Functional characterization and expression of molluscan detoxification enzymes and transporters involved in dietary allelochemical resistance

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution June 2008
Understanding how organisms deal with potentially toxic or fitness-reducing allelochemicals is important for understanding patterns of predation and herbivory in the marine environment. The ability of marine consumers to tolerate dietary toxins may involve biochemical resistance mechanisms, which increase the hydrophilicity of compounds and facilitate their active efflux out of sensitive cells and tissues. While several allelochemical-responsive detoxification enzymes have been sequenced and functionally characterized in terrestrial invertebrates feeding on chemically defended host plants, there is virtually no information concerning the role of these biotransformation enzymes that may mediate feeding tolerance in marine invertebrates. The objective of this research was to assess the diversity and dietary regulation of cytochrome P450s (CYP), glutathione S-transferases (GST) and ABC transporters in the generalist marine gastropod Cyphoma gibbosum feeding on a variety of chemically defended gorgonian corals, and to identify those dietary natural products that act as substrates for these proteins. Molecular and proteomic techniques identified both allelochemically-responsive CYPs, and constitutively expressed GSTs and transporters in Cyphoma digestive glands. Inhibition of Cyphoma GST activity by gorgonian extracts and selected allelochemicals (i.e., prostaglandins) indicated that gorgonian diets are likely to contain substrates for molluscan detoxification enzymes. In vitro metabolism studies with recombinant CYPs suggested those Cyphoma enzymes most closely related to vertebrate fatty acid hydroxylating enzymes may contribute to the detoxification of ichthyodeterrent cyclopentenone prostaglandins found in abundance in selected gorgonian species. Finally, the presence and activity of multixenobiotic resistance transporters in Cyphoma and the co-occuring specialist nudibranch, Tritonia hamnerorum, suggests these efflux transporters could function as a first line of de
Financial support for this research was provided by the following organizations and individuals: National Science Foundation Graduate Research Fellowship, Tropical Research Initiative Grant – Ocean Life Institute (WHOI), Ocean Ventures Fund (WHOI), National Undersea Research Center – Caribbean Marine Research Center, Program Development Proposal (CMRC-03-PRMN-01-03A), Seaspace Student Scholarship, Conchologists of America Student Award, NOAA National Sea Grant College Program (Grant No. NA16RG2273), Woods Hole Oceanographic Institution - Sea Grant (Project No. R/P-66), A grant from Walter A. and Hope Noyes Smith supported proteomic work at the University of California, Davis – Proteomics Center, Academic Programs Office, Woods Hole Oceanographic Institution