UC Riverside

Mosquitoes are the world’s deadliest animals due to the link between blood feeding and disease transmission. Amino acids (AA) from a blood meal are required for initiation of egg development, a process that is central to reproductive cycles. Reproduction is governed by alternating peaks of two important insect hormones: Juvenile Hormone III (JH) and 20-hydroxyecdysone (20E). The vitellogenic, 20E-controlled phase of a reproductive cycle occurs after a blood meal and is characterized by the massive production and uptake of yolk protein precursors (YPPs). Microarray studies of the female fat body, a site of YPP synthesis, have revealed that nearly half of the genes in the Aedes aegypti genome are differentially regulated in discrete waves during the vitellogenic phase of a reproductive cycle. We have determined the regulatory factors responsible for each wave of up-or down-regulation by a combination of RNA interference (RNAi) and in vitro fat body culture (IVFBC) techniques. This study contributes to our understanding of temporal regulation of transcription during reproduction and uncovers a unique role of Ecdysone receptor (EcR) as a transcriptional repressor. Transcriptional activation of the main YPP gene, vitellogenin, is directed by the 20E/EcR regulatory pathway and has been methodically worked out by molecular methods. However, transcriptional repression by the EcR regulatory pathway and the mechanisms by which it occurs remain a mystery. We examined the role of components of the 20E/EcR signaling cascade in transcriptional repression of a group of genes, as well as epistatic relationships of implicated pathway components using RNAi, IVFBC and cycloheximide (CHX) assays. We also addressed the involvement of co-repressor and promoter elements in 20E/EcR transcriptional repression using RNAi techniques, luciferase transfection assays, bioinformatics analysis and electrophoretic mobility shift assays (EMSA). The results of this study illuminate the complexity of gene regulation in the female mosquito during reproductive events. A detailed understanding of the nuances of gene expression during mosquito reproductive cycles is essential for development of novel strategies to control mosquito-borne disease.