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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.

Understanding 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.

 

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we usedAedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

 

Insects often harbour heritable symbionts that provide defence against specialized natural enemies, yet little is known about symbiont protection when hosts face simultaneous threats. In pea aphids (Acyrthosiphon pisum), the facultative endosymbiontHamiltonella defensaconfers protection against the parasitoid,Aphidius ervi, andRegiella insecticolaprotects against aphid‐specific fungal pathogens, includingPandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virusA. pisum virus(APV), and whether their antifungal and antiparasitoid services are impacted by APV infection. We found that APV imposed large fitness costs on symbiont‐free aphids and these costs were elevated in aphids also housingH. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids withR. insecticola. To our knowledge,R. insecticolais the first aphid symbiont shown to protect against a viral pathogen, and only the second arthropod symbiont reported to do so. In contrast, APV infection did not impact the protective services of eitherR. insecticolaorH. defensa. To better understand APV biology, we produced five genomes and examined transmission routes. We found that moderate rates of vertical transmission, combined with horizontal transfer through food plants, were the major route of APV spread, although lateral transfer by parasitoids also occurred. Transmission was unaffected by facultative symbionts. In summary, the presence and species identity of facultative symbionts resulted in highly divergent outcomes for aphids infected with APV, while not impacting defensive services that target other enemies. These findings add to the diverse phenotypes conferred by aphid symbionts, and to the growing body of work highlighting extensive variation in symbiont‐mediated interactions.

 

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 Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti, where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle, but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes, but few genes – including almost no transcripts for immune factors – were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny. 

Most phages infect free-living bacteria but a few have been identified that infect heritable symbionts of insects or other eukaryotes. Heritable symbionts are usually specialized and isolated from other bacteria with little known about the origins of associated phages.Hamiltonella defensais a heritable bacterial symbiont of aphids that is usually infected by a tailed, double-stranded DNA phage named APSE.

We conducted comparative genomic and phylogenetic studies to determine how APSE is related to other phages and prophages.

Each APSE genome was organized into four modules and two predicted functional units. Gene content and order were near-fully conserved in modules 1 and 2, which encode predicted DNA metabolism genes, and module 4, which encodes predicted virion assembly genes. Gene content of module 3, which contains predicted toxin, holin and lysozyme genes differed among haplotypes. Comparisons to other sequenced phages suggested APSE genomes are mosaics with modules 1 and 2 sharing similarities withBordetella-Bcep-Xylostella fastidiosa-like podoviruses, module 4 sharing similarities with P22-like podoviruses, and module 3 sharing no similarities with known phages. Comparisons to other sequenced bacterial genomes identified APSE-like elements in other heritable insect symbionts (Arsenophonusspp.) and enteric bacteria in the familyMorganellaceae.

APSEs are most closely related to phage elements in the genusArsenophonusand other bacteria in theMorganellaceae.