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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 endosymbiont Hamiltonella defensa confers protection against the parasitoid, Aphidius ervi, and Regiella insecticola protects against aphid-specific fungal pathogens, including Pandora neoaphidis. Here, we investigated whether these two common aphid symbionts protect against a specialized virus A. 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 housing H. defensa. In contrast, APV titres were significantly reduced and costs to APV infection were largely eliminated in aphids with R. insecticola. To our knowledge, R. insecticola is 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 either R. insecticola or H. 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.

Anautogenous mosquitoes must blood feed on a vertebrate host to produce eggs. Each gonadotrophic cycle is subdivided into a sugar-feeding previtellogenic phase that produces primary follicles and a blood meal-activated vitellogenic phase in which large numbers of eggs synchronously mature and are laid. Multiple endocrine factors including juvenile hormone (JH), insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH), and 20-hydroxyecdysone (20E) coordinate each gonadotrophic cycle. Egg formation also requires nutrients from feeding that are stored in the fat body. Regulation of egg formation is best understood in Aedes aegypti but the role different endocrine factors play in regulating nutrient mobilization and storage remains unclear. In this study, we report that adult female Ae. aegypti maintained triacylglycerol (TAG) stores during the previtellogenic phase of the first gonadotrophic cycle while glycogen stores declined. In contrast, TAG and glycogen stores were rapidly mobilized during the vitellogenic phase and then replenishment. Several genes encoding enzymes with functions in TAG and glycogen metabolism were differentially expressed in the fat body, which suggested regulation was mediated in part at the transcriptional level. Gain of function assays indicated that stored nutrients were primarily mobilized by adipokinetic hormone (AKH) while juvenoids and OEH regulated replenishment. ILP3 further showed evidence of negatively regulating certain lipolytic enzymes. Loss of function assays indicated AKH depends on the AKH receptor (AKHR) for function. Altogether, our results indicate that the opposing activities of different hormones regulate nutrient stores during a gonadotrophic cycle in Ae. aegypti.

Females of many mosquito species feed on vertebrate blood to produce eggs, making them effective disease vectors. In the dengue vectorAedes aegypti, blood feeding signals the brain to release ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs) that trigger ecdysteroid production by the ovaries. These ecdysteroids regulate synthesis of the yolk protein vitellogenin (Vg) that is packaged into eggs. Less is known about the reproductive biology ofAnophelesmosquitoes, which pose a greater public health threat thanAedesspp. because they are competent to transmit mammalian malaria. ILPs can triggerAn. stephensiovaries to secrete ecdysteroids. UnlikeAe. aegypti,Anophelesalso transfer ecdysteroids fromAnophelesmales to females during mating. To elucidate the role of OEH and ILPs inAn. stephensi, we decapitated blood-fed females to ablate the source of these peptides and injected them with each hormone. Yolk deposition into oocytes was abolished in decapitated females and rescued by ILP injection. ILP activity was dependent on blood feeding and little change in triglyceride and glycogen stores was observed in response to blood-feeding, suggesting this species requires nutrients from blood to form eggs. We also measured egg maturation, ecdysteroid titers, and yolk protein expression in mated and virgin females. Although yolk deposition into developing oocytes was significantly reduced in virgins compared to mated females, no differences in ecdysteroid titers or Vg transcript abundance were detected between these groups. 20-hydroxyecdysone (20E) stimulated Vg expression in female fat bodies in primary culture. Given these results, we conclude that ILPs control egg formation by regulating ecdysteroid production in the ovaries.

Bracoviruses (BVs) and ichnoviruses (IVs) evolved from different endogenized viruses but through convergence have been coopted by parasitoids in the families Braconidae and Ichneumonidae for similar functions in parasitizing hosts. Experimentally studying the role of endogenized viral genes in virion morphogenesis remains a key challenge in the study of BVs and IVs. Here we summarize how multiomics, electron microscopy, and RNA interference (RNAi) methods have provided new insights about BV and IV gene function.

Animal genomes commonly contain genes or sequences that have been acquired from different types of viruses. The vast majority of these endogenous virus elements (EVEs) are inactive or consist of only a small number of components that show no evidence of cooption for new functions or interaction. Unlike most EVEs, bracoviruses (BVs), ichnoviruses (IVs) and virus-like particles (VLPs) in parasitoid wasps have evolved through retention and interaction of many genes from virus ancestors. Here, we discuss current understanding of BV, IV and VLP evolution along with associated implications for what constitutes a virus. We suggest that BVs and IVs are domesticated endogenous viruses (DEVs) that differ in several important ways from other known EVEs.

The mosquito family Culicidae is divided into two subfamilies named the Culicinae and Anophelinae.Nix, the dominant male-determining factor, has only been found in the culicinesAedes aegyptiandAe. albopictus, two important arboviral vectors that belong to the subgenus Stegomyia. Here we performed sex-specific whole-genome sequencing and RNAseq of divergent mosquito species and explored additional male-inclusive datasets to investigate the distribution ofNix. Except for the Culex genus,Nixhomologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from three highly divergent tribes comprising 4 genera, suggestingNixoriginated at least 133-165 MYA. Heterologous expression of one of three divergentNixORFs inAe. aegyptiresulted in partial masculinization of genetic females as evidenced by morphology anddoublesexsplicing. It is not clear whether insufficient transgene expression or sequence divergence or both are responsible for the lack of phenotype for the other two. Phylogenetic analysis suggestsNixis related tofemaleless(fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes.Nixfrom all species has a conserved structure, including three RNA-recognition motifs (RRMs), as doesfle. However,Nixhas evolved at a much faster rate thanfle. The RRM3 of bothNixandfleare related to the single RRM of a widely distributed and conserved splicing factortransformer-2(tra2). RRM3-based phylogenetic analysis suggests this domain inNixandflemay have evolved fromtra2in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex-determination and sex chromosomes in mosquitoes, and will inform broad applications of mosquito-control strategies based on manipulating sex ratios towards the non-biting males.

Studying the microbial symbionts of eukaryotic hosts has revealed a range of interactions that benefit host biology. Most eukaryotes are also infected by parasites that adversely affect host biology for their own benefit. However, it is largely unclear whether the ability of parasites to develop in hosts also depends on host-associated symbionts, e.g., the gut microbiota. Here, we studied the parasitic wasp Leptopilina boulardi (Lb) and its host Drosophila melanogaster. Results showed that Lb successfully develops in conventional hosts (CN) with a gut microbiota but fails to develop in axenic hosts (AX) without a gut microbiota. We determined that developing Lb larvae consume fat body cells that store lipids. We also determined that much larger amounts of lipid accumulate in fat body cells of parasitized CN hosts than parasitized AX hosts. CN hosts parasitized by Lb exhibited large increases in the abundance of the bacterium Acetobacter pomorum in the gut, but did not affect the abundance of Lactobacillus fructivorans which is another common member of the host gut microbiota. However, AX hosts inoculated with A. pomorum and/or L. fructivorans did not rescue development of Lb. In contrast, AX larvae inoculated with A. pomorum plus other identified gut community members including a Bacillus sp. substantially rescued Lb development. Rescue was further associated with increased lipid accumulation in host fat body cells. Insulin-like peptides increased in brain neurosecretory cells of parasitized CN larvae. Lipid accumulation in the fat body of CN hosts was further associated with reduced Bmm lipase activity mediated by insulin/insulin-like growth factor signaling (IIS). Altogether, our results identify a previously unknown role for the gut microbiota in defining host permissiveness for a parasite. Our findings also identify a new paradigm for parasite manipulation of host metabolism that depends on insulin signaling and the gut microbiota.

Many factors affect the host range of baculoviruses. Chrysodeixis includens and Trichoplusia ni are two closely related members of the lepidopteran subfamily Plusiinae that are important insect pests of different crops worldwide. In Brazil, C. includens has recently become a major defoliating pest of soybean. In this study, we compared the susceptibility of C. includens and T. ni to an alphabaculovirus isolated from a C. includens population in Brazil named Chrysodeixis includens nucleopolyhedrovirus isolate GO (ChinNPV-GO). Our results showed that ChinNPV-GO infects and kills C. includens at low oral doses, whereas T. ni larvae exhibited much lower levels of susceptibility to oral infection. In contrast, ChinNPV-GO very similarly infected and killed C. includens and T. ni larvae after budded viruses were injected into the hemocoel. Altogether, we conclude that T. ni is much less susceptible to ChinNPV-GO due to a reduced ability of this virus to establish a primary infection in the midgut. Our results further support that ChinNPV-GO and other plusiinae-associated SNPVs studied to date exhibit restricted host ranges even though known hosts are phylogenetically closely related.

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.

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.

Background 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 defensa is a heritable bacterial symbiont of aphids that is usually infected by a tailed, double-stranded DNA phage named APSE. Methods We conducted comparative genomic and phylogenetic studies to determine how APSE is related to other phages and prophages. Results 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 with Bordetella-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 (Arsenophonus spp.) and enteric bacteria in the family Morganellaceae. Conclusions APSEs are most closely related to phage elements in the genus Arsenophonus and other bacteria in the Morganellaceae.

Most mosquitoes, including Aedes aegypti, only produce eggs after blood feeding on a vertebrate host. Oogenesis in A. aegypti consists of a pre-vitellogenic stage before blood feeding and a vitellogenic stage after blood feeding. Primary egg chambers remain developmentally arrested during the pre-vitellogenic stage but complete oogenesis to form mature eggs during the vitellogenic stage. In contrast, the signaling factors that maintain primary egg chambers in pre-vitellogenic arrest or that activate vitellogenic growth are largely unclear. Prior studies showed that A. aegypti females release insulin-like peptide 3 (ILP3) and ovary ecdysteroidogenic hormone (OEH) from brain neurosecretory cells after blood feeding. Here, we report that primary egg chambers exit pre-vitellogenic arrest by 8 h post-blood meal as evidenced by proliferation of follicle cells, endoreplication of nurse cells, and formation of cytoophidia. Ex vivo assays showed that ILP3 and OEH stimulate primary egg chambers to exit pre-vitellogenic arrest in the presence of nutrients but not in their absence. Characterization of associated pathways indicated that activation of insulin/insulin growth factor signaling (IIS) by ILP3 or OEH inactivated glycogen synthase kinase 3 (GSK3) via phosphorylation by phosphorylated Akt. GSK3 inactivation correlated with accumulation of the basic helix-loop-helix transcription factor Max and primary egg chambers exiting pre-vitellogenic arrest. Direct inhibition of GSK3 by CHIR-99021 also stimulated Myc/Max accumulation and primary egg chambers exiting pre-vitellogenic arrest. Collectively, our results identify GSK3 as a key factor in regulating the pre- and vitellogenic stages of oogenesis in A. aegypti.

Entomopathogenic nematodes and parasitoid wasps are used as biological control agents for management of insect pests such as the Indian meal moth, Plodia interpunctella. The parasitoid wasp Habrobracon hebetor injects a paralytic venom into P. interpunctella larvae before laying eggs. A previous study reported that the entomopathogenic nematode Heterorhabditis indica preferentially infects P. interpunctella that have been envenomed by H. hebetor while results in this study showed a similar preference by the entomopathogenic nematode, Steinernema glaseri. We therefore tested four hypotheses for why nematode infection rates are higher in envenomed hosts: (1) elevated CO2 emission from envenomed hosts attracts nematodes, (2) paralysis prevents hosts from escaping nematodes, (3) volatile chemicals emitted from envenomed hosts attract nematodes and increase infection, and (4) reduced immune defenses in envenomed hosts increase nematode survival. Results showed that envenomed P. interpunctella larvae emitted lower amounts of CO2 than non-envenomed larvae. Physical immobilization of P. interpunctella larvae did not increase infection rates by S. glaseri but did increase infection rates by H. indica. Emissions from envenomed hosts were collected and analyzed by thermal desorption gas chromatography/mass spectrometry. The most abundant compound, 3-methyl-3-buten-1-ol, was found to be an effective cue for S. glaseri attraction and infection but was not an effective stimulus for H. indica. Envenomed P. interpunctella exhibited a stronger immune response toward nematodes than non-envenomed hosts. Altogether, we conclude that different mechanisms underlie preferential infection in the two nematode species: host immobilization for H. indica and chemical cues for S. glaseri.

Gut microbes and diet can both strongly affect the biology of multicellular animals, but it is often difficult to disentangle microbiota–diet interactions due to the complex microbial communities many animals harbor and the nutritionally variable diets they consume. While theoretical and empirical studies indicate that greater microbiota diversity is beneficial for many animal hosts, there have been few tests performed in aquatic invertebrates. Most mosquito species are aquatic detritivores during their juvenile stages that harbor variable microbiotas and consume diets that range from nutrient rich to nutrient poor. In this study, we produced a gnotobiotic model that allowed us to examine how interactions between specific gut microbes and diets affect the fitness of Aedes aegypti, the yellow fever mosquito. Using a simplified seven-member community of bacteria (ALL7) and various laboratory and natural mosquito diets, we allowed larval mosquitoes to develop under different microbial and dietary conditions and measured the resulting time to adulthood and adult size. Larvae inoculated with the ALL7 or a more complex community developed similarly when fed nutrient-rich rat chow or fish food laboratory diets, whereas larvae inoculated with individual bacterial members of the ALL7 community exhibited few differences in development when fed a rat chow diet but exhibited large differences in performance when fed a fish food diet. In contrast, the ALL7 community largely failed to support the growth of larvae fed field-collected detritus diets unless supplemented with additional protein or yeast. Collectively, our results indicate that mosquito development and fitness are strongly contingent on both diet and microbial community composition.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)–like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2–like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9–TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4–MD-2–LPS pathway.

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spatzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)-like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2-like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9-TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4-MD-2-LPS pathway.

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Bracoviruses (BVs) are endogenized nudiviruses that braconid parasitoid wasps have coopted for functions in parasitizing hosts. Microplitis demolitor is a braconid wasp that produces Microplitis demolitor bracovirus (MdBV) and parasitizes the larval stage of the moth Chrysodeixis includens. Some BV core genes are homologs of genes also present in baculoviruses while others are only known from nudiviruses or other BVs. In this study, we had two main goals. The first was to separate MdBV virions into envelope and nucleocapsid fractions before proteomic analysis to identify core gene products that were preferentially associated with one fraction or the other. Results indicated that nearly all MdBV baculovirus-like gene products that were detected by our proteomic analysis had similar distributions to homologs in the occlusion-derived form of baculoviruses. Several core gene products unknown from baculoviruses were also identified as envelope or nucleocapsid components. Our second goal was to functionally characterize a core gene unknown from baculoviruses that was originally named HzNVorf64-like. Immunoblotting assays supported our proteomic data that identified HzNVorf64-like as an envelope protein. We thus renamed HzNVorf64-like as MdBVe46, which we further hypothesized was important for infection of C. includens. Knockdown of MdBVe46 by RNA interference (RNAi) greatly reduced transcript and protein abundance. Knockdown of MdBVe46 also altered virion morphogenesis, near-fully inhibited infection of C. includens, and significantly reduced the proportion of hosts that were successfully parasitized by M. demolitor.

Background Most female mosquitoes are anautogenous and must blood feed on a vertebrate host to produce eggs. Prior studies show that the number of eggs females lay per clutch correlates with the volume of blood ingested and that protein is the most important macronutrient for egg formation. In contrast, how whole blood, blood fractions and specific blood proteins from different vertebrates affect egg formation is less clear. Since egg formation is best understood in Aedes aegypti, we examined how blood and blood components from different vertebrates affect this species and two others: the malaria vector Anopheles gambiae and arbovirus vector Culex quinquefasciatus. Methods Adult female mosquitoes were fed blood, blood fractions and purified major blood proteins from different vertebrate hosts. Markers of reproductive response including ovary ecdysteroidogenesis, yolk deposition into oocytes and number of mature eggs produced were measured. Results Ae. aegypti, An. gambiae and C. quinquefasciatus responded differently to meals of whole blood, plasma or blood cells from human, rat, chicken and turkey hosts. We observed more similarities between the anthropophiles Ae. aegypti and An. gambiae than the ornithophile C. quinquefasciatus. Focusing on Ae. aegypti, the major plasma-derived proteins (serum albumin, fibrinogen and globulins) differentially stimulated egg formation as a function of vertebrate host source. The major blood cell protein, hemoglobin, stimulated yolk deposition when from pigs but not humans, cows or sheep. Serum albumins from different vertebrates also variably affected egg formation. Bovine serum albumin (BSA) stimulated ovary ecdysteroidogenesis, but more weakly induced digestive enzyme activities than whole blood. In contrast, BSA-derived peptides and free amino acids had no stimulatory effects on ecdysteroidogenesis or yolk deposition into oocytes. Conclusions Whole blood, blood fractions and specific blood proteins supported egg formation in three species of anautogenous mosquitoes but specific responses varied with the vertebrate source of the blood components tested. Graphical Abstract

This book concerns the population biology of vector-borne diseases. Vector-borne diseases of people are a perennial challenge for public health. Although recent decades have enjoyed major declines in the incidence of diseases like malaria and onchocerciasis (river blindness), vector-borne diseases continue to claim the lives of more than 700,000 people per year and exact costs of tens of billions of dollars in expenses for control and through lost productivity (...

Most species of mosquitoes are detritivores that feed on decaying plant and animal materials in their aquatic environment. Studies of several detritivorous mosquito species indicate that they host relatively low diversity communities of microbes that are acquired from the environment while feeding. Our recent results also indicate that detritivorous species normally require a living gut microbiota to grow beyond the first instar. Less well known is that some mosquitoes, including those belonging to the genus Toxorhynchites , are predators that feed on other species of mosquitoes and nektonic prey. In this study, we asked whether predaceous Toxorhynchites amboinensis larvae still require living microbes in their gut in order to develop. Using the detritivorous mosquito Aedes aegypti as prey, we found that T. amboinensis larvae harbour bacterial communities that are highly similar to that of their prey. Functional assays showed that T. amboinensis first instars provided axenic (i.e. bacteria-free) prey failed to develop, while two bacterial species present in gnotobiotic (i.e. colonized by one or more known bacterial species) prey successfully colonized the T. amboinensis gut and rescued development. Axenic T. amboinensis larvae also displayed defects in growth consistent with previously identified roles for microbe-mediated gut hypoxia in nutrient acquisition and assimilation in A. aegypti. Collectively, these results support a conserved role for gut microbes in regulating the development of mosquitoes with different feeding strategies.

Microplitis demolitor (Hymenoptera: Braconidae) is a parasitoid used as a biological control agent to control larval-stage Lepidoptera and serves as a model for studying the function and evolution of symbiotic viruses in the genus Bracovirus. Here we present the M. demolitor genome (assembly version 2.0), with a genome size of 241 Mb, and a N50 scaffold and contig size of 1.1 Mb and 14 Kb, respectively. Using RNA-Seq data and manual annotation of genes of viral origin, we produced a high-quality gene set that includes 18,586 eukaryotic and 171 virus-derived protein-coding genes. Bracoviruses are dsDNA viruses with unusual genome architecture, in which the viral genome is integrated into the wasp genome and is comprised of two distinct components: proviral segments that are amplified, circularized, and packaged into virions for export into the wasp’s host via oviposition; and replication genes. This genome assembly revealed that at least two scaffolds contain both nudivirus-like genes and proviral segments, demonstrating that at least some of these components are near each other in the genome on a single chromosome. The updated assembly and annotation are available in several publicly accessible databases; including the National Center for Biotechnology Information and the Ag Data Commons. In addition, all raw sequence data available for M. demolitor have been consolidated and are available for visualization at the i5k Workspace. This whole genome assembly and annotation represents the only genome-scale, annotated assembly from the lineage of parasitoid wasps that has associations with bracoviruses (the ‘microgastroid complex’), providing important baseline knowledge about the architecture of co-opted virus symbiont genomes.

An estimated 3500 species of mosquitoes (family Culicidae) exist worldwide of which several are known vectors of pathogens that cause disease in humans and other vertebrates. Mosquitoes also host communities of microbes in their digestive tract that form a gut microbiota. Recent studies provide important insights on how mosquitoes acquire a gut microbiota and the community of microbes that are present. Results also indicate that the gut microbiota affects several aspects of mosquito biology. Altogether, these effects impact mosquito fitness with potential consequences for disease prevalence.

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., The moth Plutella xylostella during its larval stage is the host of the endoparasitic wasp Cotesia vestalis. Here the authors show that the parasitoids deliver microRNAs to their hosts through their symbiotic virus and specialized cells leading to induced developmental delay.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.

Heritable symbionts are common in terrestrial arthropods and often provide beneficial services to hosts. Unlike obligate, nutritional symbionts that largely persist under strict host control within specialized host cells, heritable facultative symbionts exhibit large variation in within-host lifestyles and services rendered with many retaining the capacity to transition among roles. One enigmatic symbiont, Candidatus Fukatsuia symbiotica, frequently infects aphids with reported roles ranging from pathogen, defensive symbiont, mutualism exploiter and nutritional co-obligate symbiont. Here we used an in vitro culture-assisted protocol to sequence the genome of a facultative strain of Fukatsuia from pea aphids (Acyrthosiphon pisum). Phylogenetic and genomic comparisons indicate that Fukatsuia is an aerobic heterotroph, which together with Regiella insecticola and Hamiltonella defensa form a clade of heritable facultative symbionts within the Yersiniaceae (Enterobacteriales). These three heritable facultative symbionts largely share overlapping inventories of genes associated with housekeeping functions, metabolism, and nutrient acquisition, while varying in complements of mobile DNA. One unusual feature of Fukatsuia is its strong tendency to occur as a co-infection with H. defensa. However, the overall similarity of gene inventories among aphid heritable facultative symbionts suggest that metabolic complementarity is not the basis for co-infection, unless playing out on a H. defensa strain-specific basis. We also compared the pea aphid Fukatsuia with a strain from the aphid Cinara confinis (Lachninae) where it is reported to have transitioned to co-obligate status to support decaying Buchnera function. Overall the two genomes are very similar with no clear genomic signatures consistent with such a transition, which suggests co-obligate status in C. confinis was a recent event.

Polydnaviruses (PDVs) were originally viewed as large DNA viruses that are beneficial symbionts of parasitoid wasps. Two groups of PDVs were also recognized: bracoviruses (BVs), which are associated with wasps in the family Braconidae, and ichnoviruses (IVs), which are associated with wasps in the family Ichneumonidae. Results to date indicate that BVs are endogenous virus elements (EVEs) that evolved from an ancient betanudivirus. IVs are also likely EVEs but are unrelated to BVs. BVs and IVs are very unusual relative to most known EVEs because they retain many viral functions that benefit wasps in parasitizing hosts. However, BVs and IVs cannot be considered beneficial symbionts because all components of their genomes are fixed in wasps. Recent studies indicate that other nudiviruses have endogenized in insects. Each exhibits a different functional fate from BVs but shares certain architectural features. We discuss options for classifying BVs and other endogenized nudiviruses. We also discuss future directions.

Insects are frequently infected with inherited facultative symbionts known to provide a range of conditionally beneficial services, including host protection. Pea aphids (Acyrthosiphon pisum) often harbour the bacterium Hamiltonella defensa, which together with its associated bacteriophage A. pisum secondary endosymbiont (APSE) confer protection against an important natural enemy, the parasitic wasp Aphidius ervi. Previous studies showed that spontaneous loss of phage APSE resulted in the complete loss of the protective phenotype. Here, we demonstrate that APSEs can be experimentally transferred into phage-free (i.e. non-protecting) Hamiltonella strains. Unexpectedly, trials using injections of phage particles alone failed, with successful transfer occurring only when APSE and Hamiltonella were simultaneously injected. After transfer, stable establishment of APSE fully restored anti-parasitoid defenses. Thus, phages associated with heritable bacterial symbionts can move horizontally among symbiont strains facilitating the rapid transfer of ecologically important traits although natural barriers may preclude regular exchange.

Background Parasitic insects are well-known biological control agents for arthropod pests worldwide. They are capable of regulating their host’s physiology, development and behaviour. However, many of the molecular mechanisms involved in host-parasitoid interaction remain unknown. Results We sequenced the genomes of two parasitic wasps (Cotesia vestalis, and Diadromus collaris) that parasitize the diamondback moth Plutella xylostella using Illumina and Pacbio sequencing platforms. Genome assembly using SOAPdenovo produced a 178 Mb draft genome for C. vestalis and a 399 Mb draft genome for D. collaris. A total set that contained 11,278 and 15,328 protein-coding genes for C. vestalis and D. collaris, respectively, were predicted using evidence (homology-based and transcriptome-based) and de novo prediction methodology. Phylogenetic analysis showed that the braconid C. vestalis and the ichneumonid D. collaris diverged approximately 124 million years ago. These two wasps exhibit gene gains and losses that in some cases reflect their shared life history as parasitic wasps and in other cases are unique to particular species. Gene families with functions in development, nutrient acquisition from hosts, and metabolism have expanded in each wasp species, while genes required for biosynthesis of some amino acids and steroids have been lost, since these nutrients can be directly obtained from the host. Both wasp species encode a relative higher number of neprilysins (NEPs) thus far reported in arthropod genomes while several genes encoding immune-related proteins and detoxification enzymes were lost in both wasp genomes. Conclusions We present the annotated genome sequence of two parasitic wasps C. vestalis and D. collaris, which parasitize a common host, the diamondback moth, P. xylostella. These data will provide a fundamental source for studying the mechanism of host control and will be used in parasitoid comparative genomics to study the origin and diversification of the parasitic lifestyle.

Mosquitoes are insects of interest because several species vector disease-causing pathogens to humans and other vertebrates. We previously reported that mosquitoes from long-term laboratory cultures require living bacteria in their gut to develop, but development does not depend on particular species of bacteria. Here, we focused on three distinct but interrelated areas of study to better understand the role of bacteria in mosquito development by studying field and laboratory populations of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from the southeastern United States. Sequence analysis of bacterial 16S rRNA gene amplicons showed that bacterial community composition differed substantially in larvae from different collection sites, whereas larvae from the same site shared similarities. Although previously unknown to be infected by Wolbachia, results also indicated that Ae. aegypti from one field site hosted a dual infection. Regardless of collection site or factors like Wolbachia infection, however, each mosquito species required living bacteria in their digestive tract to develop. Results also identified several concerns in using antibiotics to eliminate the bacterial community in larvae in order to study its developmental consequences. Altogether, our results indicate that several mosquito species require living bacteria for development. We also hypothesize these species do not rely on particular bacteria because larvae do not reliably encounter the same bacteria in the aquatic habitats they develop in.

Polydnaviruses in the genus Bracovirus (BV) are associated with parasitoid wasps in the family Braconidae. BV-carrying wasps rely on their associated viruses to parasitize permissive hosts but also occasionally oviposit into host species that are non-permissive. Here, we studied Microplitis demolitor and M. demolitor bracovirus (MdBV) in Chrysodeixis includens, a permissive host, and Trichoplusia ni, which is usually non-permissive. M. demolitor laid eggs and injected MdBV into both hosts but almost no wasp offspring developed in T. ni. MdBV DNA similarly persisted in both host species, but deep sequencing data showed that transcript abundance for most viral genes was higher in C. includens than T. ni. Overall, our results identify lower expression of MdBV genes as an important factor in the non-permissiveness of T. ni. However, certain genes with functions in immunosuppression were sufficiently expressed to have similar effect in T. ni and C. includens.

A critical step in mosquito reproduction is the ingestion of a blood meal from a vertebrate host. In mosquitoes like Aedes aegypti, blood feeding stimulates the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3). This induces the ovaries to produce ecdysteroid hormone (ECD), which then drives egg maturation. In many immature insects, prothoracicotropic hormone (PTTH) stimulates the prothoracic glands to produce ECD that directs molting and metamorphosis. The receptors for OEH, ILP3 and PTTH are different receptor tyrosine kinases with OEH and ILP3 signaling converging downstream in the insulin pathway and PTTH activating the mitogen-activated protein kinase pathway. Calcium (Ca(2+)) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca(2+) in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca(2+) flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca(2+) flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca(2+) flux. Recombinant PTTH from Bombyx mori fully activated ECD production by B. mori PTGs, but exhibited no activity toward A. aegypti ovaries. Recombinant PTTH from A. aegypti also failed to stimulate either B. mori PTGs or A. aegypti ovaries to produce ECD. We discuss the implications of these results in the context of mosquito reproduction and ECD biosynthesis by insects generally.

The Drosophila melanogaster Toll-Spätzle pathway plays an important role in development and immunity. Drosophila genome encodes nine Toll receptors and six Spätzle (Spz) proteins, and only the canonical Toll-Spz (Toll-1-Spz-1) pathway has been well investigated. In this study, we compared the nine Drosophila Tolls and showed that similarly to Toll, Toll-7 also strongly activated drosomycin promoter. Importantly, we showed that both Toll and Toll-7 interacted with Spz, Spz-2 and Spz-5, and co-expression of Toll or Toll-7 with Spz, Spz-2 and Spz-5 activated the drosomycin promoter. Furthermore, Toll and Toll-7 both recognized vesicular stomatitis virus (VSV) by binding to the VSV glycoprotein. Septic infection in Toll and Toll-7 mutant flies suggested that Toll and Toll-7 differentially affected defense responses in adult males and females after systemic infection by Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans or VSV. Our results suggest multiple Toll family members activate the expression of antimicrobial peptides. Our results also provide evidence that Toll and Toll-7 bind multiple Spätzle proteins and differentially affect immune defense against different pathogens in adult male and female flies.

We recently reported that larval stage Aedes aegypti and several other species of mosquitoes grow when living bacteria are present in the gut but do not grow when living bacteria are absent. We further reported that living bacteria induce a hypoxia signal in the gut, which activates hypoxia-induced transcription factors and other processes larvae require for growth. In this study we assessed whether other types of organisms induce mosquito larvae to grow and asked if the density of non-living microbes or diet larvae are fed obviate the requirement for living organisms prior results indicated are required for growth. Using culture conditions identical to our own prior studies, we determined that inoculation density of living Escherichia coli positively affected growth rates of Ae. aegypti larvae, whereas non-living E. coli had no effect on growth across the same range of inoculation densities. A living yeast, alga, and insect cell line induced axenic Ae. aegypti first instars to grow, and stimulated similar levels of midgut hypoxia, HIF-α stabilization, and neutral lipid accumulation in the fat body as E. coli. However, the same organisms had no effect on larval growth if heat-killed. In addition, no axenic larvae molted when fed two other diets, when fed diets supplemented with heat-killed microbes or lysed and heat-killed microbes. Experiments conducted with An. gambiae yielded similar findings. Taken together, our results indicate that organisms from different prokaryotic and eukaryotic groups induce mosquito larvae to grow, whereas no conditions were identified that stimulated larvae to grow in the absence of living organisms., Author summary Studies conducted in our laboratory indicate that several species of mosquito larvae require living bacteria in their gut to grow. Previous results also show that living bacteria reduce gut oxygen levels below 5% and that bacteria-induced gut hypoxia functions as a signal that activates several processes in larvae with growth functions. Here we report that bacterial density affects larval growth rates and that other living organisms besides bacteria induce larval stage Aedes aegypti and Anopheles gambiae to grow. In contrast, the requirement for living organisms was not altered by the diet larvae were fed. These findings are of fundamental interest because they show that several types of organisms in the gut activate the same growth-related processes when viable but none do so when non-viable. Reported results also have applied implications for producing axenic adult mosquitoes for use in different types of studies.