Meeting Abstract
P3.39 Saturday, Jan. 5 Double-Stranded RNA Interference Lowers Expression of Molt-inhibiting Hormone in the Eyestalk of the Land Crab Gecarcinus lateralis EHLINGER, Casey C.*; COVI, Joseph A.; CHANG, Ernest S.; MYKLES, Donald L.; Colorado State University, Fort Collins; Colorado State University, Fort Collins; University of California, Davis Bodega Marine Lab; Colorado State University, Fort Collins caseyce@simla.colostate.edu
Molt-inhibiting hormone (MIH) found in the black-backed land crab, Gecarcinus lateralis, aids in the regulation of the crabs� molting. The hormone is produced in the X organ in the eyestalk ganglia (ESG) of the crab and inhibits the secretion of ecdysteriods from the Y organ located in the abdominal cavity. Two small regions of MIH have been cloned using RT-PCR on cDNA from the ESG of Gecarcinus lateralis. The first sequence is on the 5� end of the cloning region and is 240 bp, while the second sequence spans the 5� end and the MIH coding region and is 502 bp. Each sequence was cloned into the pGem T-Easy Vector in both orientations. The Promega T7 RiboMAX Express RNAi System was used to synthesize dsRNA for MIH. Concentrations of 2 micrograms dsRNA per gram, wet weight for each individual crab, and equivalent amounts of nuclease-free water for the control group were injected into Gecarcinus lateralis. Hemolymph was collected at corresponding time points to determine ecdysteriod levels and analyzed with radioimmunoassays (RIA). The animals were sacrificed after 24 hours and RNA was isolated from the ESGs of each animal. The results from the endpoint PCR done on the ESG RNA showed a partial knock-down of MIH expression in two of three crabs for the dsRNA 5� end and three of three crabs for the dsRNA sequence spanning the MIH coding region relative to MIH abundance in the control animals. The RIA results of the hemolymph analysis showed no changes in ecdysteriod concentrations after injection. Further experiments are planned that include higher concentrations of dsRNA to be injected. Supported by NSF (IBN-0342982).
