CHARACTERIZATION OF SEXUAL PHEROMONES IN APLYSIA

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The experiments outlined in this proposal will isolate and chemically characterize the pheromonal factors that attract the marine mollusc Aplysia to egg-laying conspecifics and induce them to mate. The factors will be purified from acidic extracts of the atrial gland, an exocrine organ secreting into the oviduct, and from seawater eluates of recently deposited egg cordons; pheromonal activity will be assessed in T-maze and mating experiments. The active factors will be characterized by compositional and microsequence or FAB/MS analyses, and the sequences synthesized for use in HPLC coelution studies (to verify the native sequence) and in bioassay studies (to confirm pheromonal activity). The synthetic peptide will also be used to generate rabbit antisera, which will provide an immunocytochemical approach to demonstrating release from the atrial gland onto the egg cordon; antibody specificity will be characterized by immunodot assay, immunocytochemistry and preabsorption studies. If this approach is unsuccessful, we will use a combination of pharmacological and biochemical techniques to determine whether there are alternative tissue sources of the characterized peptide; if none are detected, the peptide is likely to have originated in the atrial gland. This conclusion will be further tested by examining peptide release in vitro (i.e., by analyzing perfusates from an isolated atrial gland/large hermaphroditic duct preparation before and after stimulation with high K artificial seawater, and before and after exposure to bag cell products). These studies will demonstrate whether the atrial gland is a physiological source of sexual pheromones, and whether the pheromonal factors are products of the ELH-related genes expressed in the atrial gland. If they are, these experiments will provide an initial characterization of one facet of a potentially interesting model system in which products of different subsets of a family of genes regulate different subsets of behavior to produce a coordinated function -- products of the bag-cell ELH gene being secreted into the hemocoel to induce egg laying, and products of the atrial gland ELH-related genes being deposited onto the egg cordon to act as pheromones that attract other Aplysia to the egg layer and induce mating. The studies will also contribute to our understanding of pheromones and pheromonal function in an aquatic environment, by identifying the first peptide pheromone(s) in molluscs.