Development and validation of PCR-RFLP assay for identification of Gambierdiscus species in the Greater Caribbean Region
- 133 Downloads
The genus Gambierdiscus is a group of marine epiphytic-benthic dinoflagellates that produce the toxins that cause ciguatera fish poisoning (CFP). To date, 15 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. Species comprising the Gambierdiscus genus 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 expensive. To provide an alternative approach, a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol was developed for efficient and accurate identification of Gambierdiscus strains isolated from the Caribbean Sea. The assay targeted the D1–D2 hypervariable regions of the large subunit ribosomal RNA (LSU rDNA) gene and used a single restriction enzyme, BsrI. This method produced distinct RFLP banding patterns for the six species of Gambierdiscus reported from the Gulf of Mexico and Caribbean Sea and also distinguished them from four Pacific endemic species. This method was successfully used to type 472 clonal isolates of Gambierdiscus from the US Virgin Islands and east coast of Mexico. This BsrI PCR-RFLP method expands the tools available to researchers and managers engaged in monitoring activities and ecological studies.
KeywordsHarmful algae Benthic dinoflagellate Bsr1 restriction enzyme D1–D2 LSU Species identification
We are grateful for the assistance of Dr. Kenneth Dunton in facilitating the collection of Akumal Bay samples. This is contribution # 191 from the Center for Marine and Environmental Studies at the University of the Virgin Islands.
This study was supported by the U.S. National Oceanic and Atmospheric Administration Center for Sponsored Coastal Ocean Research ECOHAB program under award nos. NA11NOS4780061 (D.L.E.), NA11NOS4780062 (T.B.S.), and NA11NOS4780060 (D.M.A. and M.L.R.). This is publication #ECOHAB919.
- Caillaud A, De la Iglesia P, Darius HT, Pauillac S, Aligizaki K, Fraga S, Chinain M, Diogene G (2010) Update on methodologies available for ciguatoxin determination: perspectives to confront the onset of ciguatera fish poisoning in Europe. Mar Drugs 8:1838–1907PubMedPubMedCentralCrossRefGoogle Scholar
- Fleming LE, Baden DG, Bean JA, Weisman R, Blythe DG (1998) Seafood toxin diseases: issues in epidemiology and community outreach. In: Reguera B, Blanco J, Fernandez MK, Wyatt T (eds) Harmful algae. Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Paris, pp 245–248Google Scholar
- Halstead BW (1978) Poisonous and venomous marine animals of the world. Darwin Press, PrincetonGoogle Scholar
- Nishimura T, Sato S, Tawong W, Sakanari H, Uehara K, Shah MMR, Suda S, Yasumoto T, Taira Y, Yamaguchi H, Adachi M (2013) Genetic diversity and distribution of the ciguatera-causing dinoflagellate Gambierdiscus spp. (Dinophyceae) in coastal areas of Japan. PLoS One 8:e60882PubMedPubMedCentralCrossRefGoogle Scholar