Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that several mosquito species, including , 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, rescue development of axenic larvae into adults. The molecular mechanisms underlying bacteria-dependent growth are unknown. Here, we designed a genetic screen around that identified high-affinity cytochrome oxidase as an essential bacterial gene product for mosquito growth. Bioassays showed that bacteria in nonsterile larvae and gnotobiotic larvae inoculated with wild-type reduced midgut oxygen levels below 5%, whereas larvae inoculated with mutants defective for cytochrome 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.

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.

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 (Ca2+) flux and cAMP have also been implicated in PTTH signaling, but the role of Ca2+ in OEH, ILP3, and cAMP signaling in ovaries is unknown. Here, we assessed whether Ca2+ flux affects OEH, ILP3, and cAMP activity in A. aegypti ovaries and also asked whether PTTH stimulated ovaries to produce ECD. Results indicated that Ca2+ flux enhanced but was not essential for OEH or ILP3 activity, whereas cAMP signaling was dependent on Ca2+ 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.

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