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

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

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.

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.

Modulation of antiviral immunity by the ichnovirus HdIV in Spodoptera frugiperda.

Polydnaviruses (PDVs) are obligatory symbionts found in thousands of endoparasitoid species and essential for successful parasitism. The two genera of PDVs, ichnovirus (IV) and bracovirus (BV), use different sets of virulence factors to ensure successful parasitization of the host. Previous studies have shown that PDVs target apoptosis, one of the innate antiviral responses in many host organisms. However, IV and BV have been shown to have opposite effects on this process. BV induces apoptosis in host cells, whereas some IV proteins have been shown to have anti-apoptotic activity. The different biological contexts in which the assays were performed may account for this difference. In this study, we evaluated the interplay between apoptosis and the ichnovirus HdIV from the parasitoid Hyposoter didymator, in the HdIV-infected hemocytes and fat bodies of S. frugiperda larvae, and in the Sf9 insect cell line challenged with HdIV. We found that HdIV induced cell death in hemocytes and fat bodies, whereas anti-apoptotic activity was observed in HdIV-infected Sf9 cells, with and without stimulation with viral PAMPs or chemical inducers. We also used an RT-qPCR approach to determine the expression profiles of a set of genes known to encode key components of the other main antiviral immune pathways described in insects. The analysis of immune gene transcription highlighted differences in antiviral responses to HdIV as a function of host cell type. However, all these antiviral pathways appeared to be neutralized by low levels of expression for the genes encoding the key components of these pathways, in all biological contexts. Finally, we investigated the effect of HdIV on the general antiviral defenses of the lepidopteran larvae in more detail, by studying the survival of S. frugiperda co-infected with HdIV and the entomopathogenic densovirus JcDV. Coinfected S. frugiperda larvae have increased resistance to JcDV at an early phase of infection, whereas HdIV effects enhance the virulence of the virus at later stages of infection. Overall, these results reveal complex interactions between HdIV and its cellular environment.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Extensive transcription analysis of the Hyposoter didymator Ichnovirus genome in permissive and non-permissive lepidopteran host species.

Ichnoviruses are large dsDNA viruses that belong to the Polydnaviridae family. They are specifically associated with endoparasitic wasps of the family Ichneumonidae and essential for host parasitization by these wasps. We sequenced the Hyposoter didymator Ichnovirus (HdIV) encapsidated genome for further analysis of the transcription pattern of the entire set of HdIV genes following the parasitization of four different lepidopteran host species. The HdIV genome was found to consist of at least 50 circular dsDNA molecules, carrying 135 genes, 98 of which formed 18 gene families. The HdIV genome had general features typical of Ichnovirus (IV) genomes and closely resembled that of the IV carried by Hyposoter fugitivus. Subsequent transcriptomic analysis with Illumina technology during the course of Spodoptera frugiperda parasitization led to the identification of a small subset of less than 30 genes with high RPKM values in permissive hosts, consisting with these genes encoding crucial virulence proteins. Comparisons of HdIV expression profiles between host species revealed differences in transcript levels for given HdIV genes between two permissive hosts, S. frugiperda and Pseudoplusia includens. However, we found no evident intrafamily gene-specific transcription pattern consistent with the presence of multigenic families within IV genomes reflecting an ability of the wasps concerned to exploit different host species. Interestingly, in two non-permissive hosts, Mamestra brassiccae and Anticarsia gemmatalis (most of the parasitoid eggs were eliminated by the host cellular immune response), HdIV genes were generally less strongly transcribed than in permissive hosts. This suggests that successful parasitism is dependent on the expression of given HdIV genes exceeding a particular threshold value. These results raise questions about the mecanisms involved in regulating IV gene expression according to the nature of the lepidopteran host species encountered.

Specificities of ichnoviruses associated with campoplegine wasps: genome, genes and role in host-parasitoid interaction.

Ichnoviruses (IVs), unique symbiotic viruses carried by ichneumonid campoplegine wasps, derive from integration of a paleo-ichnovirus into an ancestral wasp genome. The modern 'genome' is composed of both regions that are amplified, circularized and encapsidated into viral particles and non-encapsidated viral genomic regions involved in particle morphogenesis. Packaged genomes include multiple circular dsDNAs encoding many genes mostly organized in gene families. Virus particles are assembled in specialized ovarian cells from which they exit into the oviduct lumen; mature virions are injected during oviposition into the insect host. Expression of viral proteins in infected cells correlates with physiological alterations of the host enabling success of parasitism.

Venom gland extract is not required for successful parasitism in the polydnavirus-associated endoparasitoid Hyposoter didymator (Hym. Ichneumonidae) despite the presence of numerous novel and conserved venom proteins.

The venom gland is a conserved organ in Hymenoptera that shows adaptations associated with life-style diversification. Few studies have investigated venom components and function in the highly diverse parasitic wasps and all suggest that the venom regulates host physiology. We explored the venom of the endoparasitoid Hyposoter didymator (Campopleginae), a species with an associated polydnavirus produced in the ovarian tissue. We investigated the effects of the H. didymator venom on two physiological traits of the host Spodoptera frugiperda (Noctuidae): encapsulation response and growth rate. We found that H. didymator venom had no significant effect on host cellular immunity or development, suggesting that it does not contribute to parasitism success. The host physiology seemed to be modified essentially by the ovarian fluid containing the symbiotic polydnaviruses. Proteomic analyses indicated that the H. didymator venom gland produces a large variety of proteins, consistent with the classical hymenopteran venom protein signature, including: reprolysin-like, dipeptidyl peptidase IV, hyaluronidase, arginine kinase or allergen proteins. The venom extracts also contained novel proteins, encoded by venom genes conserved in Campopleginae ichneumonids, and proteins with similarities to active molecules identified in other parasitoid species, such as calreticulin, reprolysin, superoxide dismutase and serpin. However, some of these proteins appear to be produced only in small amounts or to not be secreted. Possibly, in Campopleginae carrying polydnaviruses, the host-modifying activities of venom became redundant following the acquisition of polydnaviruses by the lineage.

Multigenic families in Ichnovirus: a tissue and host specificity study through expression analysis of vankyrins from Hyposoter didymator Ichnovirus.

The viral ankyrin (vankyrin) gene family is represented in all polydnavirus (PDVs) genomes and encodes proteins homologous to I-kappaBs, inhibitors of NF-kappaB transcription factors. The structural similarities led to the hypothesis that vankyrins mimic eukaryotic factors to subvert important physiological pathways in the infected host. Here, we identified nine vankyrin genes in the genome of the Hyposoter didymator Ichnovirus (HdIV). Time-course gene expression experiments indicate that all members are expressed throughout parasitism of Spodoptera frugiperda, as assessed using RNA extracted from whole larvae. To study tissue and/or species specificity transcriptions, the expression of HdIV vankyrin genes were compared between HdIV-injected larvae of S. frugiperda and S. littoralis. The transcriptional profiles were similar in the two species, including the largely predominant expression of Hd27-vank1 in all tissues examined. However, in various insect cell lines, the expression patterns of HdIV vankyrins differed according to species. No clear relationship between vankyrin expression patterns and abundance of vankyrin-bearing genomic segments were found in the lepidopteran cell lines. Moreover, in these cells, the amount of vankyrin-bearing genomic segments differed substantially between cytosol and nuclei of infected cells, implying the existence of an unexpected step regulating the copy number of HdIV segments in cell nuclei. Our in vitro results reveal a host-specific transcriptional profile of vankyrins that may be related to the success of parasitism in different hosts. In Spodoptera hosts, the predominant expression of Hd27-vank1 suggests that this protein might have pleiotropic functions during parasitism of these insect species.

Lepidopteran transcriptome analysis following infection by phylogenetically unrelated polydnaviruses highlights differential and common responses.

The Polydnaviridae is a family of double-stranded DNA viruses that are symbionts of parasitoid wasps. The family is currently divided into two genera, the Ichnovirus (IV) and Bracovirus (BV), which are associated with wasps in the families Ichneumonidae and Braconidae, respectively. IVs and BVs have similar immunosuppressive and developmental effects on parasitized hosts but their encapsidated genomes largely encode different genes. To assess whether IV and BV infection has similar or disparate effects on the transcriptome of shared hosts, we characterized the effects of Hyposoter didymator Ichnovirus (HdIV) and Microplitis demolitor Bracovirus (MdBV) on the fat body and hemocyte transcriptome of Spodoptera frugiperda (Lepidoptera: Noctuidae). Our results indicated that HdIV and MdBV infection alters the abundance of a relatively low proportion of S. frugiperda transcripts at 24 h post-infection. A majority of the transcripts affected by infection also differed between MdBV and HdIV. However, we did identify some host transcripts that were similarly affected by both viruses. A majority of these genes were transcribed in the fat body and most belonged to functional classes with roles in immunity, detoxification, or cell structure. Particularly prominent in this suite of transcripts were genes encoding for predicted motor-related and collagen IV-like proteins. Overall, our data suggest that the broadly similar effects that HdIV and MdBV have on host growth and immunity are not due to these viruses inducing profound changes in host gene expression. Given though that IVs and BVs encode few shared genes, the host transcripts that are similarly affected by HdIV and MdBV could indicate convergence by each virus to target a few processes at the level of transcription that are important for successful parasitism of hosts by H. didymator and M. demolitor.

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae.

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae).

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.

Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance.

The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an alpha-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1(GEO) cDNA differs from the sequence we previously reported for a susceptible strain (cpm1(IP)) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1(IP) was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1(GEO) resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1(GEO) and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.