Molecular Analysis of Juvenile Hormone Action

There has been a continuous demand for the development of insect control methods that are target-specific. Juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E is the most active form) are the major hormones that regulate development and reproduction in insects. Since these hormones are not present in vertebrates, they represent attractive targets for the development of insect control methods. Hindering this effort is the lack of understanding of the molecular basis of JH action. During the current grant period, seven proteins that are components of multi-protein complexes involved in JH signal transduction and its cross-talk with 20E were identified. Ligand binding studies performed using labeled JH showed that none of these proteins bind to JH with high affinity. While these proteins are likely involved in JH action and its cross-talk with 20E, none of them are JH receptors. Search for a better system to study JH action without interference from ecdysteroids discovered JH regulation of reproduction in Tribo/ium castaneum, an ideal system to study JH action without interference from ecdysteroids. In this insect, JH without the interference from ecdysteroids regulates vitellogenin gene expression in the fat body and vitellogenin uptake by the oocytes. These two systems provide a unique opportunity to study both genomic (fat body) and non-genomic (ovary) actions of JH in the same insect at the same time. Therefore, the renewal application proposes to study mechanism of JH action in regulation of vitellogenin gene expression in the fat body and vitellogenin uptake into the oocytes with a major goal of identifying receptors, transcription factors and protein modifiers critical for JH signal transduction. Completion of whole genome sequencing, availability of microarrays, functioning of systemic RNA interference (RNAi) and excellent response to JH and its analogs make T. castaneum an excellent model system for studies on JH action. In the first specific aim, microarray, RNAi, qRT-PCR, electrophoretic mobility shift assays and ligand-binding assays will be employed to identify and characterize receptors, transcription factors and protein modifiers involved in JH regulation of vitellogenin gene expression. In the second specific aim, a panel of in vivo, cell culture and in vitro assays will be used to determine whether any of the 51 Gprotein- coupled receptors indentified in T. castaneum as those required for egg production and embryogenesis are activated by JH to regulate vitellogenin uptake into the oocytes. JH analogs are being used for mosquito control, but some mosquitoes are already developing resistance to these analogs. The results from these studies will aid in understanding the mode of action of JH analogs, thus facilitating their judicious use. The genes identified will be useful to develop screening assays to identify new insect disease vector control agents. The receptors identified can also be used in medicine by developing gene switches for various applications such as gene therapy, tissue engineering and therapeutic protein production.