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Development and validation of PCR-RFLP assay for identification of Gambierdiscus species in the Greater Caribbean Region

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Abstract

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.

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Acknowledgements

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.

Funding

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.

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Authors and Affiliations

  1. Marine Science Institute, University of Texas at Austin, Port Aransas, TX, USA

    Yesid Lozano-Duque & Deana L. Erdner

  2. Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA

    Mindy L. Richlen & Donald M. Anderson

  3. Center for Marine and Environmental Studies, University of the Virgin Islands, St. Thomas, VI, USA

    Tyler B. Smith

Authors
  1. Yesid Lozano-Duque
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  2. Mindy L. Richlen
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  3. Tyler B. Smith
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  4. Donald M. Anderson
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  5. Deana L. Erdner
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Correspondence to Yesid Lozano-Duque.

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Lozano-Duque, Y., Richlen, M.L., Smith, T.B. et al. Development and validation of PCR-RFLP assay for identification of Gambierdiscus species in the Greater Caribbean Region. J Appl Phycol 30, 3529–3540 (2018). https://doi.org/10.1007/s10811-018-1491-5

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  • DOI: https://doi.org/10.1007/s10811-018-1491-5

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