In mosquitoes, the conversion of protein from a blood meal into yolk proteins and lipids for the developing oocytes is an essential part of the reproductive cycle. Blood feeding by a female Aedes aegypti mosquito initiates a series of events in the midgut, the fat body and the ovaries. Female mosquitoes ingest more than their own weight in blood in a short time and then spend the next 36 hours converting the amino acids from the blood proteins into the constituents of their eggs, a process termed vitellogenesis. The process begins in the midgut with the digestions of blood meal proteins and the regulation of digestive enzyme synthesis in the midgut opcurs in two phases. The early phase begins immediately after ingestion of the meal and involves activation of translation. The synthesis of early trypsin serves as.a model for this phase. The late phase begins 6-8 hours after the meal and involves activation of transcription. The synthesis of late trypsin serves as a model for this phase. In this proposal we seek funds to continue work on the mechanism of regulation of digestive enzyme synthesis in the female mosquito midgut following a blood meal. We propose the following two specific aims: . 1. Investigate the mechanism of activation of translation of the early trypsin mRNA following a protein meal. During the first 30 minutes following a blood meal, a peptide or small molecule is present in whole body extracts, which makes an in vitro midgut preparation competent for early trypsin translation. After 30 minutes, a phosphorylation cascade is initiated in the midgut which leads to activation of translation of early trypsin. We propose to isolate and characterize the factor that induces translational competency in the midgut, and to characterize the signaling processes that lead to activation of early trypsin translation. We also propose to investigate the role of the endoplasmic reticulum whorls in regulating secretory protein synthesis in the midgut. 2. Investigate the mechanism of transcriptional regulation of the late trypsin gene. About 6 hours after feeding, late trypsin transcription in the midgut is initiated. We have recently found that ecdysone, and an uncharacterized factor present in extracts prepared from fat bodies of fed mosquitoes, are involved in late trypsin gene induction. We propose to characterize the mechanism of ecdysone regulation of late trypsin expression, and to identify the fat body factor and the role of phosphoinositide 3-kinase (PI-3K) signaling in modulating late trypsin transcription.
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