Harmful algal blooms (HABs) in the Gulf of Mexico are primarily caused by dense aggregations of the dinoflagellate species, Karenia brevis. Karenia brevis produces a highly toxic neurotoxin, brevetoxin which has been shown to cause Neurotoxic Shellfish Poisoning (NSP) and respiratory distress in humans in addition to a wide range of negative impacts upon natural ecosystems. Karenia mikimotoi is a co-existing species present during K. brevis blooms. K. mikimotoi has caused major HAB events in other parts of the ocean, but has not been recognized as a major contributor to toxicity of blooms in the Gulf of Mexico. K. brevis and K. mikimotoi have both been associated with the presence of unidentified hemolytic toxins. Production of hemolysins has not previously been investigated for either species to date in the Gulf of Mexico. Presence of hemolysins may affect toxicity and the overall impact of HABs. Therefore, detection of hemolysins is imperative for accurate identification of potential harmful impacts of such blooms. The primary goal of this research is to define whether either species is capable of producing hemolytic activity independent of brevetoxin activity; and to identify if there is significant differentiation between a variety of clonal isolates regarding toxicity and growth rate when subjected to variable experimental conditions.

Karenia brevis is the major harmful bloom forming dinoflagellate in the Gulf of Mexico. The toxin produced by this dinoflagellate can cause large fish kills, marine mammal mortality, respiratory irritation, and neurotoxic shellfish poisoning in humans. Blooms can occur anywhere in the Gulf of Mexico (hereafter Gulf) but are predominantly observed off the west coast of Florida and the coast of Texas. The west coast of Florida has been hypothesized to be the origin for blooms of K. brevis in other regions within the Gulf based upon the frequent formation of blooms in this region. To investigate this possibility, microsatellite markers were used to determine the population-genetic structure of K. brevis in the Gulf of Mexico. The difficulties of culturing K. brevis required development and use of a single-cell PCR amplification protocol for preserved cells. Lugol's iodine-preserved bloom samples of K. brevis were destained with sodium thiosulfate and subjected to two rounds of PCR amplification. The destaining protocol resulted in the successful, simultaneous amplification of five microsatellite markers from single cells of K. brevis. A total of 18, highly polymorphic microsatellite markers are available for K. brevis. Each marker was amplified from 40 cultures of K. brevis isolated from water samples from Florida and Texas. Observed genetic diversity was high but similar to the genetic diversity observed in other phytoplankton species. No genetic divergence was detected between isolates from Florida and isolates from Texas. Single cells from a total of 38 field samples were analyzed at five microsatellite markers to determine if population-genetic structure was present in K. brevis in the Gulf. Significant genetic divergence between several individual samples was detected, reflecting the high genetic diversity present within the species. Observed genetic divergence was low between blooms from the west coast of Florida and the coast of Texas and supports the hypothesis of a common origin for blooms of K. brevis in the Gulf of Mexico.

Harmful algal blooms (HABs) in the Gulf of Mexico are primarily caused by dense aggregations of the dinoflagellate species, Karenia brevis. Karenia brevis produces a highly toxic neurotoxin, brevetoxin which has been shown to cause Neurotoxic Shellfish Poisoning (NSP) and respiratory distress in humans in addition to a wide range of negative impacts upon natural ecosystems. Karenia mikimotoi is a co-existing species present during K. brevis blooms. K. mikimotoi has caused major HAB events in other parts of the ocean, but has not been recognized as a major contributor to toxicity of blooms in the Gulf of Mexico. K. brevis and K. mikimotoi have both been associated with the presence of unidentified hemolytic toxins. Production of hemolysins has not previously been investigated for either species to date in the Gulf of Mexico. Presence of hemolysins may affect toxicity and the overall impact of HABs. Therefore, detection of hemolysins is imperative for accurate identification of potential harmful impacts of such blooms. The primary goal of this research is to define whether either species is capable of producing hemolytic activity independent of brevetoxin activity; and to identify if there is significant differentiation between a variety of clonal isolates regarding toxicity and growth rate when subjected to variable experimental conditions.

Dans le monde entier les introductions d'espèces dans de nouvelles régions constituent une préoccupation écologique croissante ; ces introductions peuvent conduire à des modifications drastiques des écosystèmes, entre autres du fait de la compétition avec des espèces indigènes et également par la modification des réseaux de flux d'énergie dans les écosystèmes. La moule verte Perna viridis est une espèce récemment introduite dans les eaux côtières américaines et qui s'est rapidement disséminée le long des côtes du Sud-Est des États-Unis. Cependant, il n'existe pour le moment que très peu d'informations concernant la structure des populations, et leur dynamique en réponse à la variabilité environnementale locale. Les efflorescences de marées rouges formées par le dinoflagellé toxique Karenia brevis sont fréquentes le long des côtes de Floride bordant le Golfe du Mexique, et la tolérance de P. viridis à l'égard de ces événements et des brévitoxines (PbTx) associées n'est pas connue. En outre, comme P. viridis est une espèce invasive, la potentielle concurrence (ressources trophiques, espace, …) avec des bivalves indigènes comme l'huître Crassostrea virginica, est une préoccupation majeure dans les systèmes côtiers de Floride. Cette étude vise à caractériser la dynamique des populations établies de P. viridis populations établies et leur réponse aux efflorescences naturelles de K. brevis. Les effets des efflorescences à K. brevis ont été évalués à partir des résultats d'un suivi de la croissance, de la mortalité, du recrutement, de la gamétogenèse et de la composition biochimique des tissus (protéines, glycogène et lipides) durant trois ans. En outre, les concentrations en PbTx dans les tissus ont été analysées afin de déterminer l'absorption, l'accumulation et des taux d'élimination de ces toxines. Par ailleurs, les données recueillies sur le terrain et des informations de la littérature ont été utilisées pour élaborer un modèle énergétique individuel DEB pour modéliser la croissance et la reproduction de P. viridis. Avant l'apparition de la première efflorescence à K. brevis, P. viridis présentait des taux de croissance rapide (6-11 mm mois-1) et un taux de survie élevé (mortalité <1%). Au cours des efflorescences à K. brevis, le taux de croissance a chuté de façon significative et une bioaccumulation de PbTx dans les tissus mous a été observée. Les concentrations élevées en PbTx dans les tissus ont persisté longtemps après la dissipation de l'efflorescence et les taux de mortalité élevés se sont maintenus, ce qui a réduit fortement l'abondance de P. viridis. À la fin de l'efflorescence, la concentration en PbTx dans les moules était presque le double de celle relevée chez l'huître indigène Crassostrea virginica pour des individus prélevés à la même période ; chez P. viridis, la concentration en PbTx est restée supérieure à la limite réglementaire pour la consommation humaine pendant 16 semaine, alors qu'elle est revenue en dessous de ce seuil en 2 ½ semaines chez C. virginica.La composition biochimique des tissus et la reproduction n'ont pas parues affectées par ces évènements ; P. viridis réalise sa gamétogénèse durant toute l'année et a mis en place une stratégie de ponte intermittente partielle ; elle présentait durant toute l'année une grande stabilité de la concentration en composés de réserve. L'absence de cycle saisonnier marqué de la composition biochimique suggère que la ressource trophique est suffisante pour soutenir la gamétogenèse tout au long de l'année. Cependant, la première année du suivi, deux évènements majeurs de ponte et de recrutement ont été observés au printemps et à l'automne. Au cours de la deuxième année de suivi, l'analyse histologique montre que le même patron de ponte massive est observé au printemps ; cependant, à cette période, les efflorescences toxiques ont persisté et le recrutement a été inhibé, ce qui suggère que la fécondation et / ou le développement et la survie des larves […] / Worldwide, introductions of exotic species to new regions is of rising concern which can lead to catastrophic ecosystem alterations through competition with native species and disruption in energy flow.Perna viridis is a recently introduced bivalve species to US coastal waters and has vigorously spread throughout the southeastern US. However, little information regarding population structure and response to local environmental factors has been reported. Red tide blooms formed by the toxic dinoflagellate Karenia brevis are frequent along the Gulf coast of Florida and as a recently introduced species, it is unclear what tolerance P. viridis has toward these events and associated brevetoxins (PbTx). Further, as an invasive species ecological concerns have risen regarding potential for spread and competition with native bivalve species, particularly the eastern oyster Crassostrea virginica.This study aimed to characterize the population dynamics of established P. viridis populations and their response to naturally occurring K. brevis blooms. This was completed through monitoring of growth, mortality, juvenile recruitment, gametogenesis and biochemical composition (protein, glycogen and lipid) throughout a three year monitoring period to evaluate the effects of K. brevis blooms. Additionally, tissue PbTx concentrations were analyzed to determine uptake, accumulation and elimination rates. Data collected from the field and information reported in the literature were used to create a functional DEB model to predict individual growth and reproduction of P. viridis under environmentally realistic conditions.Prior to onset of the first K. brevis bloom event, P. viridis showed rapid growth rates (6 – 11 mm month-1) and high survival (mortality <1%). However, during K. brevis blooms growth rate dropped significantly and bioaccumulation of PbTx in the soft tissue was observed. High tissue PbTx concentrations persisted long after bloom dissipation and high rates of mortality ensued, severely reducing population densities. PbTx in mussels nearly doubled that of oysters sampled during the same time and remained above the regulatory limit for significantly longer, 2 ½ weeks and 16 weeks, respectively.Biochemical composition and reproduction appeared unaffected, exhibiting year round gametogenesis with a partial, intermittent spawning strategy and stability in reserves. A lack of significant seasonal cycles in biochemical composition suggests sufficient food and energy availability to support the observed year round gametogenesis. While continuous spawning capabilities were evident two major peaks in spawning and recruitment were observed (spring and fall), suggesting reduced fertilization and / or larval development and survival due to the presence of K. brevis and associated ichthyotoxins and hemolysins.These results indicate that while high tissue PbTx concentrations may lead to reduced growth in P. viridis, gametogenesis is not inhibited, allowing the population to survive K. brevis bloom exposure and reproduce, even while individual mortality was high. Prolonged bioconcentration of PbTx may lead to increased threat of post bloom trophic transfer, resulting in negative impacts on other important fisheries and higher food web implications. While it cannot be conclusively determined that the cause of reduced growth, survival and recruitment is due to red tide events, the parallels observed suggest that K. brevis is an important factor in the drastic changes in population structure.Through the work presented here, population dynamics of locally established P. viridis populations were characterized through monthly monitoring and the development of a DEB model to accurately predict the growth and reproduction under dynamic environmental conditions. This work aims to synthesize our knowledge on the individual bioenergetics of P. viridis and to aid in understand population dynamics and potential for competition with local C. virginica [...]

The accurate identification of taxa from mixed assemblages using genetic analysis remains an important field of molecular biology research. The common principle behind the development of numerous documented genetic detection technologies is to exploit specific nucleotide sequences inherent to each taxon. This body of work focuses on practical applications of real-time nucleic acid sequence-based amplification (RT-NASBA) in marine science, and is presented in four case studies. Each study represents novel work in the genetic identification of respective taxa of interest using RT-NASBA. Two case studies documented the development of an assay targeting mitochondrial 16S rRNA to discern legally salable grouper species in the U.S. from fraudulently mislabeled surrogate fish. This technology was first validated using lab-based, benchtop instrumentation, and was then adapted into a complete field detection system. The third study documented an internally controlled RT-NASBA (IC-NASBA) assay for the detection and quantification of the harmful algal bloom-causing dinoflagellate, Karenia mikimotoi, by targeting the ribulose-1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) large-subunit gene (rbcL). The final section of this dissertation details the preliminary development of an IC-NASBA assay targeting large subunit rRNA for the quantification of Enterococcus, which is a genus of bacteria commonly used as an indicator of fecal pollution in recreational marine water. My results show that RT-NASBA provides a suitable format for the accurate identification of target species from these taxa which include prokaryotes, as well as both unicellular and multicellular eukaryotes.

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.

Laser in situ scattering and transmissometry (LISST) instruments are used to measure the particle size distributions (PSDs) and volume concentration of individual and groups of phytoplankton in water. The objective of this research was to test the LISST’s ability in detecting monospecific blooms in-situ and the ability to detect aggregation after diatoms were subjected to different temperatures and bacteria concentrations. The PSDs of ten harmful algal bloom (HAB) species were measured with the LISST characterizing the peak location, peak height, peak width, and peak range resulting in a scattering signature for each species. Each species had specific characteristics that would allow for their detection with the LISST, though microscope observations would be needed for complete accuracy. The LISST was able to detect HABs placed in natural seawater collected off the Texas coast. Blooms of four HAB species before they reached full” bloom concentrations were detected making the LISST a possible low cost, effective tool in the early detection and monitoring of HABs. The diatom, Odontella aurita, was used to test how well the LISST could monitor aggregation, an important process in the termination of many phytoplankton blooms. Increasing temperature causes an increase in transparent exopolymer particle (TEP) production in diatoms, which is a critical sticky particle that increases the probability of aggregation. An increase in temperature can also cause an increase in bacteria concentration that can positively effect TEP production and thus aggregation. O. aurita was grown at 20 °C and 28 °C and showed a significant increase in TEP abundance with temperature (p = 0.002), though no relationship between TEP production and bacteria concentration existed. Coomassie stained particles (CSP) are proteinaceous gel-like particles, which are currently understudied. CSP was consistently produced though it did not appear to be dependent upon any single factor. The increase in ocean temperatures has implications for an increase in phytoplankton blooms making the monitoring and understanding of these blooms even more important as they can affect the carbon cycle and potentially the microbial loop.

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, le dinoflagellé produisant des brevetoxines (PbTx), est la principale espèce d’efflorescences d’algues toxiques dans le Golfe du Mexique. Les effets de cette algue sur Mercenaria mercenaria et Crassostrea virginica sont méconnus tandis que les efflorescences coïncident avec la période de reproduction de ces espèces. Ce projet avait pour but de déterminer les effets i) d’une exposition à K. brevis en laboratoire et naturelle de terrain sur les processus physiologiques associés à la reproduction de M. mercenaria et C. virginica, et ii) d’une exposition à K.brevis sur la qualité et le développement des gamètes, embryons et larve de ces espèces. Suite à l'exposition des adultes de clams et d’huîtres à K. brevis, les paramètres physiologiques de la reproduction ont été affectés. La présence de PbTx dans les tissus des gamètes et le potentiel transfert maternel de PbTx à la progénie via les ovocytes, pourraient avoir entraîné les effets négatifs observés lors du développement larvaire.Les effets négatifs similaires causés par l'exposition des stades précoces à différentes préparations de cellules de K. brevis suggèrent que d'autres composés toxiques, en plus de PbTx, pourraient être impliqués dans la toxicité et, que la majorité des effets délétères se produisent durant les divisions embryonnaires.Le clam et l’huître américaine sont sensibles à K. brevis. Les effets négatifs sur les adultes et jeunes stades de vie, combinés à une exposition quasi-annuelle aux efflorescences de K. brevis, pourraient engendrer des perturbations majeures sur le recrutement des populations de ces espèces importantes, et avoir des répercussions environnementales et économiques. / The brevetoxin (PbTx) producing dinoflagellate, Karenia brevis is the most prevalent harmful algal bloom species in the Gulf of Mexico. The effects of this alga on Mercenaria mercenaria and Crassostrea virginica are poorly understood yet, blooms typically overlap with periods of reproduction and spawning in these species.The aims of this project were to determine the effects of i) laboratory and field exposure of K. brevis on the reproductive and related physiological processes of adult M. mercenaria and C. virginica and the quality of the offspring that were produced and ii) K. brevis exposure on gamete, embryo and larval development in these species.Following exposure of adult clams and oysters to K. brevis, negative effects were recorded on reproductive and physiological parameters. PbTx was recorded in gamete tissues, and maternal transfer of this PbTx to the offspring via the oocytes, may have resulted in the significant negative effects recorded on larval development up to the end of the lecithotrophic phase.The similar dose-dependent negative effects caused by direct exposure of gamete and early life stages to different cell preparations of K. brevis suggests that other toxic compounds in addition to PbTx may be involved in toxicity and, that the majority of negative effects occur during embryonic divisions.Hard clams and eastern oysters are susceptible to K. brevis exposure. The negative effects on adult and early life stages combined with the near- annual exposure to blooms of K. brevis could cause significant bottle-necks on the recruitment and population dynamics of these important species and, have wider reaching environmental and economic impacts.

Mass mortalities of Florida manatees (Trichechus manatus latirostris) and bottlenose dolphins (Tursiops truncatus) have been attributed to brevetoxins produced by the Florida red tide dinoflagellate Karenia brevis. The multiple routes through which marine mammals can be exposed to brevetoxins have complicated efforts to understand the mechanisms that lead to mass mortality events. In spring of 2002, 34 endangered Florida manatees died in southwest Florida, and in spring of 2004, 107 bottlenose dolphins died in the Florida Panhandle. These events provided unique opportunities to make clear connections between ingested brevetoxins and marine mammal mortalities without the confounding issues of concurrent exposure through direct contact or inhalation. Prior to 2002, the accumulation of brevetoxins on or in seagrass had never been previously reported, and the delayed or chronic exposure of manatees to brevetoxins through seagrass was not recognized as a threat. Brevetoxins were shown to persist in association with seagrass at high levels for weeks and at lower levels for months in the absence of K. brevis. Analyses of the epiphytes and detritus on the surface of the seagrass leaves as well as of the cleaned seagrass leaves and rhizomes revealed that during a K. brevis bloom as much as half of the toxin present in the seagrass may be associated with the leaves themselves, while after a bloom, the majority of the toxin present is associated with the epiphytes. The 2004 mass mortality of bottlenose dolphins in the Florida Panhandle clearly indicated that fish have the potential to vector brevetoxins to higher tropic levels. Analyses of fish collected live from St. Joseph Bay and southwest Florida revealed that brevetoxin accumulation in fish is a common occurrence. Planktivorous clupeid fish are capable of accumulating high concentrations of brevetoxins within their viscera, and their movement can result in spatial separation of a bloom and animal exposure. Sciaenid species and pinfish also accumulated brevetoxins but to a lower extent. These fish, as well as other omnivorous and piscivorous species, may retain brevetoxins in their tissues at significant concentrations after a bloom has dissipated, which may lead to temporal separation of blooms and animal exposure.