Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Marine Science

Major Professor

Edward Van Vleet, Ph.D.

Committee Member

Bill Baker, Ph.D.

Committee Member

Robert Byrne, Ph.D.

Committee Member

Jerome Naar, Ph.D.

Committee Member

Gabriel Vargo, Ph.D.

Keywords

brevetoxin, eutrophication, natural products, nitrogen, phytoplankton, saxitoxin

Abstract

Despite the critical role algae serve as primary producers, increases or accumulation of certain algae may result in Harmful Algal Blooms (HABs). Algal toxins from these blooms contribute significantly to incidences of food borne illness, and evidence suggests HABs are expanding in frequency and distribution. Mitigation of these HABs without knowledge of the ecological purpose and biochemical regulation of their toxins is highly unlikely. The production, function, and potential of secondary metabolites produced by the dinoflagellates Karenia brevis and Pyrodinium bahamense, were investigated.

Brevetoxins were demonstrated by two different methods to localize within the cytosol of Karenia brevis. Differential and density-dependent centrifugation followed by Enzyme Linked Immunosorbant Assays (ELISAs) indicated that brevetoxin was not contained by any cellular organelles. Light microscopy of brevetoxin immunolabeled preserved cells visually confirmed these results, showing stain to be distributed throughout the cytosol and notably absent from the nucleus. These results have implications for brevetoxin synthesis and function. The complex cyclization process of brevetoxin therefore likely occurs in the cytosol after export of a polyketide precursor from the chloroplast. Functionally, this cellular location suggests use of brevetoxin in cytosolic functions such as signaling and chelation.

Culture experiments of Pyrodinium. bahamense var. bahamense were undertaken to determine the effects of nutrients and environmental conditions on growth requirements and toxin production. HPLC analysis was employed to separate and quantify the saxitoxins. As eutrophication is a concern where this species is most problematic, in the Indian River Lagoon area of Florida, utilization of urea and ammonium were explored and compared to nitrate. While all nitrogen conditions yielded similar growth curves in P. bahamense, the cultures using urea contained a substantially lesser amount of the potent STX congener. This difference implies the urease enzyme utilized by P. bahamense is inefficient and urea based fertilizers are unlikely to create blooms with greater toxicity. Cyst production in P. bahamense was found to depend on nutrient limitation. Cultures utilizing ammonium displayed a smaller proportion of cysts, presumably attributable to the bioavailablility of ammonium. The total toxin content of P. bahamense was found to vary inversely with growth rate, although mole percents of the saxitoxins were largely unchanged over a suite of environmental parameters including temperature, salinity, and pH. Possible reasons for the reported increase in HABs include global warming, dumping of ballast water, and nutrient influx. These studies outline controls on toxin synthesis and production and conditions needed for growth and will aid in predicting environmental and human health effects pending these global changes.

Extracts of K. brevis and P. bahamense cultures were assayed against various pathogenic agents. Growth of K. pneumoniae was inhibited by extracts of both K. brevis and P. bahamense. An extract of K. brevis additionally inhibited MRSA, while a P. bahamense extract additionally inhibited both S. aureus and MRSA as well as the most common protozoan vector of malaria, P. falciparum. The activity of a dinoflagellate against an Apicomplexan (P. falciparum) found in this study is especially interesting as the phyla are closely phylogenetically related. Differences in activity of extracts against P. falciparum between a clonal culture on P. bahamense from the Indian River Lagoon and a 2011 bloom sampled from Tampa Bay were observed. Drugs are losing their effectiveness against these infectious agents, making pursuit of new drugs an important field. These results suggest that HAB dinoflagellates hold promise in drug discovery similar to other phytoplankton.

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