Characterization of components of the ecdysteroid biosynthetic pathway in the ovaries of the yellow fever mosquito, Aedes aegypti
Sieglaff, Douglas Harold
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A blood meal induces the ovaries of Aedes aegypti mosquitoes to produce ecdysteroid hormones that regulate oogenesis. The ovaries of blood-fed females begin to produce ecdysteroids 6 h post blood meal (PBM), peak around 18 h PBM, and fall to pre-blood feeding levels by 48 h PBM. Various proteins involved in the intracellular transfer and biosynthesis of ecdysteroids in other insects have been identified and characterized. To begin a study of these processes in mosquito ovaries, complete cDNAs were cloned for putative orthologs of diazepam-binding inhibitor (DBI; AedaeDBI), StAR-related lipid transfer domain containing protein (Start1; AedaeStart1), aldo/keto reductase (A/KR; AedaeA/KR), adrenodoxin reductase (AR; AedaeAR), 22-hydroxylase (CYP302a1; AedaeCYP302a1), 2-hydroxylase (CYP315a1; AedaeCYP315a1) and 20-hydroxylase (CYP314a1; AedaeCYP314a1). As shown by RT-PCR analysis, transcripts for all seven genes were present in ovaries and other tissues both before and PBM. As determined by real-time PCR analysis, gene transcript abundance for AedaeCYP302a1 and AedaeCYP315a1 was significantly greater (9 and 12 fold, respectively) in ovaries 18 h PBM relative to that in ovaries from females not blood fed (NBF) or 2 h PBM. AedaeDBI, AedaeStart1, AedaeA/KR and AedaeAR also displayed higher transcript levels in ovaries at 18h PBM; although, AedaeDBI transcripts were greatest at 48 h PBM. In contrast, gene transcript abundance for AedaeCYP314a1 decreased PBM. Two separate control points of ecdysteroid biosynthesis, enzyme modification and sterol transfer, were addressed by studying expression and localization of AedaeCYP302a1 and AedaeStart1 protein in A. aegypti ovaries of NBF and blood-fed females. AedaeCYP302a1 was present in ovaries and other tissues both before and PBM. AedaeCYP302a1 increased in ovaries of blood-fed females and remained high 48 - 60 h PBM despite a lack of ovarian ecdysteroid production at these times. The levels of AedaeStart1 did not change in ovaries PBM. AedaeCYP302a1 localized predominantly to ovariole follicle cells, whereas AedaeStart1 was observed in both ovariole nurse and follicle cells. The information provided in this dissertation suggests that ovarian ecdysteroidogenesis in A. aegypti is partially regulated at the transcript and translational levels.