Thesis (M.S.)--University of North Carolina Wilmington, 2008. / Includes appendixes. Title from PDF title page (viewed May 27, 2009) Includes bibliographical references (p. 96-102)

The West Florida Shelf (WFS) is one of the largest and most diversely-used continental shelf/slope systems in the world. The presence of paleoshorelines and scarped hardbottom outcrops (up to 4 m in relief) along the inner shelf (10-30 m depth) provide important habitat for a variety of infaunal, epifaunal, and fish assemblages that contribute to the productivity of the region. This dissertation will present a comprehensive overview of the geological, physical, and chemical settings of the inner West Florida Shelf, with particular focus on biological and ecological community dynamics of epibenthic macroinvertebrates, algae, and fish assemblages. Baseline and comparative data sets are presented in the form of historic and modern species lists, with focus on seasonal and intra-annual variations. Quantitative effects of disturbances (e.g., hurricanes, thermal stresses, and red tides) and subsequent recovery rates are discussed as they periodically perturb inner-shelf systems and can have significant effects on community structure. Benefits of and recommendations for using artificial reefs as restoration tools along the inner shelf, as mitigation for future disturbances, are presented.

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.

Karenia brevis (Davis cf. Hansen & Moestrup = Gymnodinium breve) is the non-peridinin containing dinoflagellate responsible for many harmful algal blooms (red tides) in the Gulf of Mexico. These recurrent blooms can have significant negative ecological, economic, and human health impacts including fish kills, tainting of shellfish, poisoning of marine mammals, loss of tourism revenue due to beach closures, and respiratory distress and food poisoning in humans. A method for detection of Karenia brevis was developed based upon amplification of the mRNA for the plastid-encoded gene of the carbon fixing enzyme ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). Using sequence information from a primer set targeting a 554-bp region of the Karenia rbcL gene, a small (91 bp amplicon) primer and probe set was created for TaqMan(registered trademark) real time RT-PCR of K. brevis rbcL. The primer/probe set is sensitive to as little as 0.1 fg of target transcript and as little as 1 pg of total cellular K. brevis RNA extract, corresponding to less than 1 cell reaction-1. The primer/probe set did not amplify rbcL transcript from any of the non-target algae tested. Bloom samples analyzed by this method have shown the assay to be a reliable method, with effective enumeration and a linear relationship showing good correlation to the cell counts by microscopy (r2= 0.8344). The assay has been shown to be robust and perform well even in non-ideal conditions, with pre-extraction RNA from unialgal culture stable at room temperature for up to 3 days and up to a month at -80 degrees C in Stratagene's lysis buffer. The transcription of the rbcL gene demonstrated minor variation throughout the diel period, however the variation was not linked to the diel cycle or to carbon fixation, which showed a distinct diel signal. Due to the relatively constant expression of the rbcL gene, the real-time RT-PCR assay developed should be able to reliably enumerate K. brevis populations in the natural environment, as long as the sample is placed in Stratagene's lysis buffer and processed within one or two days or frozen at -80 degrees C and processed within a month.

The species composition of planktonic communities is determined not only by abiotic factors, such as nutrient availability, temperature, and water column stratification but also by biotic interactions between hosts and parasites, predators and prey, and among competitors. Blooms of the red tide dinoflagellate, Karenia brevis, can dramatically alter the planktonic community, reaching densities of millions of cells per liter and occurring nearly monospecifically. I investigated whether K. brevis uses chemical compounds to defend against grazing or to inhibit the growth of competitors. Because K. brevis is known to produce brevetoxins which act as potent neurotoxins in mammals, I also investigated whether brevetoxins played a role in competition or predator resistance. Experiments revealed that copepods fed diets rich in Karenia brevis experienced lowered fitness, however, nutritional inadequacy, rather than toxicity, was responsible for the decrease in grazer fitness. Compounds exuded from natural samples of K. brevis blooms did, however, inhibit the growth of four of five model competitors. Compounds exuded from K. brevis cultures were similarly allelopathic to competitors. Exposure to these allelopathic compounds resulted in lowered photosynthetic efficiency of all competitors, and decreased cell membrane integrity of three competitors. The allelopathic potency of K. brevis blooms was variable between collections and years, but allelopathy did not correlate with bloom density or concentration of brevetoxins. However, the variability of allelopathy could partially be explained by the presence of specific competitors. The diatom Skeletonema costatum reduced the growth-inhibiting effects of K. brevis bloom exudates, suggesting that S. costatum has a mechanism for undermining K. brevis allelopathy. Allelopathic compounds exuded by K. brevis that inhibited the growth of the diatom Asterionellopsis glacialis were partially characterized. K. brevis produced multiple, polar, organic compounds that inhibited A. glacialis growth. Exuded brevetoxins, on the other hand, had no effect on A.glacialis growth. Taken together, these results indicate that K. brevis is not chemically defended against grazing, but does produce yet-unidentified allelopathic compounds that inhibit the growth of competing phytoplankton. Blooms of K. brevis may be facilitated by the exudation of potent allelopathic compounds, but the specific phytoplankton assemblage has the potential to alter bloom dynamics.

The following document provides insight into the uncharted process of producing a micro-budget feature length film. This paper aims to document my growth as an artist in terms of storytelling and filmmaking as well as the development and production process. Red Tide: A Feature Length Motion Picture includes elements from each phase of the production process, from story and script development to marketing and distribution. This document reflects on the obstacles we faced and the solutions we implemented during the process of creating a feature length motion picture on an undersized budget.

Commonly referred to as “red tide”, harmful algal blooms (HABs) formed by Karenia brevis occur frequently in the Gulf of Mexico (GOM). A bloom is defined as cell abundances >105 cells L-1. This thesis will focus primarily on Karenia brevis, formerly known as Gymnodinium breve, in the Gulf of Mexico. K. brevis is harmful because it produces brevetoxin, a ladder-frame polyether that acts as a potent neurotoxin in vertebrates. K. brevis commonly causes fish kills, respiratory irritation in humans, and Neurotoxic Shellfish Poisoning (NSP) if ingested. Blooms of K. brevis occur almost annually along the West Florida Shelf (WFS) in the late summer and early fall, when the coastal current is favorable for bloom initiation. Along the Texas-Louisiana shelf (TLS) however, blooms of K. brevis are infrequent and sporadic. While much is known of the blooms along the WFS due to their frequent presence, little is known of the mechanisms driving the blooms along the TLS due to their inconsistent presence. To understand the stochastic nature of HABs along the TLS, historical data of bloom occurrences from 1996 to present were compared with NOAA station PTAT2 wind, sea-level pressure, air and water temperature data and NCEP NARR-A sea-level pressure data. The difference in the monthly-mean along-shore component of the wind was statistically significant between bloom and non-bloom years in September (p<<0.001) and April (p=0.0015), with bloom years having a strong downcoast current. Monthly mean water temperature values yielded similar results between bloom and non-bloom years. Both March and September monthly-mean water temperature values were lower during non-bloom years with p-values of 0.01 and 0.048, respectively. These results suggest the possibly of forecasting for HABs along the TLS with currently measured, publicly available data.

A natural phenomenon characterized by dense aggregations of unicellular photosynthetic marine organisms has been termed colloquially as red tides because of the vivid discoloration of the water. The dinoflagellate Karenia brevis is the cause of the Florida red tide bloom. K. brevis produces the brevetoxins, a potent suite of neurotoxins responsible for substantial amounts of marine mammal and fish mortalities. When consumed by humans, the toxin causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin within the organism has remained mysterious since its discovery. There is a need to identify factors which contribute to and regulate toxin production within K. brevis. These toxins are produced and retained within the cell implicating a significant cellular role for their presence. Localization of brevetoxin and identification of a native receptor may provide insight into its native role as well as other polyether ladder type toxins such as the ciguatoxins, maitotoxins, and yessotoxins. In higher organisms these polyether ladder molecules bind to transmembrane proteins with high affinity. We anticipated the native brevetoxin receptor would also be a transmembrane protein. Photoaffinity labeling has become increasingly popular for identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification provides the protein with the ligand directly attached. A molecule that is capable of fluorescence is a fluorophore, which upon excitation is capable of re-emitting light. Fluorescent labeling uses fluorophores by attaching them covalently to biologically active compounds. The synthesis of a brevetoxin photoaffinity probe and its application in identifying a native brevetoxin receptor will be described. The preparation of a fluorescent derivative of brevetoxin will be described and its use in localizing the toxin to an organelle within K. brevis. In addition, the general utility of a synthesized photoaffinity label with other toxins having similar functionality will be described. An alternative synthetic approach to a general photoaffinity label will also be discussed whose goal was to accelerate the preparation and improve the overall synthetic yields of a multifunctional label.

Alexandrium tamarense is a harmful algal species that can produce saxitoxins, a suite of powerful neurotoxins that bioaccumulate up the food chain and can have severe economic and health impacts. With harmful algal blooms increasing temporally and spatially, it is important for us to understand the relationship between harmful algal blooms and nutrients, particularly nitrogen from anthropogenic sources. To this end, the stable nitrogen isotopic composition (δ¹⁵N) of medium nitrate, algal cells and toxin in both nitrogen-replete and nitrogen-limited batch cultures of A. tamarense were measured in order to assess the potential for using the δ¹⁵N of the toxin as a tracer of the nitrogen source used for growth. A. tamarense cells grown under nitrate-replete conditions were depleted by 1.5‰ relative to the growth medium, and saxitoxin was depleted by 1.5‰ relative to the whole cells. Under nitrate-limiting conditions, the isotopic difference between cells and saxitoxin changed as nitrate in the growth medium was depleted, indicating uncoupling of toxin synthesis and cell growth rates under changing external nutrient conditions. Determination of the absolute magnitude of the isotopic differences between the medium nitrate and either the cells or the saxitoxin was confounded by 1) using two different nitrate sources – one nitrate source was used to grow the inoculum and a different nitrate source was used for the experimental medium - with different ‰ values and 2) the presence of an unidentified, isotopically-light, nitrogen blank in the low-nitrate medium samples. I conclude that STX nitrogen isotope values have the potential to be used as nitrogen source indicators. However, overall fractionation between whole cells and STX is unknown due to the uncoupling between cell growth and STX synthesis observed during my nitrogen-limited experiment. Based on previous research on cell growth and toxin production dynamics under different nutrient regimes, it is also reasonable to assume that the observed results here may differ if a different nitrogen source was utilized by the cells for STX production. Further research could include isotope analysis of cultures grown on different nitrogen sources, such as ammonium and urea; isotopic analysis of additional compounds, such as amino acids; or use of additional stable isotopes, such as C or O. / text

Coral cover is in decline on a global scale, with increased mortality events being attributed to a number of global and regional stressors. While the impacts of global stressors (e.g. sea surface temperature rise, ocean acidification) are well documented, there is growing interest in identifying and understanding the impacts of regional stressors. The reason for this change in focus is that regional stressors can often work in combination, sometimes synergistically, with global stressors and that stressors on a regional scale tend to be more easily mitigated by management practices. One regional stressor that impacts a myriad of marine organisms in the southeastern United States is the annual red tide blooms produced by the dinoflagellate, Karenia brevis. Their impacts, along with the lipid soluble polyether neurotoxins they produce, termed brevetoxins, are well studied in economically important organisms, such as bivalves. However, little is known of their impacts on organisms that possess ecological importance, such as species of scleractinian coral. To address this gap in knowledge, a multifaceted study is discussed herein, which evaluated the effects of ecologically relevant concentrations of K. brevis and associated brevetoxins on different coral life history stages and coral species. The second chapter addresses the impacts of red tide on larval behavior, settlement and survival of the coral species Porites astreoides, as well as impacts of photochemical efficiency and oxidative stress within different coral species (P. astreoides larvae, P. astreoides adults, Acropora cervicornis, Cladocora arbuscula, and Phyllangia americana). The third chapter confers the use of broad-scale proteomic analysis to identify the cellular response of the non-model coral species, P. astreoides, following exposure to red tide. Coral larvae actively avoided both medium and high bloom conditions of K. brevis and brevetoxins, while percent larval settlement and survival were impacted following exposure to high bloom concentrations of K. brevis. Photochemical efficiency of in hospite Symbiodinium was reduced following exposure to both K. brevis and brevetoxin in P. astreoides larvae, as well as exposure to K. brevis in P. astreoides adults, while being unimpacted in A. cervicornis. Compared to controls, high bloom conditions resulted in an increase in biomarkers of lipid peroxidation in C. arbuscula. This was also seen in P. astreoides larvae at 24 hours; however, this difference was indistinguishable following 48 hours. Surprisingly, no other biomarker of oxidative stress analyzed were impacted. Broad-scale proteomic analysis of P. astreoides following exposure to red tide conditions revealed variable changes in proteome expression depending on if the corals were exposed to K. brevis or brevetoxins. Exposure to brevetoxins resulted in differential expression of proteins related to DNA organization, chromatin formation and transcription expression; while exposure to K. brevis resulted in differential expression of proteins related to redox homeostasis, protein folding, energy metabolism, and production of reactive oxygen species. The results of this study demonstrate the potential for annual red tide blooms to act as a regional stressor on coral species. They highlight the ability of red tide conditions to negatively impact coral at multiple life history stages and that the extent of these effects may be species specific. They also provide further incite of coral’s response to red tide exposure at the cellular level.