UC Riverside

Ecdysis triggering hormone (ETH) is a peptide hormone that regulates the behavioral sequence necessary for shedding of exocuticle at the end of each developmental stage, a process called ecdysis. ETH is secreted by Inka cells of the epitracheal glands of insects. Drosophila larvae have seven pairs of Inka cells. ETH acts on its receptors (ETHRs), present in the central nervous system (CNS) to regulate the behavior. Ecdysis is a stage specific behavior that does not persist in adults. Interestingly, Inka cells producing ETH are present in the adult stage of Drosophila, suggesting a possible role for ETH signaling in adults. Recently, ETHR transcripts were found to be expressed in corpora allata of fourth and fifth instar Bombyx larvae. Molting hormone, 20-hydroxy-ecdysone (20HE) and juvenile hormone (JH), through their morphogenetic action are key regulators of insect molting. In adults, they play roles in courtship and reproduction.

In this study, I determined the expression of ETH in Inka cells in the adult stage. We investigated the role of ETHRs in adult Drosophila melanogaster behavior by performing RNAi, using Aug-21-Gal-4. Aug-21-Gal-4 is reputed to be a corpora allata (CA) specific driver in larval stage. It also drives expression in larval salivary glands and gut. Expression of Aug-21-Gal-4 in adult stage was determined by driving GFP under Aug-21-Gal-4 and immunostaining using antibody against GFP. In adults, apart from CA, salivary glands and gut, Aug-21-Gal-4 drives expression in neurons of brain and thoracic neuromere. Two pairs of neurons in the brain were identified to be eclosion hormone (EH) neurons and giant fiber neurons. Silencing of ETHRs using Aug-21-Gal-4 results in elevated male-male courtship. Silencing of ETHRs using EH-Gal-4 or A307-Gal-4 does not elevate male-male courtship. Elevated male-male courtship was observed after driving ETHR-RNAi using a pan-neuronal elav-Gal-4 and fruitless neurons specific fru-Gal-4 drivers. This indicates that ETHRs in fru neurons play a role in male courtship behavior. The fruitless mutation is known to affect courtship song regulating genes. ETHR silencing does not affect female behavior. This can be explained by absence of ETHR transcripts in adult females.

Transcriptome analysis of CA, heads and whole flies was done to check the differentially expressed genes downstream of ETHR-RNAi. Genes involved in male courtship behavior, axon guidance, transcription factors and courtship song are discussed in detail. Chromatin organization genes were downregulated in heads after ETHR-RNAi. Clustering of differentially expressed genes was observed on chromosomes, indicating the role of ETHRs in chromatin organization. Interestingly, male accessory gland specific genes were found to be expressed in male corpora allata tissue. JH expression is known to affect male accessory gland proteins in accessory glands. Presence of male accessory gland specific genes in corpora allata shows direct relation of JH in regulation of male accessory gland proteins. Male courtship related genes differentially expressed downstream of ETHR-RNAi include dsx(doublesex), one of the sex determination genes. Comparison of differentially expressed genes from three tissues indicates tissue specific regulation of genes in each tissue. Overall, this is the first study to show a role for ETHRs in adult Drosophila melanogaster.