Insulin‐mediated secretion of ecdysteroids from mosquito ovaries and molecular cloning of the insulin receptor homologue from ovaries of bloodfed Aedes aegypti

Abstract

The reproductive cycle of female mosquitoes is activated by ingestion of blood from vertebrate hosts. Shortly after feeding, neurohormones are released from the brain neurosecretory system and stimulate the ovaries to secrete ecdysteroids, which are necessary for vitellogenesis by the fat body. Because bombyxins, which are insulin-like peptides, stimulate ecdysteriodgenesis in silkworm larvae, we tested porcine insulin and found that it activates ecdysteroidogenesis and protein synthesis in ovaries isolated from unfed mosquitoes. To further characterize the regulation of ecdysteroidogenesis in female mosquitoes, we cloned the mosquito insulin receptor (MIR) homologue from ovarian mRNA. The sequence of the extracellular domain shows moderate homologies with vertebrate and Drosophila insulin receptor homologues, as well as with the insulin receptor-related receptor, the latter being an "orphan" receptor with an unknown function. In the intracellular domain, high homologies are observed, particularly in those subdomains that are responsible for ATP binding and kinase activity. Northern blot analysis of MIR demonstrated a highly specific expression in ovaries, and cloning experiments indicated its presence in the brain. Recombinant MIR extracts from a baculovirus expression system contained high constitutive kinase activity in the presence of manganese or magnesium. Activation was independent of a ligand. SDS-gel analysis suggested that the recombinant receptor was not post-translationally processed into an alpha- and beta-subunit as was expected from a putative cleavage signal. Enzymatic properties of the proreceptor are presented: the Km for ATP was between 15 and 50 microM in the presence of a synthetic substrate: maximal kinase activity to 100-fold over basic activity was reached in the presence of 1 mM manganese. Stimulation of key oogenic processes by porcine insulin and identification of a MIR indicate that insulin-like neurohormones may have an important regulatory role in mosquito oogenesis.

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti

“…A. aegypti also encodes eight predicted ILP family members of which three (ILP1, ILP3 and ILP8) are specifically expressed in brains of adult females (15). Combined with the finding that ovaries express IR [mosquito IR (MIR)] and AKT (16)(17)(18) homologs, these results suggest an endogenous ILP of brain origin stimulates egg maturation by activating the insulin signaling pathway in the ovaries. Here, we report the identification of ILP3 as a regulator of these processes.…”

“…Although bovine insulin stimulates ecdysteroid production by A. aegypti ovaries, the high concentration range required (14,16) and its inability to stimulate yolk deposition by oocytes at any concentration (M.R.B. and M.R.S., unpublished observations) suggested mammalian insulins are weak agonists for the endogenous ILPs regulating these processes.…”

“…IRs and IGFRs, the MIR proreceptor is processed into an extracellular ␣-subunit that includes the predicted ligand binding domain and a ␤-subunit that contains a transmembrane region linked to a characteristic tyrosine kinase domain (16,17). The mature MIR then forms a 400-to 500-kDa homodimer comprised of two ␣-and ␤-subunits that in ovaries localizes primarily to follicle cells (17).…”

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

“…In immunocytochemical assays, α-727 labeled the follicular epithelium that surrounds primary and secondary follicles ( Fig. 3 B and C), which is where the IR is also expressed in the mosquito ovary (25,26). Higher magnification images suggested labeling of the follicular epithelium by α-727 was both apical and basal (Fig.…”

“…Cell extracts were then immunoblotted and probed with α-49 or a previously generated OEH antibody (α-OEH) (25). α-OEH detected a band of identical mass to dimeric AAEL001915 recognized by α-49 in transformed cells incubated with OEH (Fig.…”