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Abstract
The synthesis of ecdysone in insects is fundamental to their development and reproduction. Understanding how the production of this hormone occurs is vital to understanding these processes in insects, and the quantification of this hormone is a key component of this. With the dominant older method of routine ecdysteroid quantification becoming unavailable, a new, refined enzyme-linked immunoassay is developed in parallel with superior extraction methods for insect steroid hormones. Activation of ecdysteroid synthesis occurs in mosquito ovaries following a blood meal, mediated primarily by the binding of the neuropeptides ovary ecdysteroidogenic hormone (OEH) and insulin-like peptide 3 (ILP3) to their respective receptors in follicle cell membranes of the ovary. Both neuropeptides activate ecdysteroidogenesis through the protein kinase Akt, but the relationship of calcium to this activation is unknown. Given that calcium is an important component of ecdysteroid production in the prothoracic glands, it was predicted to have import for ovarian ecdysteroidogenesis. This work demonstrates a regulatory role of calcium in ecdysteroidogenesis. Another important aspect of ecdysone production by the ovaries is that of the cholesterol precursor for steroid synthesis. Since cholesterol is stored in esterified form, it is necessary to mobilize it for ecdysteroid production. The mobilization of cholesterol is relatively unexplored in insect hormone production, which is surprising, given the inability of insects to produce cholesterol de novo. The work here shows that the mosquito ovary, and specifically the follicle cells of the ovary, is proportionally richer in cholesterol than most of the other tissues in the mosquito. The mobilization of cholesterol was confirmed in that the same signals that activate ecdysteroidogenesis in the ovaries increase the amount of free cholesterol in isolated ovaries. This indicates that the source of cholesterol for such synthesis is contained within the ovaries. Additionally, a stringent requirement for follicle cell plasma membrane cholesterol was observed in cholesterol depletion and in vitro activation experiments. This suggests the role of increased free cholesterol in the follicle cell membranes is related to membrane receptor function.