Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development.

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.

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes.

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.

Hypoxia-induced transcription factor signaling is essential for larval growth of the mosquito Aedes aegypti.

Gut microbes positively affect the physiology of many animals, but the molecular mechanisms underlying these benefits remain poorly understood. We recently reported that bacteria-induced gut hypoxia functions as a signal for growth and molting of the mosquito Aedes aegypti In this study, we tested the hypothesis that transduction of a gut hypoxia signal requires hypoxia-induced transcription factors (HIFs). Expression studies showed that HIF-α was stabilized in larvae containing bacteria that induce gut hypoxia but was destabilized in larvae that exhibit normoxia. However, we could rescue growth of larvae exhibiting gut normoxia by treating them with a prolyl hydroxylase inhibitor, FG-4592, that stabilized HIF-α, and inhibit growth of larvae exhibiting gut hypoxia by treating them with an inhibitor, PX-478, that destabilized HIF-α. Using these tools, we determined that HIF signaling activated the insulin/insulin growth factor pathway plus select mitogen-activated kinases and inhibited the adenosine monophosphate-activated protein kinase pathway. HIF signaling was also required for growth of the larval midgut and storage of neutral lipids by the fat body. Altogether, our results indicate that gut hypoxia and HIF signaling activate multiple processes in A. aegypti larvae, with conserved functions in growth and metabolism.

Blood feeding activates the vitellogenic stage of oogenesis in the mosquito Aedes aegypti through inhibition of glycogen synthase kinase 3 by the insulin and TOR pathways.

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.

Predaceous Toxorhynchites mosquitoes require a living gut microbiota to develop.

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.

Bacteria-mediated hypoxia functions as a signal for mosquito development.

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that several mosquito species, including Aedes aegypti, do not develop beyond the first instar when fed a nutritionally complete diet in the absence of a gut microbiota. In contrast, several species of bacteria, including Escherichia coli, rescue development of axenic larvae into adults. The molecular mechanisms underlying bacteria-dependent growth are unknown. Here, we designed a genetic screen around E. coli that identified high-affinity cytochrome bd oxidase as an essential bacterial gene product for mosquito growth. Bioassays showed that bacteria in nonsterile larvae and gnotobiotic larvae inoculated with wild-type E. coli reduced midgut oxygen levels below 5%, whereas larvae inoculated with E. coli mutants defective for cytochrome bd oxidase did not. Experiments further supported that hypoxia leads to growth and ecdysone-induced molting. Altogether, our results identify aerobic respiration by bacteria as a previously unknown but essential process for mosquito development.

Isolation of an insulin-like peptide from the Asian malaria mosquito, Anopheles stephensi, that acts as a steroidogenic gonadotropin across diverse mosquito taxa.

Many insulin-like peptides (ILPs) have been identified in insects, yet only a few were isolated in their native form for structural and functional studies. Antiserum produced to ILP3 in Aedes aegypti was used in a radioimmunoassay to monitor the purification of an ILP from heads of adult An. stephensi and recognized the ILP in other immunoassays. The structure of the purified peptide matched that predicted for the ILP3 in this species. The native form stimulated ecdysteroid production by ovaries isolated from non-blood fed females. Synthetic forms of An. stephensi ILP3 and ILP4 similarly activated this process in a dose responsive manner. This function was first established for ILP3 and ILP4 homologs in Aedes aegypti, thus suggesting their structural and functional conservation in mosquitoes. We tested the extent of conservation by treating ovaries of An. gambiae, Ae. aegypti, and Culex quinquefasciatus with the An. stephensi ILPs, and both the native and synthetic ILP3 were stimulatory, as was the ILP4. Taken together, these results offer the first evidence for ILP functional conservation across the Anophelinae and Culicinae subfamilies.

Ovary ecdysteroidogenic hormone requires a receptor tyrosine kinase to activate egg formation in the mosquito Aedes aegypti.

Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes required for egg formation. ILPs function by binding to the insulin receptor, which activates downstream components in the canonical insulin signaling pathway. OEH in contrast belongs to a neuropeptide family called neuroparsins, whose receptor is unknown. Here we demonstrate that a previously orphanized receptor tyrosine kinase (RTK) from A. aegypti encoded by the gene AAEL001915 is an OEH receptor. Phylogenetic studies indicated that the protein encoded by this gene, designated AAEL001915, belongs to a clade of RTKs related to the insulin receptor, which are distinguished by an extracellular Venus flytrap module. Knockdown of AAEL001915 by RNAi disabled OEH-mediated egg formation in A. aegypti. AAEL001915 was primarily detected in the mosquito ovary in association with follicular epithelial cells. Both monomeric and dimeric AAEL001915 were detected in mosquito ovaries and transfected Drosophila S2 cells. Functional assays further indicated that OEH bound to dimeric AAEL001915, which resulted in downstream phosphorylation of Ak strain transforming factor (Akt). We hypothesize that orthologs of AAEL001915 in other insects are neuroparsin receptors.

Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids.

Ecdysteroid hormones regulate several aspects of insect development and reproduction. The predominant ecdysteroids produced by insects including mosquitoes are ecdysone (E) and 20-hydroxyecdysone (20E). The ability to measure E and 20E titers is essential for many studies, but few sensitive, low cost options are currently available for doing so. To address this deficiency, we developed a new enzyme-linked immunoassay (EIA). In the first part of the study, we compared the affinity of two new antisera named EAB25 and EAB27 to other available ecdysteroid antisera. EAB25 had a 27-fold higher affinity for 20E than E, while EAB27 had a four-fold higher affinity for 20E. In the second part of the study, EIA protocols were developed for analyzing E and 20E produced by the mosquito Aedes aegypti. Results indicated that pelts from fourth instar larvae and ovaries from blood-fed, adult females produced E and 20E. Methanol extraction in the presence of magnesium from whole body samples altered antibody recognition of E and 20E by EIA. However, extraction with 1-butanol and two organic/water phase separations eliminated this problem and improved assay performance. We conclude the new antisera used in the EIA provide a low-cost, flexible, and sensitive method for measuring E and 20E in insects.

Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats.

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.

Mosquitoes rely on their gut microbiota for development.

Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood-feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.

Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti.

All vector mosquito species must feed on the blood of a vertebrate host to produce eggs. Multiple cycles of blood feeding also promote frequent contacts with hosts, which enhance the risk of exposure to infectious agents and disease transmission. Blood feeding triggers the release of insulin-like peptides (ILPs) from the brain of the mosquito Aedes aegypti, which regulate blood meal digestion and egg formation. In turn, hemocytes serve as the most important constitutive defense in mosquitoes against pathogens that enter the hemocoel. Prior studies indicated that blood feeding stimulates hemocytes to increase in abundance, but how this increase in abundance is regulated is unknown. Here, we determined that phagocytic granulocytes and oenocytoids express the A. aegypti insulin receptor (AaMIR). We then showed that: 1) decapitation of mosquitoes after blood feeding inhibited hemocyte proliferation, 2) a single dose of insulin-like peptide 3 (ILP3) sufficient to stimulate egg production rescued proliferation, and 3) knockdown of the AaMIR inhibited ILP3 rescue activity. Infection studies indicated that increased hemocyte abundance enhanced clearance of the bacterium Escherichia coli at lower levels of infection. Surprisingly, however, non-blood fed females better survived intermediate and high levels of E. coli infection than blood fed females. Taken together, our results reveal a previously unrecognized role for the insulin signaling pathway in regulating hemocyte proliferation. Our results also indicate that blood feeding enhances resistance to E. coli at lower levels of infection but reduces tolerance at higher levels of infection.

Effects of the botanical insecticide, toosendanin, on blood digestion and egg production by female Aedes aegypti (Diptera: Culicidae): topical application and ingestion.

Botanical insecticides offer novel chemistries and actions that may provide effective mosquito control. Toosendanin (TSN, 95% purity) is one such insecticide used to control crop pests in China, and in this study, it was evaluated for lethal and sublethal effects on larvae and females of the yellowfever mosquito, Aedes aegypti (L.). TSN was very toxic to first instar larvae after a 24 h exposure (LC50 = 60.8 microg/ml) and to adult females up to 96 h after topical treatment (LD50 = 4.3 microg/female) or ingestion in a sugar bait (LC50 = 1.02 microg/microl). Treatment of first instars for 24 h with a range of sublethal doses (6.3-25 microg/ml) delayed development to pupae by 1 to 2 d. Egg production and larval hatching from eggs were dose dependently reduced (>45%) by TSN doses (1.25-10.0 microg) topically applied to females 24 h before or 1 h after a bloodmeal. Ingestion of TSN (0.031-0.25 microg/microl of sugar bait) by females 24 h before a bloodmeal also greatly reduced egg production and larval hatch; no eggs were oviposited by females ingesting the highest dose. Further studies revealed that topical or ingested TSN dose-dependently disrupted yolk deposition in oocytes, blood ingestion and digestion, and ovary ecdysteroid production in blood-fed females. Overall, our results indicate that TSN is an effective insecticide for Ae. aegypti larvae and adults, because of its overt toxicity at high doses and disruption of development and reproduction at sublethal doses.

Insulin-like peptides regulate oogenesis by stimulating ovarian ecdysteroid production in the Indian malaria mosquito Anopheles stephensi.

Females of many mosquito species feed on vertebrate blood to produce eggs, making them effective disease vectors. In the dengue vector Aedes 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 of Anopheles mosquitoes, which pose a greater public health threat than Aedes spp. because they are competent to transmit mammalian malaria. ILPs can trigger An. stephensi ovaries to secrete ecdysteroids. Unlike Ae. aegypti , Anopheles also transfer ecdysteroids from Anopheles males to females during mating. To elucidate the role of OEH and ILPs in An. 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.

Multiple endocrine factors regulate nutrient mobilization and storage in Aedes aegypti during a gonadotrophic cycle.

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.

Insulin-like peptides and ovary ecdysteroidogenic hormone differentially stimulate physiological processes regulating egg formation in the mosquito Aedes aegypti.

Mosquitoes including Aedes aegypti are human disease vectors because females must blood feed to produce and lay eggs. Blood feeding triggers insulin-insulin growth factor signaling (IIS) which regulates several physiological processes required for egg development. A. aegypti encodes 8 insulin-like peptides (ILPs) and one insulin-like receptor (IR) plus ovary ecdysteroidogenic hormone (OEH) that also activates IIS through the OEH receptor (OEHR). In this study, we assessed the expression of A. aegypti ILPs and OEH during a gonadotrophic cycle and produced each that were functionally characterized to further understand their roles in regulating egg formation. All A. aegypti ILPs and OEH were expressed during a gonadotrophic cycle. Five ILPs (1, 3, 4, 7, 8) and OEH were specifically expressed in the head, while antibodies to ILP3 and OEH indicated each was released after blood feeding from ventricular axons that terminate on the anterior midgut. A subset of ILP family members and OEH stimulated nutrient storage in previtellogenic females before blood feeding, whereas most IIS-dependent processes after blood feeding were activated by one or more of the brain-specific ILPs and/or OEH. ILPs and OEH with different biological activities also exhibited differences in IIS as measured by phosphorylation of the IR, phosphoinositide 3-kinase/Akt kinase (AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK). Altogether, our results provide the first results that compare the functional activities of all ILP family members and OEH produced by an insect.

Enterococcus faecium Empyema Following Extracorporeal Membrane Oxygenation for COVID-19 Acute Respiratory Distress Syndrome.

A 33-year-old male with severe COVID-19 required prolonged veno-venous extracorporeal membrane oxygenation (ECMO) support. Following decannulation, he developed an Enterococcus faecium empyema. Tube thoracostomy and broad-spectrum antibiotics were initiated, followed by an unsuccessful attempt at pleural irrigation with saline, given the patient had an increased risk of bleeding due to the concomitant requirement for systemic anticoagulation. Subsequently, intrapleural tissue plasminogen activator (tPA) and recombinant human Dornase alfa (DNase) were safely administered with the resolution of empyema. Enterococcus faecium is an uncommon cause of pleural empyema and, to our knowledge, has not previously been reported to be associated with COVID-19 or ECMO.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence.

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 used Aedes 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.

Ovary ecdysteroidogenic hormone activates egg maturation in the mosquito Georgecraigius atropalpus after adult eclosion or a blood meal.

The rockpool mosquito, Georgecraigius atropalpus, is a facultatively autogenous species that produces its first egg clutch without a blood meal shortly after emergence. Several days after depositing this clutch, females must take a blood meal to produce a second egg clutch. Decapitation of females shortly after emergence or blood ingestion prevents egg maturation. Here, we report that a single injected dose of the neuropeptide ovary ecdysteroidogenic hormone (OEH) fully restored egg maturation in decapitated females in both circumstances. This neuropeptide and two insulin-like peptides (ILPs) are potent gonadotropins in the related yellow fever mosquito, Aedes aegypti. ILP3 was marginally restorative in decapitated G. atropalpus, and ILP4 had no effect. Egg maturation in non- and blood-fed G. atropalpus was dependent on the enzymatic mobilization of amino acids from stored protein or the blood meal for yolk protein (vitellogenin, VG) synthesis and uptake by oocytes. We further show that OEH stimulates serine protease activity in the fat body of newly eclosed females or in the midgut of blood-fed ones, and ecdysteroid hormone production by the ovaries of both females. In contrast, only 20-hydroxyecdysone stimulated VG synthesis in the fat body of non- and blood-fed females. Using RNA interference to knock down expression of the insulin receptor, we found that OEH still fully restored autogenous egg maturation. In summary, our results identify OEH as a primary regulator of egg maturation in both autogenous and blood-fed G. atropalpus females and suggest the shift from blood meal-dependent to blood meal-independent release of OEH is a key factor in the evolution of autogeny in this species.