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Karenia brevis is the major harmful algal bloom-forming species in the Gulf of Mexico, and produces neurotoxins, known as brevetoxins, that cause large fish kills, neurotoxic shellfish poisoning, and human respiratory distress. Brevetoxins are polyethers that bind voltage-sensitive sodium channels, opening them for prolonged periods of time. Clonal cultures of K. brevis exhibit unique brevetoxin profiles, which not only differ from one another, but also change when subjected to different environmental conditions. The brevetoxin structures were elucidated 30 years ago without any breakthroughs for the biosynthetic pathway. These unique ladder-like polyethers have 10 (PbTx-1) or 11 (PbTx-2) rings, indicating that they are synthesized as secondary metabolites by polyketide synthases. The extensive size of the genome and the lack of histones and nucleosomes combined with the additional regulatory step of a trans-splicing spliced leader sequence make normal molecular techniques ineffective in determining the genes involved in toxin synthesis. The goal of this project is to identify a potential link between toxin, gene, and function. One objective is to take the next step towards identifying the genes associated with the synthesis and regulation of brevetoxins and to help elucidate the hypothesized gene clusters of multi-protein enzymatic complexes involved in brevetoxin production, one for each backbone. The second objective is to make an effort to determine the in vivo function of the costly brevetoxins by identifying possible ion channels, which could be osmotically regulated by the toxins. Genes for polyketide synthases (PKS) were identified in K. brevis, obtained from Expressed Sequence Tag (EST) libraries. In this work, reverse transcription polymerase chain reactions (RT-PCR) were used to generate pools of complementary DNA (cDNA), which was used in real-time quantitative polymerase chain reactions (qPCR) to give relative amounts of PKS transcripts. K. brevis clones have shown a significant increase in toxin production after a rapid shift from high salinity to low salinity, indicating a regulation of brevetoxin synthesis. To gain a better understanding of regulation of toxin production during algal blooms, we compared the toxin levels under different conditions to the transcript levels of PKS genes, as determined by quantitative RT-PCR. In a separate line of investigation, an in silico analysis of the EST library was performed to identify ion channel genes expressed by K. brevis, which may be the in vivo binding site of brevetoxin. The information generated from this project will help to elucidate the effects of environmental variations on toxin production and the biological function of toxin production -- valuable information for the shellfish industries and public health.

Polyether compounds are a subgroup of natural products with regular occurrence of multiple C-O-C motifs. The biosynthetic origin of polycylic polethers has been studied and the majority of them are derived from polyketide or terpene pathways. Normally, the polycyclic polyethers can be divided into two groups based on their structural features: the first group features multiple rings that are interconnected by carbon-carbon single bond, which are produced by a biosynthetic cascade of exo epoxide-opening reactions; the other group has multiple fused cyclic ethers and are formed by an cascade of endo epoxide-opening reactions. Karenia brevis (K. brevis) is known as principle harmful bloom (HAB) organism of the Gulf of Mexico which can cause red tides. Brevetoxins (PbTx) are a suit of cyclic polyether ladder compounds produced by K. brevis. Brevetoxins are neurotoxins that can bind to voltage-gated sodium channels in nerve and muscle cells, resulting in disruption of normal neurological processes causing the human illness which is clinically described as neurotoxic shellfish poisoning (NSP). Inspired by Cane-Celmer-Wesley’s proposal regarding monensin biosynthesis, Nakanishi and Shimizu proposed a biosynthetic pathway for brevetoxin which suggests that PKS-mediated synthesis of the polyene is followed by epoxidation to afford a polyepoxide which then undergoes an epoxide-opening cascade, catalyzed by an epoxide hydrolase (EH). To find evidence to support the hypothesis that an epoxide hydrolase from polyether ladder producing dinoflagellates will catalyze the construction of the polyether ladder framework from polyepoxide substrates, and to study the role of epoxide hydrolase in the biosynthesis of polyether ladder compounds, it is necessary to identify and produce one or more epoxide hydrolase from dinoflagellates. The methods to detect epoxide hydrolase activity in K. brevis and different techniques to obtain epoxide hydrolases from K. brevis are discussed. A microsomal EH identified from a K. brevis EST library was cloned and expressed. The characterization of this EH, including substrate selectivity and enantioselectivity as well as its potential to catalyze the critical ento-tet cyclization epoxy alcohol, is discussed.

Karenia brevis dinoflagellate blooms off the west coast of Florida can create devastating effects on marine communities when they release a neurotoxin known as a brevetoxin. These blooms, informally referred to as red tides, can cause massive fish kills, necessitate closures of shellfish fisheries, and can even leave lingering toxins that impact shelf communities long after the bloom has dissipated. As a result, much effort has been put into studying K. brevis bloom initiation and dynamics. However, how K. brevis blooms impact Florida's fisheries is not fully understood because the relationship between K. brevis cell counts and fish mortality is poorly described. To study this relationship and the ecosystem response to K. brevis blooms, Ecopath with Ecosim (EwE) modeling is used to force K. brevis bloom mortality on the shelf ecosystems by using a recently developed time series that indexes K. brevis bloom severity. This index dynamically drives K. brevis bloom mortality in EwE in a historical reconstruction scenario from 1980 to 2009. Three hypotheses on ecosystem response are explored using Gag grouper as a case study. We postulate a) that K. brevis blooms impose bottom-up and top-down effects on the food web, b) that episodic perturbations by these blooms shape the community structure and c) that fishing pressure exacerbates those effects. Results support the hypothesis that K. brevis blooms pose top-down food web pressures, which is seen by evidence of trophic cascading. Changes in community structure with bloom mortality are also evidenced by changes seen in biodiversity and richness. An exacerbation of those effects as a result of heavy fishing pressure is evident, however, is only seen during severe bloom events. Little to no changes were found in the mortality from K.brevis blooms during blooms of average severity, and less mortality was imposed on the system during blooms of particularly low severity. However, this may be an artefact of the mode of action of K. brevis in EwE. Investigation of bloom effects on Gag showed that natural mortality rates of Gag appear to be largely influenced by mortality incurred during K. brevis blooms relative to the low rate of predation on Gag. Moreover, consumption rates of Gag on its prey were found to increase under a realistic schedule of these blooms. This may be due to a combination of effects, including increased mortality on competitors (making more prey available for Gag) and a lowering of the mean age of the Gag stock, which increases population productivity.

Harmful algal blooms (HABs) are increasing in frequency and duration worldwide. Karenia brevis, the major toxic dinoflagellate in the Gulf of Mexico, produces potent neurotoxins, known as brevetoxins. For K. brevis, only minor concentrations of brevetoxins are needed to induce toxicity and environmental conditions appear to have the most direct impact on the cellular content of these toxins. A better understanding of K. brevis biology is essential to understand the mechanisms underlying toxin production and the ecology of such HABs, as well as to better anticipate and respond to such blooms. Here we present findings on the effect of salinity and availability of carbon on cellular physiology and brevetoxin and brevenal production by K. brevis. When grown at salinities of 35 and 27, but otherwise identical conditions, total brevetoxin cellular concentration varied between 0 to 18.5 pg cell-1 and brevenal varied between 0 and 1 pg cell-1. In response to hypoosmotic stress brevetoxin production was triggered, as a result, brevetoxin production increased up to 53%, while growth rates remained unchanged. A significant hypoosmotic event of >11%, was needed to trigger the response in brevetoxin production. To determine if K. brevis was sensing changes in specific ions within seawater (K+, Cl- or Ca2+), we systematically removed one ion while keeping the remaining ions at equivalent molar concentration for salinity of 35. Dilution in seawater K+ concentrations triggered the production of brevetoxins, increasing production ≥44%. Ecosystem changes due to climate change have increased the production of toxins in other HAB species; here we examined the impact on K. brevis. We have shown that modification of pCO2 level and temperature did not influence brevetoxin production; however, predicted climate change scenarios (increased temperature and pCO2) did significantly increase the growth rate of K. brevis, by 60% at 25°C and 55% at 30°C. We suggest that K. brevis blooms could benefit from predicted increase in pCO2 over the next 100 years. Overall, our findings close a critical gap in knowledge regarding the function of brevetoxin in K. brevis by identifying a connection between brevetoxin production and osmoacclimation.

The purposes of this study were to characterize the complexation of toxic metals with algal toxins and to determine the effects of arsenic and copper on the growth of Karenia brevis under specific experimental conditions. Microcystins, pahayokolides, brevetoxins and okadaic acid were used as representatives of algal toxins while arsenic, copper, cadmium, cobalt, iron, manganese and mercury were selected as typical toxic metals (including metalloids here) in the aquatic environment. The stabilities of the toxin-metal complexes were determined using equilibrium dialysis and/or centrifugal ultrafiltration technique. A direct exposure of arsenic and copper to the K. brevis was carried out to determine the effects of these metals to the growth of the algal cell. The results indicated that Cu2+, Hg2+, Co2+, Cd2+ and Fe2+ were capable of complexing with the algal toxins. Moreover, the exposure experiments demonstrated that the high concentration of arsenic and copper could affect the growth of the K. brevis.

Algae are important to marine and fresh-water ecosystems. However, some species of algae are harmful or even toxic. They can consume oxygen or block sunlight that is essential for other organisms to live. Indeed, some algae blooms can produce toxins that damage the health of the environment, plants, animals, and humans. Harmful algal blooms (HABs) which are often more green, brown, or dark-colored than red have spread along the coastlines and in the surface waters of the United States. Therefore, scientists are making great efforts to study HABs in order to maintain human and ecosystem health. Karenia brevis, the major harmful algal bloom dinoflagellate of the Gulf of Mexico, plays a destructive role in the region. Karenia brevis, responsible for Florida red tide, is the principle HAB dinoflagellate in the Gulf of Mexico. K. brevis blooms can produce brevetoxin: ladder-shaped polyether (LSP) compounds, which can lead to adverse human health effects, like reduced respiratory function through inhalation exposure, or neurotoxic shellfish poisoning through consumption of contaminated shellfish. The poisoning has been attributed to their affinity for voltage-sensitive sodium ion channels causing channel opening and depolarization of excitable cell membranes. Conservative estimate suggests that the economic impact from all harmful algal bloom events in the United States is at least $82 million/year. The public health costs occupy $37 million alone. The study presented herein utilized fluorescent and photolabile brevetoxin probes to demonstrate that brevetoxin localizes in the chloroplast of K. brevis where it binds to light harvest complex II (LHC II) and thioredoxin (Trx). It had been discovered that the TrxR/Trx system was inhibited by brevetoxin-2 (PbTx-2) with an IC50 of 25 µM. The mechanism of the inhibition was discussed in this work. The research also revealed that the K. brevis high-toxic and low-toxic strains have a significant difference in their ability, not only to produce brevetoxin, but also to perform NPQ and in the production of ROS. I compared and contrasted various metabolic and biochemical parameters in two strains of K. brevis which had a ten-fold difference in toxin content. The work could shed light on the physiological role that brevetoxin fills for K. brevis and may contribute to understanding the effect of ladder-shaped polyether compounds on both marine animals and exposed humans and shall inform improved treatments for brevetoxicosis.

The thioredoxin system is the major cellular reductant system present in the cell, whose role is to maintain cellular redox homeostasis. It does this in part, by regulating the activity of many other enzymes including ribonucleotide reductase, which is essential for DNA synthesis. It also acts as an antioxidant, reducing destructive reactive oxygen species. The thioredoxin system is comprised of thioredoxin (Trx) which reduces target protein disulfide bridges by thiol-disulfide exchange and thioredoxin reductase (TrxR) which reduces Trx back to its active state. Thioredoxin reductase is a common target for many cancer drugs including cisplatin and auranofin. Recently we have shown that the Florida red tide toxin, brevetoxin-2 (PbTx-2) can inhibit mammalian TrxR1. Brevetoxin-2 has α, β-unsaturated aldehyde moiety that was proposed to inhibit the enzyme by forming a Michael adduct. Several compounds which are similar to brevetoxin in size and functionality have a similar effect on TrxR. These compounds include antitumor and antibiotics such as manumycin A, geldanamycin, rifamycin SV and thiostrepton and toxins such as brevetoxin-3, nodularin and microcystin-LR. Manumycin A behaves as a typical TrxR1 inhibitor while other compounds screened activate the reduction of small disulfides such as DTNB (5,5’-dithiobis-(2-nitrobenzoic acid)). Mammalian thioredoxin reductase is a homodimer with two redox center viz. N-terminal dithiol buried in the enzyme and C-terminal selenosulfide located on the flexible C-terminal tail. Modification of the C-terminal tail of TrxR by these test compounds can expose N-terminal redox thiol that could reduce DTNB. The C-terminal Sec, a nucleophile can form a Michael adduct with α, β-unsaturated carbonyl moiety of test compounds. Together with point-specific mutant enzymes (C-terminal tail truncated, dead tail and Cys mutant) and enzyme assays that are specific/dependent on C-terminal Sec were used to decipher the site-specific interaction between these test compounds and TrxR. Inhibition of TrxR at the C-terminal redox center produces a pro-oxidant known as SecTRAP (Selenium Compromised Thioredoxin Reductase-derived Apoptotic Proteins), which uses NADPH to produce superoxide radical anion as observed with manumycin A. Since many cancer drugs target TrxR the present study has the potential to discover new cancer drugs.

Clay-based compounds are globally the most advanced and widely used method of direct suppression of marine harmful algal blooms, and are currently undergoing investigation as an option to control Karenia brevis blooms in Florida. Before clay may be accepted for widespread use, there are multiple concerns and challenges that must be addressed regarding the environmental safety of this method, such as effects on water quality, the fate of toxins, and potential impacts of clay treatment to non-target organisms. To contribute to ongoing assessments of clay as a potential control method for K. brevis blooms, we conducted experiments with a formulation of kaolinite clay modified with polyaluminum chloride known as Modified Clay II (MC II). In these experiments, we evaluated water chemistry dynamics and physiological responses in several bottom-dwelling marine species with ecological and economic significance, including blue crab (Callinectes sapidus), sea urchin (Lytechinus variegatus), and hard clam (Mercenaria campechiensis). First, we conducted an experiment with blue crabs in 20 L aquarium tanks (N = 48), exposing the animals to cultured K. brevis (1 x 106 cells L-1) and MC II (0.5 g L-1) and measuring mortality and reflexes over 192 hours. In our second experiment, K. brevis (1x106 cells L-1) and MC II (0.2 g L-1) were applied to 1,400 L mesocosms (N = 9) containing blue crabs, sea urchins, and hard clams, which were observed over 96 hours. In our final experiment, we modified the methods of the previous experiment to again examine K. brevis (1x106 cells L-1) and MC II (0.2 g L-1) with the same model species in 1,400 L mesocosms (N = 12) over 72 hours. In these two mesocosm experiments, we observed cell and toxin removal, changes in water quality characteristics including nutrients and carbonate chemistry, and measured mortality, respiration rate, reflexes, and internal toxin content. Our results were congruent across our three experiments. Treatment with MC II significantly reduced cell concentrations but did not reduce toxin concentrations in the water column. We found no notable impacts of clay treatment to reflexes, respiration rates, or internal toxin content for either of our three species. No significant differences in mortality were found for our three species, excluding crabs in the first mesocosm study, which were found to have pre-existing infections that confounded our results. Analyses of nutrients indicates MC II may remove dissolved phosphorus from the water column, and the potential to improve water quality which may make this formulation of clay desirable to managers. Overall, treatment with this formulation of clay did not appear to induce any significant measured effects on the model species within the observed time frames of these experiments. Clay appears to be a promising option to treat K. brevis blooms given its low cost, ease of application, and negligible impacts to the environment, and its use may relieve the damaging effects of K. brevis blooms by preventing mortalities that would otherwise occur were blooms allowed to persist. We therefore recommend that clays, including MC II, be considered for additional laboratory and field tests, with the goal of obtaining further information on potential ecological impacts so that managers and researchers can make informed decisions on the use of bloom control technologies in Florida waters.

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.