The Role of Juvenile Hormone Receptor Methoprene Tolerant in Reproductive Development of Adult Female Mosquito, Aedes aegypti

Vector mosquitoes require vertebrate blood for their egg maturation, and repeated bloodfeeding cycles result in acquisition and transmission of pathogens which course numerous devastating human diseases. Elucidation of molecular basis of blood meal activated reproductive cycles is essential for developing future mosquito control strategies. The juvenile hormone (JH) controlled post-eclosion (PE) developmental phase is required for female mosquitoes to attain competence for egg development. Time course microarray analysis of Aedes fat body revealed a high transcriptional activity in this tissue during the PE development. The hierarchical clustering identified two major gene clusters: 1843 early (EPE) genes maximally expressed at 6 h PE and 1815 late (LPE) genes at 66 h PE. Functionally, the EPE and LPE clusters are markedly different, with the former genes involved in metabolic pathways and the latter those regulating transcription and translation processes. RNA interference screen of the JH receptor Methoprene-tolerant (MET) resulted in up-regulation of EPE gene expression and down-regulation of LPE genes. Overrepresentation of putative e-box-like MET binding sites was observed in upstream regions of MET-activated (LPE), but not MET-repressed genes (EPE). Electrophoretic mobility shift assays (EMSA), utilizing a combination of mutational and anti-Met antibody super-shift analyses, confirmed binding properties of the MET consensus motif variants in a subset of MET activated genes. We tested an indirect model of gene repression by JH/MET hierarchy. The JH/MET-activated bHLH protein Hairy has been established as an intermediate factor mediating the JH/MET gene repression by means of RNAi/RNA-sequencing. 79% of Hairy-repressed genes showed an overlap with MET-repressed transcriptome. Upstream regions of 20% of these MET/Hairy repressedgenes harbored a Hairy interacting putative cis-regulatory module (hCRM) containingHairy-binding sites. Lastly, Cycle (CYC) was identified as a bHLH-PAS binding partnerof MET. Here, we characterize the MET/CYC heterodimer mediated JH-dependant circadian expression of Krüppel homolog-1 and Hairy. The activation of these genes not only depends on JH but on the ratio of light:dark periods as well. These studies have elucidated the molecular mechanism of gene regulation by JH/MET signaling pathway