ECOHAB 2001: Project Summaries
Anderson, D.M. (WHOI).
Harmful algal blooms (HABs) are a serious economic and public health problem throughout the world. In the U.S., the most widespread HAB problem is paralytic shellfish poisoning (PSP), a potentially fatal neurological disorder caused by human ingestion of shellfish that accumulate saxitoxins produced by dinoflagellates in the genus Alexandrium. PSP closures affect thousands of miles of U.S. coastline and numerous fisheries resources annually.
Understanding the processes that lead to the growth, accumulation, and negative impacts of toxic dinoflagellates is a fundamental goal of HAB research, and an essential element of prevention, control, and mitigation (PCM) strategies. In particular, the factors that regulate dinoflagellate toxicity need to be elucidated, as these directly affect the nature and quantity of toxins that enter the food chain. For PSP toxins, this requires knowledge of not only the abundance of the toxic cells, but of their total toxicity (toxin content) and the chemical nature of that toxicity (toxin composition), since more than 20 saxitoxins exist, with widely varying potencies. Furthermore, efforts to model or predict toxicity require a detailed parameterization of the manner in which these toxicity characteristics are affected by environmental factors such as temperature and nutrients. There have been studies of Alexandrium toxicity as it is affected by environmental factors, but that database proved inadequate when it was recently used to interpret field observations during a major Alexandrium bloom in the Gulf of Maine. In particular, some of the patterns that were expected based on the laboratory data were not observed, such as a decrease in toxin content or a significant increase in C toxins as the cells became nutrient limited, both of which had been considered robust indicators of nitrogen limitation. Part of the problem in extrapolating laboratory culture studies to field populations is that the former did not use Alexandrium isolates representative of the region under study, nor has a "generic" response or diagnostic indicator been identified that applies to all Alexandrium populations under all conditions. In addition, the experimental procedures did not adequately simulate conditions in the natural environment, especially the transition to sexuality and cyst formation that occurs under nutrient limitation. The overall goal of the project proposed here is to use semi-continuous cultures and field-deployed mesocosms to characterize and parameterize the manner in which Alexandrium populations vary in toxicity in response to environmental and nutritional fluctuations. Specific project objectives are to: 1) Establish a set of Alexandrium isolates that represent the extremes of regional and temporal population variability for the Gulf of Maine in particular, and for globally distributed Alexandrium, in general; 2) Document the extent to which toxin content and composition vary in these isolates under different nutritional conditions using semi-continuous cultures; 3) Determine the extent to which nutrient-driven toxicity variations are modulated by temperature; 4) Study the effects of sexual induction and cyst formation on patterns of toxin accumulation; 5) Identify robust indicators of nutrient limitation, considering trends in toxin content and a variety of toxin composition ratios; 6) Evaluate the validity of these nutritional indicators using mesocosm deployments and controlled nutrient additions at sites of natural Alexandrium blooms; 7) Use the observed patterns of toxin composition variability to infer saxitoxin biosynthetic and interconversion pathways and mechanisms and identify generic responses to nutrient limitation; and 8) Incorporate nutrient and toxin variability into existing physical/biological coupled models for Alexandrium in the Gulf of Maine.
The information to be gained from this study will add much to our ability to understand, model and predict PSP outbreaks caused by Alexandrium, and to assess the extent to which natural populations are nutrient limited using an indicator which is unique to this group of saxitoxin-producing organisms.
Buskey, E.J. (UT-Austin).
The objective of this proposal is to better understand the potential role of zooplankton grazers in the initiation and maintenance of harmful algal blooms, and to better define their role in the transfer of toxins through the food chain where they can potentially be ingested by humans or endangered wildlife species. This project will focus on the toxic dinoflagellate Gymnodinium breve which causes extensive fish kills, human health risk and economic loss along the Gulf of Mexico coast. Protozoan grazers have rapid growth rates and are the most likely grazers on harmful algal species to be capable of controlling blooms during their initial phase. Metazoan planktonic grazers such as copepods may also graze on harmful species, but their longer generation times make them poor candidates for controlling blooms. However, since some copepods tend to avoid consuming toxic phytoplankton species, they may switch to alternate prey, including protozoan grazers of harmful algal species, which may indirectly aid the formation or maintenance of blooms. A series of laboratory experiments is proposed to measure the grazing and growth rate of zooplankton grazers on harmful algal blooms and the impact of metazoan planktonic grazers on the interaction between protozoa and harmful algae species, to determine the role of planktonic grazers in harmful algal bloom dynamics and as vectors to transfer toxins to higher trophic levels. Although several studies have shown that some species of harmful algae are not acutely toxic to zooplankton, there has been less study of sublethal effects of toxins on zooplankton. If these toxins, many of which act as neurotoxins, affect the behavior of zooplankton, this might make them more likely prey for visual predators. Changes in behavior could both make the zooplankton more conspicuous to their predators and might also reduce the efficacy of their escape behaviors. In this study we propose to use video computer motion analysis techniques to see if toxins produce abnormal swimming behavior in zooplankton that ingest them. We will also use high speed video to carefully examine the kinetics of copepod escape behaviors to see if zooplankton that ingest toxic algae have impaired escape responses, making them more likely prey and increasing the chances of biological magnification of toxins. If the opportunity presents itself, we also propose to sample zooplankton during an outbreak of red tide along the Texas coast to examine feeding behavior of zooplankton under natural conditions and to test for copepod growth and toxin content within bloom areas.
Connell, L. (UMaine), V. Trainer (NOAA/NWFSC), and V.M. Bricelj (IMB).
The proposed multidisciplinary research collaboration will characterize the complex mechanism underlying bivalve susceptibility to paralytic shellfish toxins (PSTs) and species-specific toxin accumulation. In mammals, PSTs affect nerve function via specific block of the voltage-sensitive Na+ channel. Bivalves, however, clearly have adaptations that permit them to tolerate toxins in their algal food. Specifically, "insensitive" bivalve species are known to harbor, without apparent harm, high concentrations of PSTs, while more "sensitive" species attain relatively low toxin levels and can suffer sublethal or lethal effects from harmful algal blooms (HABs). This susceptibility to ingested toxins and thus, ability to accumulate toxins, varies markedly both within and among bivalve species.
Although many examples of resistant and sensitive Na+ channels have been described in nature, little is known about the genetic basis of these different subtypes in molluscan shellfish. For example rat and human cardiac Na+ channels are resistant to toxins, whereas their skeletal muscle Na+ channels are sensitive. Additionally, natural resistance to pesticides has developed in the Na+ channel gene of some insect populations. Our current ECOHAB research has characterized up to 50-fold differences in toxin affinity among populations of Mya arenaria, providing evidence for the presence of a Na+ channel mutation that confers resistance to PSP. A key goal of this proposal is to more completely characterize the basis (at the molecular level) for inter- and intraspecific variation in toxin uptake and sensitivity in bivalves. Specific objectives of this research will be to characterize toxin tolerance (and thus also toxin accumulation) at the level of (1) the Na+ channel gene, with specific focus on the known sites of PST action; (2) the isolated nerve; (3) behavior (burrowing); and (4) feeding physiology and toxin uptake. Interspecies differences will shed light on protective mechanisms used by various shellfish to resist the effects of toxins using both short-term behavioral and long-term genetic strategies, as well as on the fate of toxins through the ecosystem.
This research will help to develop genetic markers for identification of bivalves that have natural mutations in the Na+ channel gene. These will be related to the organisms¹ ability to tolerate (and thus concentrate) toxin. Interspecific differences in shellfish susceptibility to toxins will be explored in oysters (Crassostrea gigas, C. virginica), and clams (Mya arenaria, Siliqua patula, and Saxidomus giganteus) from historically toxic and non-toxic areas on the Pacific (including Alaska) and Atlantic coasts of N. America. Genetic markers will allow selection of non-accumulating bivalve stocks in a given region, thereby reducing harvest losses. Identification of inter- and intraspecific genetic differences will contribute to our fundamental understanding of toxin resistance mechanisms and perhaps open future avenues for selective breeding. Regional characterization of bivalve responses to toxic algae will help to predict the impacts of paralytic shellfish poisoning (PSP) at the level of primary consumers over a wide geographical range. Understanding of the relationship of specific toxin vectors to the intensity and frequency of HABs in a given area, will contribute to improved management of commercially important shellfisheries and assessment of human health risks associated with HABs.
Dam, H.G. (UCONN-Avery Point)
Doucette, G.J. (NOAA/CCEHBR).
Blooms of the toxic dinoflagellate Gymnodinium breve occur in coastal waters throughout much of the Gulf of Mexico, but are especially problematic along the Florida coast where these annual events last at least 3-4 months and exact a large toll on the regional economy with losses estimated at about $20 million per episode. As a result, there is a growing interest in exploring strategies for the prevention, control, and/or mitigation of G. breve blooms. Understanding the population dynamics of harmful algal blooms (HABs) and elucidating the factors controlling transitions between bloom phases are critical for effectively dealing with HABs such as Florida¹s red tides, and ultimately minimizing their socio-economic impacts. Thus, a logical approach for efforts aimed at identifying potential strategies for managing G. breve blooms and their negative consequences is to acquire knowledge of natural mechanisms involved in regulating population growth of this species.
Several reports of algicidal bacteria in high concentrations at the onset of HAB termination have led to the suggestion that such microbes may directly or indirectly lead to the decline of blooms. The issue of bacterial involvement in terminating algal blooms has only recently been addressed in relation to HABs occurring in U.S. waters. In a previous ECOHAB project, our group initiated a search for algicidal bacteria active against G. breve in waters of the west Florida shelf. This effort led to the first discovery of algicidal bacteria targeting a HAB species resident along the U.S. coast. In fact, two bacterial strains lethal to G. breve were isolated, classified phylogenetically using 16S rDNA sequences, and their killing activity partially characterized. Moreover, we have used the sequence data to develop taxon-specific rRNA probes, which have been used as tools to begin examining the population dynamics of these algicidal bacteria. Additionally, study of the killing activity revealed that both algicidal strains released a dissolved bioactive compound(s) into the growth medium, causing the death and lysis of G. breve.
We have developed a conceptual model for the population dynamics of algicidal bacteria over the course of a G. breve bloom event based on our previous findings, and propose to test these ideas in laboratory cultures, microcosms, and field populations with the work described herein. A principle hypothesis is that algicidal bacteria targeting G. breve, initially a background component of the ambient bacterial community, increase in both absolute and relative abundance over the course of a bloom. Taxon-specific rRNA probes and other molecular tools (e.g., DGGE) employed in our previous work will provide the means to track populations of these algicidal bacteria within mixed microbial assemblages in laboratory cultures and microcosms, as well as samples taken from G. breve blooms, and thus to better assess their involvement in promoting bloom decline. The other focus of this project will be identification of the bacterially produced algicidal compound(s) and elucidation of its mode of action, thereby permitting development of an assay for the detection and measurement of this activity.
These complimentary lines of investigation involving both the organism and its biologically active compound(s) will yield a better understanding of the role played by algicidal bacteria in terminating G. breve blooms, as well as further insights into the potential use of these bacteria as part of an effective management strategy for HABs and their frequently devastating impacts.
Durbin, E.G. (URI), G.J. Teegarden (Bowdoin College), R. Campbell (URI), and A.D. Cembella (Institute for Marine Biosciences, NS).
Harmful algal blooms threaten public health and economic activities (fisheries, aquaculture, and tourism) in coastal environments. The biological interactions of harmful species and their competitors and predators are significant and may determine bloom dynamics and ecosystem impacts. Results from our recent ECOHAB study suggest that grazer response to harmful algal blooms will vary with spatial and temporal changes in grazer communities and prey field assemblages. Based on these results, we propose a study of the role of grazing by zooplankton in the population dynamics of Alexandrium, and the transfer and fate of toxins in marine food webs. Field and laboratory experiments will test these principal hypotheses: 1) Grazer response to toxic Alexandrium in natural assemblages is dependent on Alexandriumconcentration and abundance relative to co-occurring phytoplankton (e.g. stage of bloom development). 2) Grazing pressure on Alexandrium depends on the composition and abundance of the co-occurring phytoplankton community. 3) Grazing pressure on Alexandrium depends on the composition, abundance, and selective feeding ecology of the grazing community. 4) Significant losses of both toxin and carbon from ingested Alexandrium cells result from destructive rejection of toxic cells, with implications for toxin transfer and secondary production. Laboratory experiments will: 1) Examine the feeding behavior and selectivity of dominant zooplankton from the Gulf of Maine in gradients of Alexandrium cell concentrations and alternate prey. 2) Investigate the acute and chronic effects of toxic Alexandrium exposure on grazing behavior, toxin assimilation, detoxification, growth, and reproduction. Field studies in the Grand Manan Basin, an upstream source region of Alexandrium for the Gulf of Maine, will use fine-scale drifter studies to: 1) Determine the feeding behavior of zooplankton in this region of higher abundance of Alexandrium. 2) Investigate the role of grazing by zooplankton in controlling Alexandriumblooms. 3) Determine fine-scale distribution of zooplankton, potential co-existence of zooplankton and layers of Alexandrium, and the temporal dynamics of zooplankton feeding processes in layers, using process studies in distinct water masses. 4) Describe the accumulation of toxin in zooplankton tissues and determine whether toxin accumulation in zooplankton poses risks to higher trophic levels. The results will address several program objectives. Characterization of grazer response over a continuum of bloom conditions will provide basic information on the significant role of grazers in the regulation (or lack thereof) of harmful algal blooms. A grazer deterrent effect (which would promote bloom formation), apparent under some but not all conditions, is a hypothesized ecophysiological role for phycotoxin production, and our proposed study will directly address the validity of this hypothesis. Zooplankton grazers can act as vectors of toxin in food webs; the proposed study will provide quantitative assessment of this threat to ecosystems over a variety of bloom conditions. The results will expand on our recent ECOHAB study to provide quantitative estimates of grazing impact over the range of Alexandrium bloom conditions, currently lacking, but necessary for accurate modeling of harmful bloom dynamics.
Kamykowski, D. (NCSU) and G. S. Janowitz (NCSU).
Abbreviated versions of the hypotheses to be tested are: 1) populations of Gymnodinium breve can be reliably quantized (all cells divide together every 3rd day) by isolating the cells that vertically migrate to form a population aggregate in the mid-afternoon surface layer into a separate culture; 2) in nutrient replete water columns, unequal cell division by Heterocapsa illdefina and G. breve parent cells yields a high variance of biochemical content among daughter cells at all depths immediately after cell division; 3) all G. breve cells in a nutrient replete water column have similar generation times irrespective of the depth occupied; 4) G. breve cells that grow in a water column where nitrogen is vertically patchy undertake diel vertical migrations influenced by the vertical location of the nitrogen source; 5) the toxin content of aggregating and non-aggregating G. breve cells differs because of unequal allocation at division and/or because of different, behaviorally determined light/nutrient exposure in the water column; and, 6) environmentally and behaviorally realistic physical-biological models are required for accurate HAB prediction. These hypotheses will be tested using proven experiment protocols applied to 225 L cultures of G. breve and will be extended to natural populations by refining our evolving physical-biological numerical models.
Predicting when and where different dinoflagellate species significantly contribute to HABs presently is compromised by the incomplete information on how cells use their motility. This information is difficult to obtain with field populations because of unknown population history associated with patchy events in time and space that occur in a dynamic fluid environment. The proposed laboratory work, under conditions where the population history is controlled and the same population is continuously available, will help define the biological context within which cell behavior influences vertical position in the water column and, thus, cell growth/division rates and horizontal transport.
Kudela, R.M. (UCSC), Smith G.J. (SJSU) and Armbrust, V.E. (UW).
The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used to interrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested:
H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia.
H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development.
H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer.
Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.
Maranda, L. (URI), S.L. Morton (NOAA), and P.E. Hargraves (URI).
The overall goal of this proposal is to determine the potential for diarrhetic toxins to contaminate shellfish resources along the coast of New England. This research will contribute to a better understanding of the population dynamics of the toxin producer, Prorocentrum lima, within the epibiotic communities of wild and cultured shellfish and examine the relationship between the abundance of P. lima and shellfish contamination with diarrhetic toxins. Our specific objectives are: 1) to determine the seasonal distribution of P. lima associated with wild and cultured shellfish, 2) to elucidate the quantitative relationships between P. lima and the epibiotic community fouling cultured shellfish, and 3) to determine the toxin load and composition of the epibiotic community, and of wild and cultured shellfish over time. Three hypotheses will be tested: 1) P. lima from New England coastal waters is toxigenic with respect to production of okadaic acid and derivative active compounds, 2) P. lima is more abundant within the epibiotic community associated with suspended shellfish rather than with wild shellfish, and 3) Shellfish grown in suspension culture become contaminated with DSP toxins at a faster rate and at a higher level than wild shellfish.
The seasonal distribution of P. lima and epibiota will be assessed at eight sites along the New England coast where P. lima is either known or suspected to occur; four sampling stations will be located at shellfish aquaculture facilities and four will be in coves open to wild shellfish harvesting when in season. Toxin analysis will be performed on epibiota < 90 µm) and on the digestive glands of shellfish at given locations. Toxin will be reported in terms of protein phosphatase inhibition activity and toxin composition.
We know P. lima is widespread in New England coastal waters. This presence signals the potential for diarrhetic shellfish poisoning. We know that low levels of okadaic acid-like activity have been detected in blue mussels along the coast of Maine. We now need to find out whether and to which extent this DSP potential can be realized and under what conditions. This proposal attempts to fill some of these gaps (first area of emphasis of the ECOHAB solicitation), especially in light of changes in aquaculture practices from on-bottom to suspension mode (specific topic #2), creating conditions favorable for the growth of P. lima. We expect to provide results which will allow us to verify whether the pattern observed in eastern Canadian waters may repeat and affect shellfish and public health in the U. S. Northeast.
Mulholland, M.R. (ODU) and E. Minor (ODU).
09/01/01 - 08/31/03.
Dissolved organic material (DOM) has been implicated as a causative agent promoting the growth of harmful algal species and initiating blooms in the inland waterways of the Eastern United States. In particular, the Brown Tide chrysophyte, Aureococcus anophagefferens, occurs at bloom densities in the inland waterways of NY, DE and MD. It is likely that its geographical range extends into VA. These areas tend to be shallow and have restricted flushing and therefore are impacted by nutrient inputs from terrestrial runoff, groundwater inputs and sediment resuspension. The objectives of this project are to: identify compounds and compound classes that stimulate the growth of A. anophagefferens, determine possible sources of DOM and its importance to the nutrition of this species, assess how competition for DOM and nutrients affects growth of A. anophagefferens relative to co-occurring taxa, and determine the nutrient conditions that promote blooms.
To meet our project goals, we have selected field sites that have similar physical attributes (e.g., circulation and morphology) but different densities of A. anophagefferens. We will conduct intensive process studies at a site in Chincoteague Bay, where A. anophagefferens is known to occur at high densities, and at more southerly sites that are less urbanized, have different sources of DOM and have not previously experienced high abundances of this species (e.g., Hog Island Bay), but may be vulnerable to blooms if there are changes in nutrient conditions. These sites have the advantage of being near Norfolk and the Virginia Institute of Marine Science¹s Eastern Shore Laboratory (ESL) in Wachapreague, VA. In addition, there are long-term monitoring data available for both sites. At each site, and one near to Wachapreague, we will employ a variety of new techniques to characterize DOM, identify and measure the primary pathways of C and N cycling and determine the competitive interactions that affect the cycling of DOM and its use by mixotrophs, such as A. anophagefferens. In addition, we will conduct outdoor mesocosm experiments at ESL using natural populations. This will enable us to determine the effects of longer-term (14 - 21 days) nutrient enrichments on competition for DOM and nutrients and competition among co-occurring species. This work will answer basic questions regarding the extent to which organic N and C supports the growth of A. anophagefferens, the nutrient conditions that stimulate blooms and mixotrophy, how competition for DOM varies depending on its source and composition, and differences among areas that experience blooms and those that do not.
Paul, J.H. (USF).
By conservative estimates, harmful algal blooms cost the U.S. over $50 million/year. In the Gulf of Mexico, K. brevis has caused economic loss and massive marine animal (including mammal) mortalities for over a century. Methods for rapid detection of blooms and monitoring of specific K. brevis strains are greatly needed. We have recently cloned and sequenced a portion of the K. brevis carbon fixation gene, rbcL. As a coding gene (in contrast to rRNA genes) we hypothesize that there might be enough genetic diversity present in rbcL to differentiate K. brevis strains. Additionally, as a functional gene which is expressed in viable cells, we can detect its expression (as mRNA) as an indicator of viable (transcriptionally active) cells. Our overall objective is to understand the function of this gene in this organism, its regulation, its organization within the plastid genome, and its ability to serve as a marker for K. brevis detection in the field. Our specific objectives are: 1) To design Real Time PCR probes for detection, quantitation, and differentiation of Karenia strains based upon rbcL sequence data 2) To assess the performance of the probes with Karenia strains in cultures and in field samples and 3) To determine the molecular regulation of the carbon fixation genes in K. brevis. For the first objective, we will sequence strains provided by Karen Steidinger of the Florida Marine Research Laboratory and design probes based upon sequence alignments. For the second objective, we will test probes against these strains and natural seawater samples containing bloom and non-bloom levels of K. brevis. For the third objective, carbon fixation and rbcL transcript abundance will be determined in diel studies in cultures to understand regulation of the carbon fixation operon. Collectively these studies will yield a functional approach to the issue of carbon fixation in K. brevis, as well as provide probes for the specific detection of active strains in the field.
Frances M. Van Dolah, F.M. (NOAA/CCEHBR).
Development of effective management strategies for addressing the occurrence and impacts of harmful algal blooms is dependent upon adequate insight into the mechanisms that control bloom initiation, growth and termination. Dinoflagellates are the major group of microalgae responsible for the production of toxins that impact human and environmental health. Among these, the Florida red tide dinoflagellate, Gymnodinium breve, is among the most notorious. G. breve blooms occur almost annually off the west coast of Florida, where they cause extensive fish kills, marine mammal mortalities, and human illness due to both respiratory irritiation and neurotoxic shellfish poisoning. Blooms of G. breve initiate offshore, and become a threat to coastal ecosystems and humans only when carried inshore by prevailing wind and oceanographic conditions. Once in coastal waters, G. breve cells may experience dramatic changes in environment, resulting in temperature, salinity, pH, light, turbulence, and oxidative stresses. As a consequence, blooms may either rapidly die out, or they may adapt and persist for many months within coastal embayments, where their impacts on the coastal populations of marine animals and coastal economies may be devastating.
A goal of the ECOHAB Program is to develop scientifically sound approaches to prevention, control and mitigation of HABs. The processes that result alternatively in cellular adaptation and bloom persistence, or cell death and bloom termination, may be suitable targets for developing predictive indicators or control strategies applicable to limited, sensitive areas. Analogous to cancer research, we must understand the cellular processes driving the consequences of this "decision point" in the fate of a bloom, before we can potentially modulate its outcome. In this project, we propose to elucidate cellular mechanisms in G. breve that mediate adaptation of G. breve cells to environmental stressors, and mechanisms that mediate G. breve cell death.
All eukaryotic cells possess stress proteins that are activated in response to denaturing cellular stress, including heat shock proteins (sHsps), molecular chaperones (Hsp60, Hsp70), and antioxidant systems (e.g., GSH, SOD). In the current project we propose to identify stress proteins present in G. breve and characterize their expression in response to relevant environmental stressors likely to be encountered by coastal blooms. Failure of cells to adapt to such stressors results in cell death. The pathways present in dinoflagellates that trigger cell death are largely uninvestigated. Programmed cell death (PCD) is a highly conserved mechanism by which eukaryotic cells selectively die. Although previously thought to have evolved in multicellular organisms, recent evidence indicates the presence of at least certain components of this pathway in unicellular protists. We propose to determine the presence of the PCD in G. breve, evaluate its activity during the growth, stationary phase, decline phases of laboratory cultures, and determine the triggers for its activation.
Following characterization of stress responses and cell death pathways in laboratory investigations, we will evaluate their expression in growth vs termination phase blooms of G. breve on the west coast of Florida. This proposal represents the first attempt to define cellular mechanisms responsible for bloom termination in a toxic dinoflagellate. The results of this project will yield molecular biomarkers of physiological status in naturally occurring blooms and may identify novel targets for control measures. Although this work will be carried out in G. breve, we anticipate that insight gained in this species, and tools developed for this species, will be broadly applicable to other toxic dinoflagellate species.
Vogelbein, W.K. (VIMS), L.W. Haas (VIMS), K.S. Reece (VIMS), and J.D. Shields (VIMS).
The toxic ambush-predator dinoflagellate, Pfiesteria piscicida, is considered a serious emerging fish and human health problem in some Mid-Atlantic U.S. estuaries. Adverse health effects are thought to be caused by potent exotoxins, the production of which appears intimately tied to life cycle transformations and specific environmental cues that regulate them. The life history of P. piscicida is complex and reported to consist of 24 stages including flagellated, amoeboid and cyst forms, several of which are reportedly toxic. However, many aspects of the life history and factors that influence toxigenicity remain poorly documented and controversial. Recently a second species, soon to be described and named Pfiesteria shumwayae, was discovered. Little published information is available on this undescribed species other than that it is believed to be similar to P. piscicida in "toxigenicity" and complex life history. We recently developed a 96 hr larval fish bioassay that provides, for the first time, a sound experimental approach to critically investigate life history and toxigenicity issues of Pfiesteria spp. under rigorously controlled laboratory conditions. This bioassay is the center piece of our proposed studies. Preliminary tests of the assay using our "pathogenic" P. shumwayae cultures, suggest that this species differs substantially from P. piscicida in life cycle complexity and "toxigenicity". We propose several refinements to the larval fish bioassay that will allow critical investigation of the role of environmental cues in life stage transformations and toxigenicity of P. shumwayae including: development and application of "gnotobiotic" fish, exposure studies in sterile membrane-compartmentalized culture flasks, one- and two-cell exposures with sterilized fish tissues and application of our recently-developed, highly specific suite of molecular probes. The objectives of the proposed project are to conduct laboratory and field studies to: (1) document the life cycle of P. shumwayae, (2) determine the biotic and abiotic environmental cues that regulate life cycle transformations and toxigenicity, (3) identify which of the life history stages are pathogenic or "toxigenic", and (4) examine the role of filter feeding organisms (e.g., menhaden and oysters) in the regulating life history events and expression of toxigenicity in this new dinoflagellate. Our approach is to examine different life cycle stages using flow cytometry for ploidy, lectin biomarkers for strain or stage specificity, histopathology, SEM and TEM for toxigenicity and life stage studies, and labeled molecular probes to verify participation of the various observed life forms (e.g., amoebae) in the P. shumwayae life cycle. We have observed P. shumwayae and other PCOs in the alimentary tracts of fishes from the laboratory and the field. Thus, do menhaden and oysters serve as vectors in the life cycle of Pfiesteria spp., or does passage through the alimentary tract of these filtering organisms induce life stage transformations directly? Can we develop them as biomonitors of environmental outbreaks of Pfiesteria spp.? We outline feasibility studies using in situ hybridization methodology and real-time PCR technology to identify and quantify life cycle stages in the tissues of these model filter-feeders and their possible important role in modulating stage transformations and toxigenicity.