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.

Metabolic relationships of uncultured bacteria associated with the microalgae Gambierdiscus

“…Cultures were maintained in this way until DNA extraction and sequencing in 2016. Species were identified by a combination of morphology (light microscopy) and sequencing of the D1–D2 region of the large subunit rDNA gene (for GHCG2‐C6, Cheeca1 and Tenn11; Lyu et al ., ) or PCR‐RFLP (GTBNAC2; Lozano‐Duque et al ., ).…”

Effect of Salinity on DMSP Production in Gambierdiscus belizeanus (Dinophyceae)

“…Established cultures of seven strains were kindly donated by Dr. Deana Erdner (University of Texas, Marine Sciences Institute) and identified by polymerase chain reaction‐restriction fragment length polymorphism analysis (Lozano‐Duque et al. ). Strains were isolated by Yesid Lozano‐Duque and Dr. Ingrid (University of Texas, Marine Sciences Institute) from field samples of epiphytes harvested from Dictyota sp., collected by Dr. Tyler Smith (University of the Virgin Islands) at field sites off the coast of St. Thomas, U.S. Virgin Islands.…”

Comparison of Spatial and Temporal Genetic Differentiation in a Harmful Dinoflagellate Species Emphasizes Impact of Local Processes

“…Macroalgal epiphyte communities were collected at St. Thomas, USVI, in 2006USVI, in , 2007USVI, in and 2008, and at the Florida Keys (FlK) in 2013 to 2014, as described previously in Xu et al (2014) and Lozano-Duque et al (2018). SCUBA divers collected multiple samples of dominant macroalgal species, which varied by sampling date and site, along with some sea water in one plastic bag per site.…”

“…After three more weeks, they were transferred into the final culture volume of 25 ml and maintained under the conditions described above. As six different Gambierdiscus species are reported from the Caribbean region, which cannot be microscopically distinguished, all strains were taxonomically identified using Restriction Fragment Length Polymorphisms of amplified fragments from 23S rDNA (Lozano-Duque et al, 2018). Isolates of the most common species G. caribaeus were used in all further analyses.…”