Ecdysterone and juvenile hormone (JH) are the primary endocrine regulators of many aspects of reproduction, development, and physiology in insects. The mechanism of action of JH is much more poorly understood than that of ecdysterone, due primarily to our dismal knowledge of the JH receptor and JH-regulated genes. The Methoprene-tolerant (Met) and recently identified germ-cell expressed (gce) bHLH-PAS genes of Drosophila melanogaster are involved in the action of JH and will serve as the key to our long-term objective of understanding the molecular action of this hormone in both D. melanogaster and mosquitoes. The proposed work is a genetic and biochemical approach that will accomplish three specific aims: First, GCE from the well-studied disease vector mosquito Aedes aegypti and from D. melanogaster will be isolated and tested for high affinity JH binding. Mosquitoes have no JH-binding MET homolog as do higher Diptera, and the hormone-binding capacity of GCE must be established to understand the receptor. Second, high-level methoprene resistance found in the salt-marsh mosquito Ochlerotatus nigromaculis in California is hypothesized to be due to mutant gce and provides an opportunity to identify the resistance gene in mosquitoes. Genomic gce from both susceptible and resistant populations will be sequenced and compared to determine gene differences that could define the resistance seen. JH binding by in vitro synthesized GCE will be compared between resistant and susceptible populations to determine if poor JH binding by resistant Oc. nigromaculis can explain the resistance. Third, JH target genes need to be firmly and extensively identified. Using D. melanogaster polytene chromosomes, we will carry out an 'umbrella' search for gene targets of MET and GCE by in situ antibody identification of these targets, which will permit immunohistochemical identification of target gene regions from which the genes can be identified using our prior microarray analysis. This methodology will also permit identification of those genes regulated by both/either MET and/or GCE in both the presence and absence of JH.
This work will have significance for understanding components of the juvenile hormone receptor in mosquitoes and genetically amenable Drosophila melanogaster. It will lead to a better understanding of the action and insect resistance to methoprene, which is widely used to control mosquito vectors of disease. Knowledge of the methoprene-resistance gene in mosquitoes will facilitate population monitoring for resistance. Finally, a better understanding of gce and Met as bHLH-PAS genes will further our knowledge of this family of transcriptional regulators, which includes homologous genes in humans.
|Baumann, Aaron; Barry, Joshua; Wang, Shaoli et al. (2010) Paralogous genes involved in juvenile hormone action in Drosophila melanogaster. Genetics 185:1327-36|