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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. [ABSTRACT FROM AUTHOR]

The mosquito family Culicidae is divided into 2 subfamilies named the Culicinae and Anophelinae. Nix , the dominant male-determining factor, has only been found in the culicines Aedes aegypti and Aedes albopictus , 2 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 of Nix. Except for the Culex genus, Nix homologs were found in all species surveyed from the Culicinae subfamily, including 12 additional species from 3 highly divergent tribes comprising 4 genera, suggesting Nix originated at least 133 to 165 million years ago (MYA). Heterologous expression of 1 of 3 divergent Nix open reading frames (ORFs) in Ae. aegypti resulted in partial masculinization of genetic females as evidenced by morphology and doublesex splicing. Phylogenetic analysis suggests Nix is related to femaleless (fle), a recently described intermediate sex-determining factor found exclusively in anopheline mosquitoes. Nix from all species has a conserved structure, including 3 RNA-recognition motifs (RRMs), as does fle. However, Nix has evolved at a much faster rate than fle. The RRM3 of both Nix and fle are distantly related to the single RRM of a widely distributed and conserved splicing factor transformer-2 (tra2). The RRM3-based phylogenetic analysis suggests this domain in Nix and fle may have evolved from tra2 or a tra2 -related gene in a common ancestor of mosquitoes. Our results provide insights into the evolution of sex determination in mosquitoes and will inform broad applications of mosquito-control strategies based on manipulating sex ratios toward nonbiting males. [ABSTRACT FROM AUTHOR]

Background: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus. Methods: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured. Results: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior. Conclusions: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 wildtype 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. [ABSTRACT FROM AUTHOR]

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that Aedes aegypti larvae colonized by a native community of bacteria and gnotobiotic larvae colonized by only Escherichia coli develop very similarly into adults, whereas axenic larvae never molt and die as first instars. In this study, we extended these findings by first comparing the growth and abundance of bacteria in conventional, gnotobiotic, and axenic larvae during the first instar. Results showed that conventional and gnotobiotic larvae exhibited no differences in growth, timing of molting, or number of bacteria in their digestive tract. Axenic larvae in contrast grew minimally and never achieved the critical size associated with molting by conventional and gnotobiotic larvae. In the second part of the study we compared patterns of gene expression in conventional, gnotobiotic and axenic larvae by conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially expressed in axenic versus conventional or gnotobiotic larvae. However, this profile consisted primarily of transcripts in seven categories that included the down-regulation of select peptidases in the gut and up-regulation of several genes in the gut and carcass with roles in amino acid transport, hormonal signaling, and metabolism. Overall, our results indicate that axenic larvae exhibit alterations in gene expression consistent with defects in acquisition and assimilation of nutrients required for growth. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus- derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Image 1 • Oogenesis is arrested in non-blood fed female A. aegypti and most other mosquitoes. • Blood feeding stimulates the release of two hormones, ILP3 and OEH, in A. aegypti. • OEH and ILP3 activate the IIS and TOR pathways in ovaries which inhibit GSK3. • Inhibition of GSK3 activates oogenesis. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • All ILPs and OEH are expressed during a gonadotrophic cycle. • Brain-produced ILPs, OEH stimulate nutrient storage during the previtellogenic phase. • Brain-produced ILPs, OEH and 20-hydroxyecdysone regulate vitellogenesis. • ILPs and OEH differentially signal in ovaries, pelts, and guts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aedes aegypti is an anautogenous mosquito that must blood feed on a vertebrate host to produce and lay a clutch of eggs. The rockpool mosquito, Georgecraigius atropalpus , is related to A. aegypti but is a facultatively autogenous species that produces its first clutch of eggs shortly after emerging without blood feeding. Consumption of a blood meal by A. aegypti triggers the release of ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3) from the brain, which stimulate egg formation . OEH and ILP3 also stimulate egg formation in G. atropalpus but are released at eclosion independently of blood feeding. These results collectively suggest that blood meal dependent release of OEH and ILP3 is one factor that prevents A. aegypti from reproducing autogenously. Here, we examined two other factors that potentially inhibit autogeny in A. aegypti : teneral nutrient reserves and the ability of OEH and ILP3 to stimulate egg formation in the absence of blood feeding. Measures of nutrient reserves showed that newly emerged A. aegypti females had similar wet weights but significantly lower protein and glycogen reserves than G. atropalpus females when larvae were reared under identical conditions. OEH stimulated non-blood fed A. aegypti females to produce ecdysteroid hormone and package yolk into oocytes more strongly than ILP3. OEH also reduced host seeking and blood feeding behavior, yet females produced few mature eggs. Overall, our results indicate that multiple factors prevent A. aegypti from reproducing autogenously. [ABSTRACT FROM AUTHOR]