Development and Validation of PCR-RFLP Assay for Identification of Gambierdiscus species in the Greater Caribbean region.

Abstract

The genus Gambierdiscus is a recognized group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, thirteen species and six ribotypes of Gambierdiscus have been identified, and multiple species commonly co-occur within a single site or epiphyte community. Toxicity can vary by species, thus it is important to be able to differentiate among species for research and monitoring purposes. Gambierdiscus species have very similar morphological characteristics and are difficult or impossible to distinguish using light microscopy. DNA sequencing has been an important tool in the definition of Gambierdiscus species, but it can be time-consuming and relatively expensive. To provide an alternative approach, a PCR-RFLP protocol was developed for efficient, rapid, and cost-effective identification of Gambierdiscus strains isolated from the Gulf of Mexico and Caribbean Sea, where CFP cases and Gambierdiscus spp. have been reported. The assay targets the D1-D2 hypervariable regions of the large subunit ribosomal RNA gene and uses a single restriction enzyme, BsrI. This method produces distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea, and also distinguishes them from four Pacific endemic species. This method was successfully used to type 465 clonal isolates of Gambierdiscus from the U.S. Virgin Islands and Akumal Beach - Mexico This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.