Meeting Abstract
P2.17 Tuesday, Jan. 5 Characterization of the ecdysteroid receptor in the American lobster (Homarus americanus) and development of an in vitro screening assay TARRANT, AM*; BEHRENDT, L; STEGEMAN, JJ; VERSLYCKE, T; Woods Hole Oceanographic Inst.; WHOI; WHOI; WHOI, Gradient Corp atarrant@whoi.edu
The ecydsteroid receptor (EcR) and retinoid X receptor (RXR) together mediate ecdysteroid regulation of molting, development and reproduction in ecdysozoans. Insect growth regulators are chemicals designed to disrupt ecdysteroid signaling in insect pests, but effects of such chemicals on ecdysteroid signaling in non-target species, especially crustaceans, have not been determined. Coastal crustaceans are increasingly exposed to potential ecdysteroid-disrupting chemicals due to growing use of insect growth regulators in many applications over the last decade, as coastal human populations continue to expand. There is an urgent need to better understand the potential for exposure and effects of these chemicals in coastal crustacean populations. To this end, we have cloned the full length EcR and RXR from an ecologically and economically important crustacean, the American lobster (Homarus americanus). We identified two forms of the EcR that differ in 15 of 542 amino acid residues. qPCR showed that EcR expression was elevated in the muscle of lobsters exhibiting symptoms of epizootic shell disease. We also identified two splice variants of the RXR, which differed by a 15 base pair insert. We expressed these proteins in vitro and characterized their binding to tritiated ponasterone. Both forms of EcR bound ponasterone with high affinity, and binding was enhanced in the presence of RXR. Competitive binding assays were used to measure the relative affinity of a suite of chemicals for the EcR/RXR including steroids, pesticides, phthalates and alkylphenols. This assay is being further developed to screen environmental extracts for their capacity to disrupt ecdysteroid signaling in crustaceans. (Supported by MIT Sea Grant and Rhode Island Sea Grant).
