Widespread Genome Reorganization of an Obligate Virus Mutualist

The family Polydnaviridae is of interest because it provides the best example of viruses that have evolved a mutualistic association with their animal hosts. Polydnaviruses in the genus Bracovirus are strictly associated with parasitoid wasps in the family Braconidae, and evolved ∼100 million years ago from a nudivirus. Each wasp species relies on its associated bracovirus to parasitize hosts, while each bracovirus relies on its wasp for vertical transmission. Prior studies establish that bracovirus genomes consist of proviral segments and nudivirus-like replication genes, but how these components are organized in the genomes of wasps is unknown. Here, we sequenced the genome of the wasp Microplitis demolitor to characterize the proviral genome of M. demolitor bracovirus (MdBV). Unlike nudiviruses, bracoviruses produce virions that package multiple circular, double-stranded DNAs. DNA segments packaged into MdBV virions resided in eight dispersed loci in the M. demolitor genome. Each proviral segment was bounded by homologous motifs that guide processing to form mature viral DNAs. Rapid evolution of proviral segments obscured homology between other bracovirus-carrying wasps and MdBV. However, some domains flanking MdBV proviral loci were shared with other species. All MdBV genes previously identified to encode proteins required for replication were identified. Some of these genes resided in a multigene cluster but others, including subunits of the RNA polymerase that transcribes structural genes and integrases that process proviral segments, were widely dispersed in the M. demolitor genome. Overall, our results indicate that genome dispersal is a key feature in the evolution of bracoviruses into mutualists.     

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

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

Systematic analysis of a wasp parasitism arsenal

Parasitoid wasps are among the most diverse insects on earth with many species causing major mortality in host populations. Parasitoids introduce a variety of factors into hosts to promote parasitism, including symbiotic viruses, venom, teratocytes and wasp larvae. Polydnavirus‐carrying wasps use viruses to globally suppress host immunity and prevent rejection of developing parasites. Although prior results provide detailed insights into the genes viruses deliver to hosts, little is known about other products. RNAseq and proteomics were used to characterize the proteins secreted by venom glands, teratocytes and larvae from Microplitis demolitor, which carries M. demolitor bracovirus (MdBV). These data revealed that venom glands and teratocytes secrete large amounts of a small number of products relative to ovaries and larvae. Venom and teratocyte products exhibited almost no overlap with one another or MdBV genes, which suggested that M. demolitor effector molecules are functionally partitioned according to their source. This finding was well illustrated in the case of MdBV and teratocytes. Many viral proteins have immunosuppressive functions that include disruption of antimicrobial peptide production, yet this study showed that teratocytes express high levels of the antimicrobial peptide hymenoptaecin, which likely compensates for MdBV‐mediated immunosuppression. A second key finding was the prevalence of duplications among genes encoding venom and teratocyte molecules. Several of these gene families share similarities with proteins from other species, while also showing specificity of expression in venom glands or teratocytes. Overall, these results provide the first comprehensive analysis of the proteins a polydnavirus‐carrying wasp introduces into its host.     

Microplitis demolitor bracovirus inhibits phagocytosis by hemocytes from Pseudoplusia includens

The braconid wasp Microplitis demolitor carries Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of several noctuid moths. A key function of MdBV in parasitism is suppression of the host's cellular immune response. Prior studies in the host Pseudoplusia includens indicated that MdBV blocks encapsulation by preventing two types of hemocytes, plasmatocytes and granulocytes, from adhering to foreign targets. The other main immune response mediated by insect hemocytes is phagocytosis. The goal of this study was to determine which hemocyte types were phagocytic in P. includens and to assess whether MdBV infection affects this defense response. Using the bacterium Escherichia coli and inert polystyrene beads as targets, our results indicated that the professional phagocyte in P. includens is granulocytes. The phagocytic responses of granulocytes were very similar to those of High Five cells that prior studies have suggested are a granulocyte-like cell line. MdBV infection dose-dependently disrupted phagocytosis in both cell types by inhibiting adhesion of targets to the cell surface. The MdBV glc1.8 gene encodes a cell surface glycoprotein that had previously been implicated in disruption of adhesion and encapsulation responses by immune cells. Knockdown of glc1.8 expression by RNA interference (RNAi) during the current study rescued the ability of MdBV-infected High Five cells to phagocytize targets. Collectively, these results indicate that glc1.8 is a key virulence determinant in disruption of both adhesion and phagocytosis by insect immune cells.     

When parasitic wasps hijacked viruses: genomic and functional evolution of polydnaviruses

The Polydnaviridae (PDV), including the Bracovirus (BV) and Ichnovirus genera, originated from the integration of unrelated viruses in the genomes of two parasitoid wasp lineages, in a remarkable example of convergent evolution. Functionally active PDVs represent the most compelling evolutionary success among endogenous viral elements (EVEs). BV evolved from the domestication by braconid wasps of a nudivirus 100 Ma. The nudivirus genome has become an EVE involved in BV particle production but is not encapsidated. Instead, BV genomes have co-opted virulence genes, used by the wasps to control the immunity and development of their hosts. Gene transfers and duplications have shaped BV genomes, now encoding hundreds of genes. Phylogenomic studies suggest that BVs contribute largely to wasp diversification and adaptation to their hosts. A genome evolution model explains how multidirectional wasp adaptation to different host species could have fostered PDV genome extension. Integrative studies linking ecological data on the wasp to genomic analyses should provide new insights into the adaptive role of particular BV genes. Forthcoming genomic advances should also indicate if the associations between endoparasitoid wasps and symbiotic viruses evolved because of their particularly intimate interactions with their hosts, or if similar domesticated EVEs could be uncovered in other parasites.     

Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera: Braconidae) suggest a high level of specificity in their associated bracoviruses

The immunological and developmental effects of bracoviruses (BVs) from three parasitoids in the genus Microplitis (Braconidae: Microgastrinae) were compared in the hosts Pseudoplusia includens and Heliothis virescens (Lepidoptera: Noctuidae). Southern blotting experiments indicated that viral DNAs from Microplitis demolitor bracovirus (MdBV) cross-hybridized with viral DNAs from Microplitis croceipes bracovirus (McBV) and Microplitis mediator bracovirus (MmBV) under conditions of high stringency. Injection of calyx fluid plus venom from each parasitoid species dose-dependently delayed development of P. includens and H. virescens. Each virus also inhibited pupation of P. includens but not H. virescens. In situ hybridization experiments indicated that MdBV and McBV persistently infect hemocytes in both hosts while MmBV persistently infects hemocytes in P. includens but not H. virescens. While MdBV infection induced a loss of adhesion by most plasmatocytes, McBV and MmBV infection induced a loss of adhesion in less than 50% of cells. Cross-protection experiments indicated that calyx fluid plus venom from one species usually protected progeny of another species from encapsulation but did not always promote successful development.     

Glc1.8 from Microplitis demolitor bracovirus induces a loss of adhesion and phagocytosis in insect high five and S2 cells

Polydnaviridae is a unique family of DNA viruses that are symbiotically associated with parasitoid wasps. Upon oviposition, wasps inject these viruses into their hosts, where they cause several physiological alterations, including suppression of the cellular immune response. Here we report that expression of the glc1.8 gene from Microplitis demolitor bracovirus (MdBV) causes a loss of adhesion by two hemocyte-like cell lines, namely, High Five cells from the lepidopteran Trichoplusia ni and S2 cells from the dipteran Drosophila melanogaster. The expression of recombinant Glc1.8 also greatly reduced the ability of these cells to phagocytize foreign targets. Glc1.8 is characterized by a signal peptide at its N terminus, an extracellular domain comprised of five nearly perfect tandem repeats of 78 amino acids, and a C-terminal hydrophobic domain that encodes a putative membrane anchor sequence. The expression of a Glc1.8 mutant lacking the anchor sequence resulted in a secreted protein that had no effect on adhesion or phagocytosis. In contrast, sequential deletion of the repeats in the extracellular domain resulted in a progressive reduction in immunosuppressive activity. Since each repeat and its associated glycosylation sites are nearly identical, these results suggested that adhesion-blocking activity depends more on the overall number of repeats in the extracellular domain than on the specific determinants within each repeat. While it severely compromised adhesion and phagocytic functions, Glc1.8 did not cause cell death. Collectively, these results indicate that Glc1.8 is a major pathogenic determinant of MdBV that is involved in suppression of the insect cellular immune response.     

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.     

Rho 1 participates in parasitoid wasp eggs maturation and host cellular immunity inhibition

Endoparasitoid wasps introduce venom into their host insects during the egg-laying stage. Venom proteins play various roles in the host physiology, development, immunity, and behavior manipulation and regulation. In this study, we identified a venom protein, MmRho1, a small guanine nucleotide-binding protein derived from ovary in the endoparasitoid wasp Microplitis mediator and found that knockdown of its expression by RNA interference caused down-regulation of vitellogenin and juvenile hormone, egg production, and cocoons formation in the female wasps. We demonstrated that MmRho1 entered the cotton bollworm's (host) hemocytes and suppressed cellular immune responses after parasitism using immunofluorescence staining. Furthermore, wasp MmRho1 interacted with the cotton bollworm's actin cytoskeleton rearrangement regulator diaphanous by yeast 2-hybrid and glutathione s-transferase pull-down. In conclusion, this study indicates that MmRho1 plays dual roles in wasp development and the suppression of the host insect cellular immune responses.     

Differences in larval feeding behavior correlate with altered developmental strategies in two parasitic wasps: implications for the size-fitness hypothesis

Parasitoid wasps have long been favored organisms for fundamental studies on reproductive strategies and life-history evolution. Progeny allocation models designed with parasitoids in mind assume that offspring develop by consuming most or all of the resources available from a single host, and that size is the most important factor affecting offspring fitness. Many parasitoids exhibit host usage patterns consistent with these assumptions, but our recent observations suggested that endoparasitic wasps in the family Braconidae often do not. To investigate how differences in host usage patterns might affect developmental strategies, we compared two related braconids with contrasting host usage patterns. Apanteles carpatus consumed virtually all host tissues during immature development, whereas Microplitis demolitor fed exclusively on host hemolymph and consumed a relatively small proportion of available host resources. Development time of M. demolitor was unaffected by host size, whereas development time of A. carpatus was much longer in small hosts than in large hosts. On the other hand, offspring size in M. demolitor correlated strongly with host size, but it correlated only weakly with host size in A. carpatus . Our results collectively suggest that selection has favored rapid development at the potential cost of reduced size in M. demolitor , and increased size at the potential cost of increased development time in A. carpatus . Tissue feeding appears to be more prevalent among parasitoids overall, but hemolymph feeding is the predominant pattern of host usage in several subfamilies of endoparasitic braconids. We argue that the relative importance of offspring size and development time will be influenced by host ecology and the effects of selected traits on parasitoid survival.     

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.     

Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae)

Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded “antennal IRs” and seven encoded “divergent IRs”. Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception.     

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.     

Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps

During oviposition, many endoparasitic wasps inject virus-like particles into their insect hosts that enable these parasitoids to evade or directly suppress their hosts' immune system, especially encapsulation by hemocytes. These particles are defined as virions that belong to viruses of the two genera that comprise the family Polydnaviridae, bracoviruses (genus Bracovirus) transmitted by braconid wasps, and ichnoviruses (genus Ichnovirus) transmitted by ichneumonid wasps. Structurally, bracovirus virions resemble nudivirus and baculovirus virions (family Baculoviridae), and ichnovirus virions resemble those of ascoviruses (family Ascoviridae). Whereas nudiviruses, baculoviruses and ascoviruses replicate their DNA and produce progeny virions, polydnavirus DNA is integrated into and replicated from the wasp genome, which also directs virion synthesis. The structural similarity of polydnavirus virions to those of viruses that attack the wasps' lepidopteran hosts, along with polydnavirus transmission and replication biology, suggest that these viruses evolved from insect DNA viruses by symbiogenesis, the same process by which mitochondia and chloroplasts evolved from bacteria. Molecular evidence supporting this hypothesis comes from similarities among structural proteins of ascoviruses and the Campoletis sonorensis ichnovirus. Implications of this hypothesis are that polydnaviruses evolved from viruses, but are no longer viruses, and that DNA packaged into polydnavirus virions is not viral genomic DNA per se, but rather wasp genomic DNA consisting primarily of wasp genes and non-coding DNA. Thus, we suggest that a better understanding of polydnaviruses would result by viewing these not as viruses, but rather as a wasp organelle system that evolved to shuttle wasp genes and proteins into hosts to evade and suppress their immune response.     

Developmental disruption of Pseudoplusia includens and Heliothis virescens larvae by the calyx fluid and venom of Microplitis demolitor

Calyx fluid and venom from the braconid parasitoid Microplitis demolitor differentially affected the development of Pseudoplusia includens and Heliothis virescens. P. includens exhibited delays in larval development, supernumerary instars, and formed larval-pupal intermediates when injected with 0.01-0.10 wasp equivalents of calyx fluid. In contrast, H. virescens was relatively unaffected by calyx fluid regardless of dose. Venom did not affect the development of either host species, but appeared to synergize the activity of calyx fluid. This was particularly evident in H. virescens, where injection of 0.10-0.20 wasp equivalents of calyx fluid and venom induced the formation of a large number of intermediates while the same amount of calyx fluid did not. The particulate portion of M. demolitor calyx fluid was the only component that caused developmental delays and the formation of intermediates in both host species. Purified virus caused developmental alterations in P. includens, while trioxsalen treated calyx fluid did not affect development of P. includens or H. virescens. These data suggest the requirement for venom in parasitism may differ between host species, and that dosage plays an important role in interpreting the interaction between calyx and venom components.