A series of molecular and physiological investigations are proposed to investigate the neurohormonal regulation of ecdysteroid hormone secretions a key process of oogenesis, in female Aedes aegypti mosquitoes. Ecdysteroids modulate fat body secretion of yolk proteins, which are selectively stored in the maturing oocytes for embryonic development. Our research has shown that three different peptide hormones, two ovarian ecdysteroidogenic hormones (OEH I and II) and a putative insulin-like peptide, stimulate ovarian ecdysteroidogenesis in mosquitoes. All three hormones have the same function, but they are structurally unrelated. With molecular techniques, an OEH I cDNA has been obtained that encodes a peptide, with a signal sequence, containing sequences of the amino-terminal and enzymatic fragments and processing sites to yield a native molecular weight of 8800 known for OEH I. This sequence is related to that of neuroparsin A, a multi-functional neurohormone in locusts. With an OEH I antiserum, immunocytochemical methods have confirmed that neurosecretory cells, with axons to the corpora cardiaca, in the female brain are the source of this hormone; immunoreactive cells in brains of larvae indicate that OEH I may have an important role throughout mosquito development. OEH II has a different amino acid sequence and a lower molecular weight (6500) than that of OEH I. The OEHs are structurally unrelated to vertebrate insulins, which also stimulate ecdysteroidogenesis in mosquito ovaries. The characterization of an insulin receptor from mosquito ovaries and the stimulation of ecdysteroidogenesis in silkworm larvae by bombyxin, an endogenous insulin-like peptide, support further efforts to identify a mosquito insulin. Only with detailed investigations will the coordinated or synergistic regulation of ovarian ecdysteroidogenesis by the OEHs and insulin-like peptides be elucidated for mosquito females. Most importantly for this research to progress, large amounts of active peptide must be obtained. Therefore, the baculovirus vector-insect cell expression system will be used to produce the necessary amounts of readily-purified active peptide, as has been demonstrated for other insect hormones. The same techniques needed to implement this expression system will be used to begin characterization of the genes encoding these peptides and to delineate hormone expression during development and oogenesis. These objectives will be the foci of our research program on the neuroendocrine regulation of ovarian ecdysteroidogenesis in mosquitoes. Structural and molecular characterization of ecdysteroidogenic hormones will lead to the identification of cell receptors and may aid in the design of new insecticides that are environmentally-stable, readily deliverable, and potent mimics. As well, both the ecdysteroidogenic hormones and their receptors will provide promising targets for genetic manipulation, once it is possible to genetically transform mosquitoes.
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