A new gene family (BAPs) of Cotesia bracovirus induces apoptosis of host hemocytes.

Polydnaviruses (PDVs), obligatory symbionts with parasitoid wasps, function as host immune suppressors and growth and development regulator. PDVs can induce host haemocyte apoptosis, but the underlying mechanism remains largely unknown. Here, we provided evidence that, during the early stages of parasitism, the activated Cotesia vestalis bracovirus (CvBV) reduced the overall number of host haemocytes by inducing apoptosis. We found that one haemocyte-highly expressed CvBV gene, CvBV-26-4, could induce haemocyte apoptosis. Further analyses showed that CvBV-26-4 has four homologs from other Cotesia bracoviruses and BV from wasps in the genus Glyptapanteles, and all four of them possessed a similar structure containing 3 copies of a well-conserved motif (Gly-Tyr-Pro-Tyr, GYPY). Mass spectrometry analysis revealed that CvBV-26-4 was secreted into plasma by haemocytes and then degraded into peptides that induced the apoptosis of haemocytes. Moreover, ectopic expression of CvBV-26-4 caused fly haemocyte apoptosis and increased the susceptibility of flies to bacteria. Based on this research, a new family of bracovirus genes, Bracovirus apoptosis-inducing proteins (BAPs), was proposed. Furthermore, it was discovered that the development of wasp larvae was affected when the function of CvBV BAP was obstructed in the parasitized hosts. The results of our study indicate that the BAP gene family from the bracoviruses group is crucial for immunosuppression during the early stages of parasitism.

A high-quality genome of the dobsonfly Neoneuromus ignobilis reveals molecular convergences in aquatic insects.

Neoneuromus ignobilis is an archaic holometabolous aquatic predatory insect. However, a lack of genomic resources hinders the use of whole genome sequencing to explore their genetic basis and molecular mechanisms for adaptive evolution. Here, we provided a high-contiguity, chromosome-level genome assembly of N. ignobilis using high coverage Nanopore and PacBio reads with the Hi-C technique. The final assembly is 480.67 MB in size, containing 12 telomere-ended pseudochromosomes with only 17 gaps. We compared 42 hexapod species genomes including six independent lineages comprising 11 aquatic insects, and found convergent expansions of long wavelength-sensitive and blue-sensitive opsins, thermal stress response TRP channels, and sulfotransferases in aquatic insects, which may be related to their aquatic adaptation. We also detected strong nonrandom signals of convergent amino acid substitutions in aquatic insects. Collectively, our comparative genomic analysis revealed the evidence of molecular convergences in aquatic insects during both gene family evolution and convergent amino acid substitutions.

Virus and endogenous viral element-derived small non-coding RNAs and their roles in insect-virus interaction.

RNA interference pathways mediated by different types of small non-coding RNAs (siRNAs, miRNAs and piRNAs) are conserved biological responses to exotic stresses, including viral infection. Aside from the well-established siRNA pathway, the miRNA pathway and the piRNA pathway process viral sequences, exogenously or endogenously, into miRNAs and piRNAs, respectively. During the host-virus interaction, viral sequences, including both coding and non-coding sequences, can be integrated as endogenous viral elements (EVEs) and thereby become present within the germline of a non-viral organism. In recent years, significant progress has been made in characterizing the biogenesis and function of viruses and EVEs associated with snRNAs. Overall, the siRNA pathway acts as the primarily antiviral defense against a wide range of exogenous viruses; the miRNA pathways associated with viruses or EVEs function in antiviral response and host gene regulation; EVE derived piRNAs with a ping-pong signature have the potential to limit cognate viral infection.

The mitochondrial genome of Chelonus formosanus (Hymenoptera: Braconidae) with novel gene orders and phylogenetic implications.

Chelonus formosanus Sonan is an important egg-larval parasitoid of noctuid moths and a potential candidate for understanding interactions between host and parasitoid mediated by polydnavirues (PDVs). We sequenced and annotated the mitochondrial genome of C. formosanus, which is 15,466 bp in length and possesses 38 mitochondrial genes. However, unlike most animal mitochondrial genomes, it contains one extra trnF gene. There are five transfer RNA (tRNA) rearrangement events compared with the ancestral gene order, which is a novel rearrangement type in Hymenoptera for all published mitogenomes so far. Phylogenetic trees supported C. formosanus from the subfamily Cheloninae was closely related to the subfamily Cardiochilinae and Microgastrinae.

A Polydnavirus Protein Tyrosine Phosphatase Negatively Regulates the Host Phenoloxidase Pathway.

Polydnavirus (PDV) is a parasitic factor of endoparasitic wasps and contributes greatly to overcoming the immune response of parasitized hosts. Protein tyrosine phosphatases (PTPs) regulate a wide variety of biological processes at the post-transcriptional level in mammals, but knowledge of PDV PTP action during a parasitoid−host interaction is limited. In this study, we characterized a PTP gene, CvBV_12-6, derived from Cotesia vestalis bracovirus (CvBV), and explored its possible regulatory role in the immune response of the host Plutella xylostella. Our results from qPCR show that CvBV_12-6 was highly expressed in hemocytes at an early stage of parasitization. To explore CvBV_12-6 function, we specifically expressed CvBV_12-6 in Drosophila melanogaster hemocytes. The results show that Hml-Gal4 > CvBV_12-6 suppressed the phenoloxidase activity of hemolymph in D. melanogaster, but exerted no effect on the total count or the viability of the hemocytes. In addition, the Hml-Gal4 > CvBV_12-6 flies exhibited decreased antibacterial abilities against Staphylococcus aureus. Similarly, we found that CvBV_12-6 significantly suppressed the melanization of the host P. xylostella 24 h post parasitization and reduced the viability, but not the number, of hemocytes. In conclusion, CvBV_12-6 negatively regulated both cellular and humoral immunity in P. xylostella, and the related molecular mechanism may be universal to insects.

CLP gene family, a new gene family of Cotesia vestalis bracovirus inhibits melanization of Plutella xylostella hemolymph.

Polydnaviruses (PDVs) are obligatory symbionts of parasitoid wasps and play an important role in suppressing host immune defenses. Although PDV genes that inhibit host melanization are known in Microplitis bracovirus, the functional homologs in Cotesia bracoviruses remain unknown. Here, we find that Cotesia vestalis bracovirus (CvBV) can inhibit hemolymph melanization of its host, Plutella xylostella larvae, during the early stages of parasitization, and that overexpression of highly expressed CvBV genes reduced host phenoloxidase activity. Furthermore, CvBV-7-1 in particular reduced host phenoloxidase activity within 12 h, and the injection of anti-CvBV-7-1 antibody increased the melanization of parasitized host larvae. Further analyses showed that CvBV-7-1 and three homologs from other Cotesia bracoviruses possessed a C-terminal leucine/isoleucine-rich region and had a similar function in inhibiting melanization. Therefore, a new family of bracovirus genes was proposed and named as C-terminal Leucine/isoleucine-rich Protein (CLP). Ectopic expression of CvBV-7-1 in Drosophila hemocytes increased susceptibility to bacterial repression of melanization and reduced the melanotic encapsulation of parasitized D. melanogaster by the parasitoid Leptopilina boulardi. The formation rate of wasp pupae and the eclosion rate of C. vestalis were affected when the function of CvBV-7-1 was blocked. Our findings suggest that CLP genes from Cotesia bracoviruses encoded proteins that contain a C-terminal leucine/isoleucine-rich region and function as melanization inhibitors during the early stage of parasitization, which is important for successful parasitization.

Genome-Wide Profiling of Diadegma semiclausum Ichnovirus Integration in Parasitized Plutella xylostella Hemocytes Identifies Host Integration Motifs and Insertion Sites.

Polydnaviruses (PDVs), classified into two genera, bracoviruses (BVs) and ichnoviruses (IVs), are large, double-stranded DNA viruses, which are beneficial symbionts of parasitoid wasps. PDVs do not replicate in their infected lepidopteran hosts. BV circles have been demonstrated to be integrated into host genomic DNA after natural parasitization. However, the integrations of IV circles in vivo remain largely unknown. Here, we analyzed the integration of Diadegma semiclausum ichnovirus (DsIV) in the genomic DNA of parasitized Plutella xylostella hemocytes. We found that DsIV circles are present in host hemocytes with non-integrated and integrated forms. Moreover, DsIV integrates its DNA circles into the host genome by two distinct strategies, conservatively, and randomly. We also found that four conserved-broken circles share similar motifs containing two reverse complementary repeats at their breaking sites, which were host integration motifs (HIMs). We also predicted HIMs of eight circles from other ichnoviruses, indicating that a HIM-mediated specific mechanism was conserved in IV integrations. Investigation of DsIV circle insertion sites of the host genome revealed the enrichment of microhomologies between the host genome and the DsIV circles at integration breakpoints. These findings will deepen our understanding of the infections of PDVs, especially IVs.

Parasitic insect-derived miRNAs modulate host development.

Parasitic wasps produce several factors including venom, polydnaviruses (PDVs) and specialized wasp cells named teratocytes that benefit the survival of offspring by altering the physiology of hosts. However, the underlying molecular mechanisms for the alterations remain unclear. Here we find that the teratocytes of Cotesia vestalis, an endoparasitoid of the diamondback moth Plutella xylostella, and its associated bracovirus (CvBV) can produce miRNAs and deliver the products into the host via different ways. Certain miRNAs in the parasitized host are mainly produced by teratocytes, while the expression level of miRNAs encoded by CvBV can be 100-fold greater in parasitized hosts than non-parasitized ones. We further show that one teratocyte-produced miRNA (Cve-miR-281-3p) and one CvBV-produced miRNA (Cve-miR-novel22-5p-1) arrest host growth by modulating expression of the host ecdysone receptor (EcR). Altogether, our results show the first evidence of cross-species regulation by miRNAs in animal parasitism and their possible function in the alteration of host physiology during parasitism.

Laccase 1 gene from Plutella xylostella (PxLac1) and its functions in humoral immune response.

Laccase (EC 1.10.3.2) is a phenoloxidase found in many insect species. The Laccase 1 gene from Plutella xylostella (PxLac1) was cloned, and its expression patterns and functions were determined using qPCR and RNAi methods. The results showed that the expression levels of PxLac1 were consistently high in all larval stages, and the most abundant was in the midgut during the 4th instar stage. Moreover, the expression of PxLac1 was up-regulated in response to bacterial infection, and decreased 24 h after being parasitized by Cotesia vestalis. Further analyses indicated that the effect of parasitization on PxLac1 was induced by active C. vestalis Bracovirus (CvBV). Haemocyte-free hemolymph phenoloxidase (PO) activity was suppressed when PxLac1 was treated with RNAi. Our results provide evidence for a connection between the Laccase 1 gene and insect immunity, and revealed that parasitoid polydnavirus suppresses host PO activity via PxLac1 regulation.

Parasitoid polydnaviruses and immune interaction with secondary hosts.

Polydnaviruses (PDVs) are obligatory symbionts with parasitoid wasps. The PDV virions are produced solely in wasp (the primary host) calyx cells. They are injected into caterpillar hosts (the secondary host) during parasitoid oviposition, where they express irreplaceable actions to ensure survival and development of wasp larvae. Some of PDV gene products suppress host immune responses while others alter host growth, metabolism or endocrine system. Here, we treat new findings on PDV gene products and their action on immunity within secondary hosts.

Comparative transcriptome analysis of venom glands from Cotesia vestalis and Diadromus collaris, two endoparasitoids of the host Plutella xylostella.

Venoms secreted by the venom gland (VG) of parasitoid wasp help ensure successful parasitism by host immune suppression and developmental regulation. Cotesia vestalis, a larval endoparasitoid, and Diadromus collaris, a pupal endoparasitoid, parasitize the diamondback moth (DBM), Plutella xylostella. To explore and compare the venom components of two endoparasitoids, we sequenced transcriptomes of the VGs and wasp bodies without VGs (BWVGs) of the two endoparasitoids. Statistically enriched GO terms and KEGG pathways of the two VGs compared to respective whole-body background were similar and reflected active protein biosynthesis activities in the two VGs. 1,595 VG specific genes of the D. collaris VG and 1,461 VG specific genes of the C. vestalis VG were identified by comparative transcript profiling. A total of 444 and 513 genes encoding potential secretory proteins were identified and defined as putative venom genes in D. collaris VG and C. vestalis VG, respectively. The putative venom genes of the two wasps showed no significant similarity or convergence. More venom genes were predicted in D. collaris VG than C. vestalis VG, especially hydrolase-coding genes. Differences in the types and quantities of putative venom genes shed light on different venom functions.

Deep sequencing of Cotesia vestalis bracovirus reveals the complexity of a polydnavirus genome.

Here we completed the whole genome sequence of Cotesia vestalis bracovirus (CvBV) by deep sequencing and compared the genome features of CvBV to those of other polydnaviruses (PDVs). The genome is 540,215 base pairs divided into 35 genomic segments that range from 2.6 to 39.2kb. Comparison of CvBV with other PDVs shows that more segments are found, including new segments that have no corresponding segments in other phylogenetically related PDVs, which suggests that there might be still more segments not being sequenced in the present known PDVs. We identified eight gene families and five genes in CvBV, including new genes which were first found in PDVs. Strikingly, we identified a putative helicase protein displaying similarity to human Pif1 helicase, which has never been reported for other PDVs. This finding will bring new insights in research of these special viruses.