| Ecdysis triggering hormone (ETH) released from endocrine Inka cells acts on the central nervous system (CNS) to initiate stereotypic ecdysis behaviors. In order to elucidate molecular and cellular mechanisms underlying ecdysis behaviors, I identified a gene encoding ETH receptors (ETHR) in the moth Manduca sexta. Similar to the fly Drosophila melanogaster , alternative mRNA splicing produces two different receptor subtypes (MasETHR-A, -B) that are highly sensitive and selective for ETHs. In situ hybridization revealed each subtype is expressed in a separate set of central neurons. In particular, MasETHR-A receptors are expressed by multiple classes of peptidergic neurons producing eclosion hormone (EH), diuretic hormones, kinins, statins, myoinhibitory peptides, small neuropeptides F, FMRFamides or the second messenger cyclic guanosine monophosphate.; Similar to M. sexta, the Drosophila ETHR-A is expressed in multiple classes of peptidergic neurons producing kinins, FMRFamide, EH and crustacean cardioactive peptide (CCAP). Using optical imaging techniques, we monitored intracellular Ca2+ dynamics of major DrmETHR-A neurons following ETH exposure. We discovered that FMRFamide neurons (Tv1-3) become active early during pre-ecdysis, while EH and subsets of CCAP neurons are activated later at the onset of ecdysis and post-ecdysis. Behavioral roles for ETHR neurons were confirmed in transgenic flies carrying targeted ablations of neurons expressing FMRFamide, EH or CCAP.; Finally, we identified the brain neuropeptide, corazonin as a releasing factor responsible for secretion of ETH and PETH prior to and during pre-ecdysis in M. sexta. In vivo and in vitro experiments showed that corazonin elicits release of ETH peptides from Inka cells. Subsequently, I identified the corazonin receptor from M. sexta (MasCRZR) and located it in Inka cells, the source of ETH peptides. Using MasCRZR as a biosensor, I showed that corazonin circulates in the hemolymph at 20 min prior to pre-ecdysis, when initial release of ETH peptides takes place. These results suggest that corazonin elicits an initial low level release of ETH and PETH appropriate for pre-ecdysis, while EH causes the subsequent massive release of these peptides necessary for the transition to ecdysis.; In summary, our data in the moth and fly demonstrate that direct interactions between peripheral Inka cells and multiple peptidergic networks in the CNS regulate consecutive phases of the ecdysis behavioral sequence. |
