Identification and Characterization of Genes Implicated in Saxitoxin Biosynthesis in Dinoflagellates

Woods Hole Oceanographic Institution, Woods Hole MA

Investigators

Abstract

"Blooms" of toxic dinoflagellates from several different genera result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global marine phenomena collectively termed harmful algal blooms (HABs). The economic, public health, and ecosystem impacts of PSP outbreaks take a variety of forms, and include human intoxications and death from contaminated shellfish or fish, alterations of marine trophic structure, and death of marine mammals, fish, and seabirds. These impacts are caused by saxitoxins (STXs), a family of neurotoxins produced by some dinoflagellates (and cyanobacteria) that are accumulated in zooplankton, shellfish, or fish during feeding. The chemical structure and activity of the saxitoxins have been well characterized, but their metabolic role within the dinoflagellate remains unknown. Likewise, the factors that cause variability in toxicity between isolates or in a single isolate under different growth conditions are poorly understood. These issues are best addressed through the study of saxitoxin production at a molecular or genetic level, but that approach faces several major hurdles, in particular a lack of mutant strains that are identical to toxic forms except for their ability to produce toxins. This project will identify and characterize genes associated with STX production in dinoflagellates, continuing directly from significant results obtained in previous studies. In the initial project a powerful technique called Representational Difference Analysis (RDA), a method for finding differences between complex genomes that was developed in the search for genetic abnormalities in human cancer cells, was used to generate two gene fragments (called RDA1 and RDA2) that are expressed in a toxic Alexandrium isolate but not in a non-toxic strain. One of these fragments was screened against mRNA from 15 cultures and correctly hybridized to the only six organisms that produce STXs, even though these represent five different species from two dinoflagellate genera, as well as a freshwater cyanobacterium. This strongly suggests that RDA1 is associated with saxitoxin production or regulation. In the present project this exciting and productive line of research, the ultimate objectives of which are to identify and characterize the gene(s)involved in saxitoxin biosynthesis in dinoflagellates and to study their regulation under changing environmental conditions, will be continued. Specific project objectives are to: 1) utilize rapid amplification of cDNA ends (RACE) and genomic cloning to obtain full sequences of the putative toxin-specific gene fragments, RDA1 and RDA2; 2) identify and characterize the genes encoded by RDA1 and RDA2; 3) conduct inter-and intraspecific screening of toxic and non-toxic dinoflagellates with the RDA1 and RDA2 genes from Alexandrium; 4) conduct physiological experiments to study the genetic regulation of RDA1 and RDA2; 5) perform additional Representational Difference Analysis on toxic and non-toxic Gymnodinium catenatum and Alexandrium isolates, if necessary; and 6)develop and test molecular probes which can distinguish toxic from non-toxic dinoflagellates. The methodological groundwork has been established to allow this project to proceed with a high probability of success. It is expected that the project will isolate and identify genes required for STX production, but any marker that can differentiate toxic from non-toxic strains is of great scientific and practical value. With either result, probes can be designed that unequivocally identify only toxic cells in a complex natural assemblage of plankton, and these are of use in academic field research as well as in commercial and governmental shellfish toxicity monitoring programs.