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ABSTRACT Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (order Hymenoptera: Family Braconidae). BVs produce replication-defective virions that adult female wasps use to transfer DNAs encoding virulence genes to parasitized hosts. Some BV genes are shared with nudiviruses and baculoviruses with studies of the latter providing insights on function, whereas other genes are only known from nudiviruses or other BVs which provide no functional insights. A proteomic analysis ofMicroplitis demolitorbracovirus (MdBV) virions recently identified 16 genes encoding nucleocapsid components. In this study, we further characterized most of these genes. Some nucleocapsid genes exhibited early or intermediate expression profiles, while others exhibited late expression profiles. RNA interference (RNAi) assays together with transmission electron microscopy indicatedvp39,HzNVorf9-like2,HzNVorf93-like,HzNVorf106-like,HzNVorf118-like,and 27bare required to produce capsids with a normal barrel-shaped morphology. RNAi knockdown ofvlf-1a,vlf-1b-1,vlf-1b-2,int-1,andp6.9-1did not alter the formation of barrel-shaped capsids but each reduced processing of amplified proviral segments and DNA packaging as evidenced by the formation of electron translucent capsids. All of the genes required for normal capsid assembly were also required for proviral segment processing and DNA packaging. Collectively, our results deorphanize several BV genes with previously unknown roles in virion morphogenesis. IMPORTANCEUnderstanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of theMicroplitis demolitorbracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding proteinp6.9-1are required for DNA processing and packaging into nucleocapsids. 

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor domesticated endogenous viruses (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly forHyposoter didymatorthat harborsH. didymatorichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene,U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown ofU16by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicatedU16knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis. 

Abstract The adelgids (Adelgidae) are a small family of sap-feeding insects, which, together with true aphids (Aphididae) and phylloxerans (Phylloxeridae), make up the infraorder Aphidomorpha. Some adelgid species are highly destructive to forest ecosystems such as Adelges tsugae, Adelges piceae, Adelges laricis, Pineus pini, and Pineus boerneri. Despite this, there are no high-quality genomic resources for adelgids, hindering advanced genomic analyses within Adelgidae and among Aphidomorpha. Here, we used PacBio continuous long-read and Illumina RNA-sequencing to construct a high-quality draft genome assembly for the Cooley spruce gall adelgid, Adelges cooleyi (Gillette), a gall-forming species endemic to North America. The assembled genome is 270.2 Mb in total size and has scaffold and contig N50 statistics of 14.87 and 7.18 Mb, respectively. There are 24,967 predicted coding sequences, and the assembly completeness is estimated at 98.1 and 99.6% with core BUSCO gene sets of Arthropoda and Hemiptera, respectively. Phylogenomic analysis using the A. cooleyi genome, 3 publicly available adelgid transcriptomes, 4 phylloxera transcriptomes, the Daktulosphaira vitifoliae (grape phylloxera) genome, 4 aphid genomes, and 2 outgroup coccoid genomes fully resolves adelgids and phylloxerans as sister taxa. The mitochondrial genome is 24 kb, among the largest in insects sampled to date, with 39.4% composed of noncoding regions. This genome assembly is currently the only genome-scale, annotated assembly for adelgids and will be a valuable resource for understanding the ecology and evolution of Aphidomorpha. 

Abstract The parasitoid wasp Venturia canescens is an important biological control agent of stored products moth pests and serves as a model to study the function and evolution of domesticated endogenous viruses (DEVs). The DEVs discovered in V. canescens are known as virus-like particles (VcVLPs), which are produced using nudivirus-derived components and incorporate wasp-derived virulence proteins instead of packaged nucleic acids. Previous studies of virus-derived components in the V. canescens genome identified 53 nudivirus-like genes organized in six gene clusters and several viral pseudogenes, but how VcVLP genes are organized among wasp chromosomes following their integration in the ancestral wasp genome is largely unknown. Here, we present a chromosomal scale genome of V. canescens consisting of 11 chromosomes and 56 unplaced small scaffolds. The genome size is 290.8 Mbp with a N50 scaffold size of 24.99 Mbp. A high-quality gene set including 11,831 protein-coding genes were produced using RNA-Seq data as well as publicly available peptide sequences from related Hymenoptera. A manual annotation of genes of viral origin produced 61 intact and 19 pseudogenized nudivirus-derived genes. The genome assembly revealed that two previously identified clusters were joined into a single cluster and a total of 5 gene clusters comprising of 60 intact nudivirus-derived genes were located in three chromosomes. In contrast, pseudogenes are dispersed among 8 chromosomes with only 4 pseudogenes associated with nudivirus gene clusters. The architecture of genes encoding VcVLP components suggests it originates from a recent virus acquisition and there is a link between the processes of dispersal and pseudogenization. This high-quality genome assembly and annotation represents the first chromosome-scale assembly for parasitoid wasps associated with VLPs, and is publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a valuable resource for future studies of DEVs in parasitoid wasps. 

Heritable symbionts display a wide variety of transmission strategies to travel from one insect generation to the next. Parasitoid wasps, one of the most diverse insect groups, maintain several heritable associations with viruses that are beneficial for wasp survival during their development as parasites of other insects. Most of these beneficial viral entities are strictly transmitted through the wasp germline as endogenous viral elements within wasp genomes. However, a beneficial poxvirus inherited by Diachasmimorpha longicaudata wasps, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), is not integrated into the wasp genome and therefore may employ different tactics to infect future wasp generations. Here, we demonstrated that transmission of DlEPV is primarily dependent on parasitoid wasps, since viral transmission within fruit fly hosts of the wasps was limited to injection of the virus directly into the larval fly body cavity. Additionally, we uncovered a previously undocumented form of posthatch transmission for a mutualistic virus that entails external acquisition and localization of the virus within the adult wasp venom gland. We showed that this route is extremely effective for vertical and horizontal transmission of the virus within D. longicaudata wasps. Furthermore, the beneficial phenotype provided by DlEPV during parasitism was also transmitted with perfect efficiency, indicating an effective mode of symbiont spread to the advantage of infected wasps. These results provide insight into the transmission of beneficial viruses among insects and indicate that viruses can share features with cellular microbes during their evolutionary transitions into symbionts. 

ABSTRACT Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae ( Microplitis demolitor and Cotesia congregata ) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis , which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized compared to M. demolitor and C. congregata . Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. IMPORTANCE Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This work characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis , which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function. 

Abstract The Ichneumonoidea (Ichneumonidae and Braconidae) is an incredibly diverse superfamily of parasitoid wasps that includes species that produce virus-like entities in their reproductive tracts to promote successful parasitism of host insects. Research on these entities has traditionally focused upon two viral genera Bracovirus (in Braconidae) and Ichnovirus (in Ichneumonidae). These viruses are produced using genes known collectively as endogenous viral elements (EVEs) that represent historical, now heritable viral integration events in wasp genomes. Here, new genome sequence assemblies for 11 species and 6 publicly available genomes from the Ichneumonoidea were screened with the goal of identifying novel EVEs and characterizing the breadth of species in lineages with known EVEs. Exhaustive similarity searches combined with the identification of ancient core genes revealed sequences from both known and novel EVEs. One species harbored a novel, independently derived EVE related to a divergent large double-stranded DNA (dsDNA) virus that manipulates behavior in other hymenopteran species. Although bracovirus or ichnovirus EVEs were identified as expected in three species, the absence of ichnoviruses in several species suggests that they are independently derived and present in two younger, less widespread lineages than previously thought. Overall, this study presents a novel bioinformatic approach for EVE discovery in genomes and shows that three divergent virus families (nudiviruses, the ancestors of ichnoviruses, and Leptopilina boulardi Filamentous Virus-like viruses) are recurrently acquired as EVEs in parasitoid wasps. Virus acquisition in the parasitoid wasps is a common process that has occurred in many more than two lineages from a diverse range of arthropod-infecting dsDNA viruses.