Cotesia congregata Bracovirus Circles Encoding PTP and Ankyrin Genes Integrate into the DNA of Parasitized Manduca sexta Hemocytes.

Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as the template for the production of double-stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production, thereby impeding viral replication in caterpillar hosts during parasitism. Here, we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata Sequences sharing similarities with host integration motifs (HIMs) of Microplitis demolitor bracovirus (MdBV) circles involved in integration into DNA could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore, by using a primer extension capture method based on these HIMs and high-throughput sequencing, we could show that 8 out of 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM, indicating that an HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.IMPORTANCE The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in braconid parasitoid wasp parasitism success. Indeed, results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins within newly dividing cells of caterpillar hosts that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

Transcriptomic response of Manduca sexta immune tissues to parasitization by the bracovirus associated wasp Cotesia congregata.

During oviposition, Cotesia congregata parasitoid wasps inject into their host, Manduca sexta, some biological factors such as venom, ovarian fluid and a symbiotic polydnavirus (PDV) named Cotesia congregata bracovirus (CcBV). During parasitism, complex interactions occur between wasp-derived factors and host targets that lead to important modifications in host physiology. In particular, the immune response leading to wasp egg encapsulation is inhibited allowing wasp survival. To date, the regulation of host genes during the interaction had only been studied for a limited number of genes. In this study, we analysed the global impact of parasitism on host gene regulation 24 h post oviposition by high throughput 454 transcriptomic analyses of two tissues known to be involved in the host immune response (hemocytes and fat body). To identify specific effects of parasitism on host transcription at this time point, transcriptomes were obtained from non-treated and parasitized larvae, and also from larvae injected with heat-killed bacteria and double stimulated larvae that were parasitized prior to bacterial challenge. Results showed that, immune challenge by bacteria leads to induction of certain antimicrobial peptide (AMP) genes in M. sexta larvae whether they were parasitized or not prior to bacterial challenge. These results show that at 24 h post oviposition pathways leading to expression of AMP genes are not all inactivated suggesting wasps are in an antiseptic environment. In contrast, at this time point genes involved in phenoloxidase activation and cellular immune responses were globally down-regulated after parasitism in accordance with the observed inhibition of wasp egg encapsulation.

Functional annotation of Cotesia congregata bracovirus: identification of viral genes expressed in parasitized host immune tissues.

UNLABELLED: Bracoviruses (BVs) from the Polydnaviridae family are symbiotic viruses used as biological weapons by parasitoid wasps to manipulate lepidopteran host physiology and induce parasitism success. BV particles are produced by wasp ovaries and injected along with the eggs into the caterpillar host body, where viral gene expression is necessary for wasp development. Recent sequencing of the proviral genome of Cotesia congregata BV (CcBV) identified 222 predicted virulence genes present on 35 proviral segments integrated into the wasp genome. To date, the expressions of only a few selected candidate virulence genes have been studied in the caterpillar host, and we lacked a global vision of viral gene expression. In this study, a large-scale transcriptomic analysis by 454 sequencing of two immune tissues (fat body and hemocytes) of parasitized Manduca sexta caterpillar hosts allowed the detection of expression of 88 CcBV genes expressed 24 h after the onset of parasitism. We linked the expression profiles of these genes to several factors, showing that different regulatory mechanisms control viral gene expression in the host. These factors include the presence of signal peptides in encoded proteins, diversification of promoter regions, and, more surprisingly, gene position on the proviral genome. Indeed, most genes for which expression could be detected are localized in particular proviral regions globally producing higher numbers of circles. Moreover, this polydnavirus (PDV) transcriptomic analysis also reveals that a majority of CcBV genes possess at least one intron and an arthropod transcription start site, consistent with an insect origin of these virulence genes. IMPORTANCE: Bracoviruses (BVs) are symbiotic polydnaviruses used by parasitoid wasps to manipulate lepidopteran host physiology, ensuring wasp offspring survival. To date, the expressions of only a few selected candidate BV virulence genes have been studied in caterpillar hosts. We performed a large-scale analysis of BV gene expression in two immune tissues of Manduca sexta caterpillars parasitized by Cotesia congregata wasps. Genes for which expression could be detected corresponded to genes localized in particular regions of the viral genome globally producing higher numbers of circles. Our study thus brings an original global vision of viral gene expression and paves the way to the determination of the regulatory mechanisms enabling the expression of BV genes in targeted organisms, such as major insect pests. In addition, we identify sequence features suggesting that most BV virulence genes were acquired from insect genomes.

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.

Differential inhibition of BmRelish1-dependent transcription in lepidopteran cells by bracovirus ankyrin-repeat proteins.

In the tripartite parasitization system of the lepidopteran host Manduca sexta, the endoparasitoid wasp Cotesia congregata and its endosymbiotic virus, C. congregata Bracovirus (CcBV), the expression of viral proteins is necessary for successful parasitization. Here we have examined the in vitro effects of six members of the ankyrin-repeat protein family (Ank) of CcBV, which are thought to interfere with the host's induced innate immune responses, on the transcriptional activity of a heterologous lepidopteran Rel/NFκB transcription factor, Relish1 of Bombyx mori. Using as transcriptional activator BmRelish1-d2 (R1d2), a constitutively active mutant of the major regulator of the Imd pathway, BmRelish1, in conjunction with a reporter gene controlled by a B. mori antimicrobial peptide gene promoter, we have found that 5 of the 6 examined Anks suppress R1d2-dependent transcriptional activity to various degrees. Immunofluorescence studies have also revealed that while some of the Ank proteins have a rather strict cytoplasmic localization, others are detected both in the cytoplasm and the nucleus of the expressing cells and that colocalization with R1d2 occurs exclusively in the nucleus. Thus, our results suggest that functional and spatial differences among the various CcBV Ank family members may be responsible for the observed differential inhibition of R1d2 activity.

Egf1.5 is a second phenoloxidase cascade inhibitor encoded by Microplitis demolitor bracovirus.

The three-member Egf gene family from the polydnavirus Microplitis demolitor bracovirus (MdBV) encodes novel proteins distinguished by a shared cysteine-rich motif. Prior studies determined that one family member, Egf1.0, inhibits melanization of hemolymph from the moth Manduca sexta by disabling phenoloxidase activating proteinases (PAPs). Here we characterized a second family member, Egf1.5, which shares an identical cysteine-rich motif with Egf1.0, but possesses an extended C-terminal repeat domain. Similar to Egf1.0, Egf1.5 inhibited processing and the amidolytic activity of PAP1 and PAP3 from M. sexta. Egf1.5 also bound PAP1, PAP3 and serine proteinase homolog 2 (SPH2). Comparative studies indicated that Egf1.5 and Egf1.0 similarly inhibited melanization of plasma from two lepidopterans (Pseudoplusia includens and Helicoverpa zea) that are permissive hosts for M. demolitor and MdBV, and two lepidopterans (M. sexta and Bombyx mori) that are nonpermissive hosts. Expression studies showed that transcript abundance of egf1.5 and egf1.0 was also similar in MdBV-infected P. includens and H. zea. Taken together, our results indicate that Egf1.5 and Egf1.0 are functionally similar paralogs.

Genome sequence of a polydnavirus: insights into symbiotic virus evolution.

Little is known of the fate of viruses involved in long-term obligatory associations with eukaryotes. For example, many species of parasitoid wasps have symbiotic viruses to manipulate host defenses and to allow development of parasitoid larvae. The complete nucleotide sequence of the DNA enclosed in the virus particles injected by a parasitoid wasp revealed a complex organization, resembling a eukaryote genomic region more than a viral genome. Although endocellular symbiont genomes have undergone a dramatic loss of genes, the evolution of symbiotic viruses appears to be characterized by extensive duplication of virulence genes coding for truncated versions of cellular proteins.

Persistence and expression of Cotesia congregata polydnavirus in host larvae of the tobacco hornworm, Manduca sexta.

The gregarious braconid wasp Cotesia congregata parasitizes host larvae of Manduca sexta, and several other sphingid species. Parasitism induces host immunosuppression due to the disruptive action of the wasp's polydnavirus (PDV) on host blood cells. During the initial stages of parasitism, these cells undergo apoptosis followed by cell clumping, which clears the hemolymph of a large number of cells. In this study, the persistence and expression of Cotesia congregata PDV (CcPDV) were examined using Southern and Northern blots, respectively. Digoxygenin-labelled total polydnaviral DNA was used to probe genomic DNA isolated from fat body and brains of hosts with emerged wasps taken 6 days following egress of the parasitoids, and significant cross-hybridization between the host fat body genomic DNA with viral DNA was seen. Thus, the virus persists in the host for the duration of parasitism, even during the post-emergence period, and may even be integrated in the host caterpillar DNA. Viral gene expression was examined using Northern blots and probes to the Cotesia rubecula CrV1 homolog, and the CrV1-like mRNAs were expressed as early as 4 h post-parasitization for at least 72 h and faint hybrization is even seen at the time the wasps eclose. In contrast, in Pieris rapae larvae the CrV1 transcript is expressed only for a brief time, during which time hemocyte function is disrupted. The effect is transitory, and hemocytes regain their normal functions after the parasites emerge as first instars.The genome of CcPDV contains one copy of the CrV1-like homolog as shown on Southern blots of viral genomic DNA. In conjunction with our earlier studies of the PDV-encoded early protein 1, the current work suggests multiple viral transcripts are produced following parasitization of the host, and likely target host hemocytes to induce their apoptosis, thereby preventing encapsulation of the parasitoid's eggs. Whether viral DNAs are integrated in the host's genomic DNA remains to be proven, but our results provide preliminary evidence that viral DNAs are detected in the host's fat body cells examined at the time of wasp emergence and several days later.

Infection of lepidoptera with a pseudotyped retroviral vector.

Studies requiring the introduction and expression of manipulated gene constructs have been technically difficult in non-drosophilid insects. Retroviruses can be engineered to be replication defective and to serve as vectors for gene constructs of interest. In this study, pseudotyped MoMLV(VSV-G) retroviral vectors are shown to successfully infect lepidopteran cells in vitro and in vivo. In Spodoptera frugiperda cells in vitro and in Manduca sexta in vivo, infection and conversion to proviral DNA were confirmed by PCR amplification and Southern blot hybridization of vector-specific sequences. Gene expression and integration of proviral DNA were also documented in vitro. This is the first report of retroviral infection in lepidoptera and suggests that pseudotyped retroviral vectors could be powerful tools in gene manipulation studies of non-drosophilid insects.

The plasma protein scolexin from Manduca sexta is induced by baculovirus infection and other immune challenges.

Manduca sexta larvae infected per os with a sublethal dose of Erinnyis ello granulosis virus (EeGV) contain an induced plasma protein of ca 33-36 kDa [Finnerty et al. (1994) J. Invertebr. Pathol. 63, 140-144]. This virus-induced protein shares characteristics with scolexin, a bacteria-induced plasma protein from M. sexta, so in this study we compared the two proteins. Two-dimensional gel electrophoresis reveals the induction of polypeptides corresponding to the subunits of scolexin in the plasma from EeGV-infected, as well as bacteria-injected, larvae. Immunoblots of these two-dimensional polyacrylamide gels show that antiserum against the virus-induced protein cross-reacts with the putative scolexin polypeptides induced by either EeGV infection or bacteria injection. Amino acid analysis of the virus-induced protein shows it to be very similar to scolexin, and the N-terminal sequences of the two proteins are nearly identical. From these data, we conclude that the virus-induced protein and scolexin are identical, or at least isoforms of the same protein. Using immunoblot, we also demonstrate that scolexin is induced in M. sexta plasma by injection with yeast or lipopolysaccharide from Serratia marcescens.