Occurrence, Origin and Function of Secondary Metabolites in Marine Pulmonate Molluscs, Siphonaria denticulata, Siphonaria virgulata

The occurrence of secondary metabolites in several families of marine pulmonate molluscs was investigated with a focus on the origin and function of the natural products. The members of the family Siphonaridae are reported to contain polypropionate metabolites and this was found to be the case for most of the species considered in this study. Five new polypropionate secondary metabolites were isolated from the Costa Rican species, Siphonaria maura. The maurapyrones A-D and maurenone exhibited mild antimicrobial activity against the marine bacterium Vibrio anguillarum but did not appear to act as a feeding inhibitor toward fish or crabs. Another population of S. maura from Puerto Vallarta, Mexico, was found to contain the vallartanones A and B. These homologues were not found to be antimicrobial but did exhibit activity in several bioassays. Both vallartanones A and B inhibited feeding by the fish Thallasoma lunare but vallartanone B was found to be more potent Vallartanone A induced settling in the larvae of the reef building worm Phragmatopoma californica while vallartanone B did not A small species from the Northeast coast of North America, S. thersites, contained the previously reported polypropionate pectinatone and two new homofuranones which are geometrical isomers. Pectinatone is known to be antimicrobial against the bacterium Bacillus subtilis and against the human pathogen Staphylococcus aureus. It was also found to inhibit cell division in fertilized sea urchin eggs and to be toxic to the larvae of P. californica. The homofuranones were also larvicidal. Four new polypropionates and one known compound, siphonarin A, were isolated from an Australian species, S. baconi. The baconipyrones A-D were not found to be antimicrobial and were not isolated in sufficient quantities for testing in other bioassays. Extracts of a large, high intertidal species from Costa Rica, S. gigas, did not contain polypropionate or other secondary metabolites.A second family of pulmonate limpets, the Trimusculidae, was investigated and two species were found to contain terpenoid metabolites. Trimusculus reticu/atus from the California coast contains 6β-isovaleroxylabda-8,13-dien-7α,l5-diol and 2α,7α-diacetoxy- 6β-isovaleroxylabda-8,13-dien-15-ol in high concentrations in the foot, mantle and mucus. These diterpenes did not exhibit antimicrobial activity and did not inhibit fish feeding. The compounds were found to be larvicidal to Phragmatapoma californica at natural concentrations and the presence of the limpet or its mucus is alsv larvicidal. The New Zealand species, T. conica, produced a closely related diterpene acid and a tetra acetoxy cholesterol derivative. These metabolites were localized in the foot, mantle and mucus of the limpets and conicoic acid was found to be present in large concentrations. The compounds did not appear to be active in any of the bioassays. Three species of the family Onchidacea were investigated for the presence of secondary metabolites. Onchidella borealis, collected in Northern California was found to contain onchidal, a known sesquiterpene acetate. An Australian species, O. patelloides and a New Zealand species, O. nigricans were also found to contain onchidal. Onchidal inhibits the growth of Staphylococcus aureus and appears to bind to acetylcholinesterase.To investigate the origin of polypropionate metabolites in siphonariids, labelling experiments were carried out by injecting 14C labelled acetate or propionate into Siphonaria denticulata and S. virgulata, two species common on the coast of New South Wales, Australia The siphonariid limpets were found to incorporate propionate but not acetate into the purified polypropionate metabolites. Degradation studies demonstrated that the propionate precursor is incorporated without randomization of the label and therefore is not broken down before utilization in the biosynthesis of polypropionate metabolites. This is the first demonstration that polypropionate secondary metabolites are not dietary in origin, but are biosynthesized by the siphonariid pulmonate limpets