Studies On The Physiological Functions And Transcriptional Regulation Of Aedes Aegypti Sterol Carrier Protein-2

Cholesterol is an important component of all animal cellular membranes and is the precursor for steroid hormone biosynthesis. Insects, unlike the vertebrates, are not able to synthesize cholesterol de novo due to the lack of key enzymes in the cholesterol synthesis pathway. Therefore, insects must depend on dietary and/or symbiotic microbes to meet their physiological requirements for cholesterol. Sterol carrier protein-2(SCP-2) is a small intracellular protein involved in cholesterol and lipid intracellular trafficking in insects. To date, most of the studies of SCP-2are performed in vertebrates, but reports on insect SCP-2are a few. Aedes aegypti Sterol carrier protein-2(AeSCP-2), the first studied insect SCP-2, plays an important role in cholesterol uptake, mosquito development and reproduction. Therefore, the important protein like SCP-2involved in the nutrition metabolism can be used as the nutrition target for developing the new pesticides to control the insects and pests.A simple and efficient DNA delivery method to introduce extrachromosomal DNA into mosquito embryos would significantly aid functional genomic studies. In this study, taking advantage of the unique aspects of mosquito reproductive physiology during vitellogenesis and an in vivo transfection reagent that mediate DNA uptake in cells via endocytosis, we have developed a new method to introduce DNA into mosquito embryos vertically via microinjection of DNA vectors in vitellogenic females without directly manipulating the embryos. This method of DNA vector delivery was able to introduce inducible DNA vector transiently into FO mosquitoes, and will allow us to perform inducible over-expression or expression knockdown in mosquito larval and adult stages without transgenic lines. This new method will be a simple and efficient tool for functional genomic studies in vivo in mosquitoes.Using the newly developed method, in vivo functional genomic studies of SCP-2gene in the Aedes aegypti was performed and the results further confirmed the important role of AeSCP-2in the development and reproduction of mosquitoes.To further investigate the mechanism of AeSCP-2gene expression regulation. Based on the newly developed gene delivery method in Aedes aegypti, the AeSCP-2promoter regulatory sequence and potential promoter regulatory proteins were studied in vivo in this study. Results showed that the sequence between-1.6kb to-1.3kb5’-upstream of the transcription start site is the promoter’s spatial/temporal regulatory sequence. The results also indicate that two transcription factors, Thanatos-associated protein (THAP) and activating transcription factor-2(ATF-2) antagonistically control AeSCP-2transcriptional activity via binding to the promoter regulatory sequence. In vivo expression knockdown of THAP and ATF-2resulted in significant changes in mosquito developmental progression, which suggested the potential function of these two transcription factors. THAP could be a new target for vector control. This study will facilitate the potential nutrition target study in insects and more effective pesticides could be developed based on the new targets.