NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY(1).

Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4-fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient-replete and nitrogen (N)-limited semi-continuous cultures. N-limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two-fold increase (1%-4% to 5%-9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4-fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

Ciguatera fish poisoning and sea surface temperatures in the Caribbean Sea and the West Indies.

Ciguatera fish poisoning (CFP) is a circumtropical disease caused by ingestion of a variety of reef fish that bioaccumulate algal toxins. Distribution and abundance of the organisms that produce these toxins, chiefly dinoflagellates of the genus Gambierdiscus, are reported to correlate positively with water temperature. Consequently, there is growing concern that increasing temperatures associated with climate change could increase the incidence of CFP. This concern prompted experiments on the growth rates of six Gambierdiscus species at temperatures between 18 degrees C and 33 degrees C and the examination of sea surface temperatures in the Caribbean and West Indies for areas that could sustain rapid Gambierdiscus growth rates year-round. The thermal optimum for five of six Gambierdiscus species tested was >/=29 degrees C. Long-term SST data from the southern Gulf of Mexico indicate the number of days with sea surface temperatures >/=29 degrees C has nearly doubled (44 to 86) in the last three decades. To determine how the sea surface temperatures and Gambierdiscus growth data correlate with CFP incidences in the Caribbean, a literature review and a uniform, region-wide survey (1996-2006) of CFP cases were conducted. The highest CFP incidence rates were in the eastern Caribbean where water temperatures are warmest and least variable.