symbiont with dual functionality in parasitoid wasps and their hosts

Insects are known to host a wide variety of beneficial microbes that are fundamental to many aspects of their biology and have substantially shaped their evolution. Notably, parasitoid wasps have repeatedly evolved beneficial associations with viruses that enable developing wasps to survive as parasites that feed from other insects. Ongoing genomic sequencing efforts have revealed that most of these virus-derived entities are fully integrated into the genomes of parasitoid wasp lineages, representing endogenous viral elements (EVEs) that retain the ability to produce virus or virus-like particles within wasp reproductive tissues. All documented parasitoid EVEs have undergone similar genomic rearrangements compared to their viral ancestors characterized by viral genes scattered across wasp genomes and specific viral gene losses. The recurrent presence of viral endogenization and genomic reorganization in beneficial virus systems identified to date suggest that these features are crucial to forming heritable alliances between parasitoid wasps and viruses. Here, our genomic characterization of a mutualistic poxvirus associated with the wasp Diachasmimorpha longicaudata, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), has uncovered the first instance of beneficial virus evolution that does not conform to the genomic architecture shared by parasitoid EVEs with which it displays evolutionary convergence. Rather, DlEPV retains the exogenous viral genome of its poxvirus ancestor and the majority of conserved poxvirus core genes. Additional comparative analyses indicate that DlEPV is related to a fly pathogen and contains a novel gene expansion that may be adaptive to its symbiotic role. Finally, differential expression analysis during virus replication in wasps and fly hosts demonstrates a unique mechanism of functional partitioning that allows DlEPV to persist within and provide benefit to its parasitoid wasp host.     

Polydnavirus hidden face: The genes producing virus particles of parasitic wasps

Very few obligatory relationships involve viruses to the remarkable exception of polydnaviruses (PDVs) associated with tens of thousands species of parasitic wasps that develop within the body of lepidopteran larvae. PDV particles, injected along with parasite eggs into the host body, act by manipulating host immune defences, development and physiology, thereby enabling wasp larvae to survive in a potentially harmful environment. Particle production does not occur in infected tissues of parasitized caterpillars, but is restricted to specialized cells of the wasp ovaries. Moreover, the genome enclosed in the particles encodes almost no viral structural protein, but mostly factors used to manipulate the physiology of the parasitized host. We recently unravelled the viral nature of PDVs associated with braconid wasps by characterizing a large set of nudivirus genes residing permanently in the wasp chromosome(s). Many of these genes encode structural components of the bracovirus particles and their expression pattern correlates with particle production. They constitute a viral machinery comprising a large number of core genes shared by nudiviruses and baculoviruses. Thus bracoviruses do not appear to be nudiviruses remnants, but instead complex nudiviral devices carrying DNA for the delivery of virulence genes into lepidopteran hosts. This highlights the fact that viruses should no longer be exclusively considered obligatory parasites, and that in certain cases they are obligatory symbionts.     

Does Manipulation by the Parasitoid Wasp Cotesia glomerata (L.) Cause Attachment Behaviour of Host Caterpillars on Cocoon Clusters?

Some parasitoid wasps appear to control the behaviour of their hosts. However, altered behaviours of parasitised hosts are not necessarily caused by parasitoids but are sometimes the result of traumatic side effects of parasitism. However, it was difficult for us to discriminate the cause of host's behaviours between manipulation by parasitoids and traumatic side effects. Larvae of the parasitoid wasp Cotesia glomerata form cocoon clusters after egression from the parasitised host caterpillar Pieris brassicae . Following parasitoid egression, host caterpillars survive for several days and remain near the cocoon clusters. These caterpillars may repel solitary pteromalid hyperparasitoid wasps, Trichomalopsis apanteloctena , that attempt to parasitise fresh C. glomerata pupae. We allowed hyperparasitoids to attack cocoon clusters in the field and laboratory and then assessed the costs and benefits to C. glomerata of attachment by the parasitised caterpillars. The eclosion success of C. glomerata in cocoon clusters with attached caterpillars was higher than that in clusters without attached caterpillars in both field and laboratory experiments. This difference was attributed to shorter hyperparasitoid visits to cocoon clusters with attached host caterpillars. However, large cluster size was potentially costly for host attachment, because the duration of host caterpillar attachment decreased with increasing numbers of C. glomerata per caterpillar. This trade-off may be related to shortages of fat body resources, which are shared between the development of wasp larvae and the survival of host caterpillars. Therefore, we concluded that caterpillar attachment satisfied some requirements of host manipulation by C. glomerata .     

Endoparasitoid wasp bracovirus-mediated inhibition of hemolin function and lepidopteran host immunosuppression

Successful embryonic development of parasitoid wasps in lepidopteran hosts is achieved through co-injection of polydna viruses whose gene products are thought to target the immune responses of the host. One gene product of the endosymbiont bracovirus of the parasitic wasp Cotesia rubecula, CrV1, has been reported to inhibit the immune responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. Here we establish that CcV1, the Cotesia congregata bracovirus orthologue of CrV1, is also uptaken by lepidopteran haemocytes and haemocyte-like established cell lines, but we also report on a different function of CcV1, which is highly relevant to the inhibition of the host immune responses and is based on its direct interaction with the pattern recognition molecule hemolin. Recombinant CcV1 inhibits hemolin functions, such as lipopolysaccharide binding and bacterial agglutination as well as bacterial phagocytosis by haemocytes and haemocyte-like cell lines, producing functional phenotypes equivalent to those observed to arise from RNAi-based inhibition of hemolin gene expression. Finally, we show that CcV1 and hemolin colocalize on the membrane surface of hemolin-expressing cells, a finding suggesting that CcV1 may be uptaken by haemocytes and inhibit haemocyte function as a result of its interaction with membrane-anchored hemolin.     

High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster

Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction. Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D. melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D. melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest, identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.     

Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus

Background

Bracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome.

Results

Eighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses.

Conclusion

Contrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.     

Parasitism-induced Effects on Host Growth and Metabolic Efficiency in Tobacco Hornworm Larvae Parasitized by Cotesia congregata

Parasitism by the braconid wasp Cotesia congregata affects the growth of Manduca sexta larvae in a parasitoid 'dose-dependent' fashion. Following parasitization of fourth-instar larvae, more heavily parasitized larvae grew larger compared to those containing fewer parasitoids due to an increase in host dry weight. The differences in host mass appeared to arise after oviposition. A 'dose-dependent' enhancement of host dry weight would appear nutritionally beneficial for the parasitoids developing in more 'crowded' hosts. The efficiencies of conversion of ingested and digested food to body mass and the approximate digestibility of the diet ingested by the host caterpillar did not vary significantly with clutch size although parasitoids took slightly longer to develop in the more heavily parasitized hosts. Larval parasitoids developing in the presence of many competitors weighed up to 50% less than those developing in hosts with fewer endoparasitoids, although the weight of adult female parasitoids did not vary significantly with wasp clutch size. The maximum number of emerging wasps was 200 parasitoids, possibly representing the host's 'carrying capacity' for larvae parasitized in the fourth-instar. The ratio of emerging to non-emerging parasitoids decreased as parasitoid clutch size increased, with few or none emerging from very heavily parasitized hosts containing more than 400 parasitoids. Copyright 1997 Elsevier Science Ltd. All right reserved