GOMEZ, A.M.*; LEE, K.J.; CHANG, E.S.; MYKLES, D.L.; Colorado State University, Fort Collins; Colorado State University, Fort Collins; Bodega Bay Marine Lab, Bodega Bay, CA; Colorado State University, Fort Collins: Establishment of an in vitro Y-Organ Assay to Investigate the Molt Inhibiting Hormone Signaling Pathway

Ecdysis, commonly known as molting, is a process crustaceans must undergo in order to grow and develop. The hormones responsible for this process are subsequently named ecdysteroids, which are synthesized in the Y-organ (YO) located in the cephalothorax. The YO is regulated by molt-inhibiting hormone (MIH), a neuropeptide secreted by the X-organ/sinus gland complex in the eyestalk that inhibits ecdysteroid synthesis. So far details of the MIH signaling pathway within the YO tissue have yet to be elucidated. Recently, research has proposed a pathway indicating possible components involved in this mechanism. One component thought to be involved is Nitric Oxide, which is formed by the action of Nitric Oxide Synthase (NOS). NOS has been detected in YO tissue by immunocytochemistry, although its involvement in the MIH pathway has not been confirmed. In order to investigate NO involvement, the conditions for culturing YOs were established. Tropical land crabs, Gecarcinus lateralis, had their eyestalks ablated 2 days prior to removal of the YOs, removing the source of MIH and thus stimulating ecdysteroid synthesis. Initially YOs were extirpated from the animal, and experiments were performed to determine a time course of secretion in vitro for 12 hours in culture media with samples taken every 2-4 hours, for determination of ecdysteroid by radioimmunoassay. The next part of the investigation will involve examining the effects of NOS inhibitors and activators on ecdysteroid secretion. Supported by NSF (IBN-0342982).