ECOHAB Research Projects (1997 - 2006): Project Summaries

ECOHAB 2006	ECOHAB 2005	ECOHAB 2004	ECOHAB 2003	ECOHAB 2002
ECOHAB 2001	ECOHAB 1999	ECOHAB 1998	ECOHAB 1997

ECOHAB2006: PROJECT SUMMARIES

Investigators: Dr. Lisa Campbell and Dr. John R. Gold
Title: Intraspecific Variation in a Toxin-producing Dinoflagellate
Institution: Texas A&M University
Project Period:  9/1/06 to 8/31/09
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
Toxic dinoflagellates of the genus Karenia are a serious economic and public health concern worldwide.  The major HAB species in the Gulf of Mexico is Karenia brevis, a dinoflagellate that produces a suite of potent neurotoxins (brevetoxins) that can cause fish kills, shellfish toxicity, and respiratory distress in humans.  Cell counts alone are not a good predictor of potential toxicity of HABs because the quantity of toxin can vary with species composition, stage of growth, and/or environmental conditions.  There also is evidence that variation in cellular toxin content and toxin profiles exist among clones of K. brevis.  Factors influencing production of brevenal, the naturally occurring antagonist for brevetoxins, among clones of K. brevis also are unknown.  A more detailed understanding of both genetic diversity and intraspecific toxin composition within and among blooms is needed so that the dynamics and potential potency of toxic dinoflagellate populations can be linked to environmental heterogeneity and change. Objectives of this project are as follows: (1) Establish clonal cultures of K. brevis isolated during the onset, bloom, and decline of a Karenia bloom in order to assess genetic and physiological variability within a bloom population; (2) Determine environmental conditions under which K. brevis cells attain maximal potential toxicity by examining variation of toxin content and toxin profiles among clones and how toxin profiles may be altered by perturbations in the environment; (3) Establish indicators/markers linking genetic profiles and intraspecific variation in toxin production in order to predict potency of a bloom. Approach: Conduct field sampling in conjunction with the ongoing monitoring program for Karenia at the Fish and Wildlife Research Institute (FWRI) in St. Petersburg, Florida. A suite of nuclear-encoded microsatellite markers developed from a K. brevis genomic library will be employed as tools to characterize genetic composition of bloom populations.  For each clonal isolate established during the course of a bloom event, allele and genotype distributions at 10 microsatellites will form the basis for tests of spatial and temporal (genetic) homogeneity.  Bench-scale studies will be performed to evaluate differences in toxin profiles among clones when grown under identical conditions. Experiments with selected clones acclimated to a range of salinities and nutrients in semi-continuous growth and with cultures subjected to rapid changes in growth conditions will be conducted to evaluate effects of environmental conditions on toxin profiles and quantity of brevetoxins and brevenal produced. Data analysis primarily will include tests of spatial and temporal homogeneity (including molecular analysis of variance or amova) of allele (haplotype) and genotype distributions (frequencies).  Estimates of haplotype diversity and intrapopulational nucleotide diversity also will be generated. Neighbor-joining topologies of genetic-distance matrices will be used as a means to assess genetic and evolutionary relationships among spatial/temporal samples and to link diversity and structure of isolates of K. brevis with the intraspecific variation in toxin production. Expected Results: This study will provide critical and much needed information on the variation in toxin composition and production among K brevis clones and over the course of a Karenia bloom.  The database of dinoflagellate microsatellite alleles for the Gulf will be expanded and the extent of diversity in toxin profiles together with genetic profiles will allow development of realistic predictive models.  Linking allelic profiles and toxicity will allow prediction of the response of HAB populations to changes in environmental factors.  Ultimately, this will result in the capability to predict how environmental factors influence toxicity or potency of a Karenia bloom.

Investigators: L. Connell, V.M. Bricelj, P. Rawson
Title: Spread of a sodium channel mutation in softshell clam, Mya arenaria, populations: implications for risk assessment and management of PSP toxins.
Institutions: University of Maine, National Research Council Canada
Project period: 1 Oct 2006- 30 Sept 2009
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by dinoflagellates, Alexandrium spp. on the eastern seaboard of North America, and are accumulated by filter feeding shellfish. Human consumption of toxic shellfish (paralytic shellfish poisoning (PSP)) can result in serious illness or death.  Shellfish that consume PSTs may also be affected, leading to an inability to burrow and a high mortality rate. The softshell clam, Mya arenaria, is a commercially important bivalve with wide latitudinal distribution in North America.  Populations of clams with a history of repeated exposure to toxic Alexandrium spp. have developed a natural resistance to the PSTs produced by these algae. Our previous work has identified a mutation in some M. arenaria conferring resistance to PSTs. The clams bearing this mutation display a resistance to toxic levels of Alexandrium spp and accumulate up to 100-Fold  toxin as compared to wild-type clams. These toxins may act as potent natural selection agents, leading to a spread of toxin resistance to PSTs in M. arenaria populations and accompanying higher toxin accumulation.   Higher accumulation of PSTs in clams can increase the risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to shellfish communities and ecosystems.
Objectives: This project will focus on establishing the range and extent of the mutation currently found in wild populations as well as determining the selective pressure blooms of Alexandrium spp. places on these populations, thereby, altering the amount of toxin entering the food web.  Correlations will be explored between areas with historical PSP exposure and those with the probability of new blooms. In addition to these population studies we will explore the physiological mechanism for toxin-induced mortality though anoxia of the mantle cavity in young clams (spat).
Approach: The methods used for this project have already been well developed.  Those methods include a nerve trunk assay for the determination of potential toxin binding in individual clams, established cDNA and DNA sequencing protocols to conduct a phylogeographic survey of the prevalence of Na+ channel mutations. Selectively bred M. arenaria will be exposed to Alexandrium spp. containing various amounts of toxin and with a range of cell concentrations both in the laboratory and in filed situations to determine the effects on both individual clams and the genetic structure of the population as a whole. Oxygen microprobes will be used to determine the level of anoxia in both resistant and sensitive clams that have been exposed to PST in order to determine if anoxia is a primary mechanism of mortality.
Expect results: The increase of clams carrying a toxin resistant mutation can significantly effect the toxin transfer in other areas of the food web. Genotype information can be used to predict potential toxin load of an individual clam after a highly toxic Alexandrium spp. bloom and clam seed can be set accordingly to limit the overall impact of toxic blooms.  Information about the population structure and its ability to sequester toxin will be useful for shellfish resource managers.

Investigator: Hans G. Dam
Title: Relation Between Grazer Toxin Dynamics and Resistance to Toxic Dinoflagellates
Institution: University of Connecticut
Project Period: 9/1/2006-8/31/2009
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
Description:   Harmful algal blooms (HAB) pose a serious threat to public health, aquaculture and fisheries.  However, the ecological and evolutionary consequences of HAB to grazers, the ramifying effects on food web structure and function, and on the transfer of toxins are not well understood. Toxic dinoflagellates of the genus Alexandrium bloom along eastern Canada and New England. In previous work, we have demonstrated local adaptation (resistance) to toxic Alexandrium in one species of copepod, Acartia hudsonica. This new information is the first documented case of resistance in marine pelagic grazers, and has helped explain disparate and sometimes contradictory results from other previously published studies. Resistance has two important consequences in food-web dynamics: 1) Potential bloom control, and 2) Potentially higher toxin transfer to upper trophic levels. Here, we propose to expand our studies to examine how resistance affects grazer toxin dynamics.
a) Objectives: To determine whether there are differences in toxin accumulation, retention, depuration, and biotransformation between resistant and nonresistant phenotypes of Acartia hudsonica to toxic Alexandrium. We will test the null hypothesis that there is no difference in the ability of resistant and nonresistant phenotype to deal with toxins.
b) Approach: We will continue our comparative studies and expose individuals of resistant and nonresistant phenotypes to diets containing toxic Alexandrium for sufficiently-long periods of time to achieve steady state in toxin accumulation.  In both kinds of phenotypes, we will measure time-dependent toxin ingestion rates, accumulation, retention, and depuration and toxin profile in the grazers relative to the food source.
c) Expected results:  We expect to see differences in all or some of the processes mentioned above involved in toxin dynamics between resistant and nonresistant phenotypes. This new information is directly relevant to two of the ECOHAB study areas: trophic transfer of toxins, and impacts on higher trophic levels. An immediate outcome of this project will be to answer the question of whether resistant grazer phenotypes enhance toxin transfer up the food web. Such information will be useful in constructing more accurate models of food web dynamics, and in predicting the impact of  HAB for higher trophic levels.

 

Investigator:  Christopher J. Gobler
Title: The impact of nutrients, zooplankton, and temperature on growth of, and toxin production by, cyanobacteria blooms in the upper reaches of Chesapeake Bay
Institution: Stony Brook University
Project Period:  03/28/07 – 03/27/10
Funded by EPA NCER STAR ECOHAB
Abstract:
The frequency and intensity of toxic cyanobacteria blooms has increased in recent decades, causing a plethora of acute, chronic and fatal illnesses in animals and humans.  A clear understanding of factors promoting bloom growth and toxicity has remained elusive, partly because blooms are comprised of toxic and non-toxic strains of the same species which cannot be resolved microscopically.  An additional confounding aspect of toxic cyanobacteria ecology is that the presence of toxic strains does not necessarily indicate toxins are being actively synthesized by a bloom.  While research of toxic cyanobacteria blooms in the Great Lakes has intensified in recent years, blooms in the upper reaches of US estuaries have been largely ignored, despite the severity of these events.  For example, recent blooms in the upper reaches of Chesapeake Bay have covered over 50 km and have had Microcystis cell densities (106 ml-1) and toxin levels (> 650 µg microcystin L-1) which exceed levels documented anywhere in the US.  The objectives of this project are to elucidate the role of nutrients, zooplankton grazing, and climatic warming on the growth and toxicity of cyanobacteria blooms in the upper reaches of Chesapeake Bay.  We will utilize quantitative polymerase chain reactions (QPCR) to establish the spatial and temporal dynamics of toxic and non-toxic strains of cyanobacteria and will use reverse transcriptase QPCR to quantify changes in microcystin synthetase gene expression.  We will place the dynamics of these populations and gene expression in the context of physiochemical water column characteristics (e.g. nutrients, T), phytoplankton and zooplankton community structure, and cyanotoxin concentrations.  We will conduct experiments to examine the individual and combined effects of nutrients and temperature on the growth of, and toxin production by, cyanobacteria.  In addition, we will concurrently examine the ability of wild and cultured micro- and mesozooplankton to graze on toxic and non-toxic strains of cyanobacteria.  Finally, we will determine the extent to which current models which forecast total Microcystis densities in the Potomac River can be used to predict densities of toxic Microcystis cells and cyanotoxins in this system.  Thus, this project will provide managers with both an enhanced forecast of blooms in this system and information needed to formulate bloom management and prevention strategies.  Our results will additionally determine how trophic interactions (zooplankton grazing) may be altered by toxic cyanobacteria blooms and how nutrient loading may affect such alterations.

Investigators: T.B. Henry, G.S. Sayler, S.W. Wilhelm, R. J. Strange
Title: Investigating chronic toxicity and bioaccumulation of microcystins in freshwater fish using toxicogenomics and histopathology
Institution: The University of Tennessee
Project Period:  9/1/06 - 8/31/09
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
Objectives/hypothesis: During the last 10 years, Microcystis spp. blooms have occurred in Western Lake Erie, and elevated levels of microcystins have become a concern for both human and ecosystem health.  Our objective is to investigate the predominant microcystin found in this system (microcystin-LR) in model fish species and to relate laboratory results to chronic low-level toxin exposure and bioaccumulation found in higher trophic level fish in W. Lake Erie.  We hypothesize that (1) specific genes that respond to microcystin-LR exposure in larval and adult zebrafish can be identified and selected as biomarkers; (2) effects of chronic, low concentration exposure of microcystin-LR can be detected by changes in biomarker gene expression, tissue histology, and reproduction in zebrafish; (3) bioaccumulation of microcystin in channel catfish is affected by route of exposure and effects can be detected in biomarker gene expression and histopathology; and (4) bioaccumulation and effects of chronic low, concentration exposure to microcystins can be detected in higher trophic level fish collected from W. Lake Erie by tissue analysis and the evaluation of biomarkers resolved from lab and mesocosm experiments. 
b. Approach:  Commercially available microarrays will be used to interpret differences in global gene expression for nearly 15,000 genetic transcripts in zebrafish exposed to microcystin-LR.  A subset of differentially expressed biomarker genes (≈20-40) will be selected for larval and adult fish and adapted to a quantitative real-time PCR format for monitoring specific exposure variables.  Subsequently, zebrafish will be exposed to chronic low concentrations of microcystin-LR throughout development (age 2-150 days), and survival, biomarker gene expression, histopathological lesions, and reproductive success will be evaluated.  Selected biomarker genes will be adapted for use in channel catfish to evaluate effects of bioaccumulation of microcystin in channel catfish after aqueous and dietary exposure.  Fish from higher trophic levels (including channel catfish) will be collected from W. Lake Erie to assess bioaccumulation of microcystin and effects on biomarkers resolved in lab experiments.
c. Expected results:  Genes selected from microarray experiments will improve our understanding of the mechanisms of microcystin toxicity and enable more specific probing into the factors that influence bioaccumulation and toxicity in fish via in vitro, mesocosm, and in situ approaches.  Our focus on chronic, low-concentration exposures to will begin to address an important knowledge gap regarding the long-term effects of algal toxins on ecological health.  We expect to determine toxin concentrations that cause negative effects in fish during chronic exposure and to demonstrate toxicogenetic and histopathological approaches that can be employed in ecological forecasting of system health.

Investigators: David A. Hutchins, Kathryn J. Coyne, Mark A. Warner
Title:  The future of harmful algal blooms: an empirical approach to predicting the combined impacts of rising CO2, temperature, and eutrophication.
Institution: University of Southern California, University of Delaware
Project Period:  03/15/07 – 03/14/10
Funded by EPA NCER STAR ECOHAB  
Abstract
Description:  Recent worldwide increases in harmful algal blooms (HABs) are almost certainly linked to cultural eutrophication of coastal environments. Virtually no attention has been given, however, to how other major anthropogenic impacts such as rising CO2 and greenhouse warming could affect HABs. The combination of nutrient enrichment with rapidly increasing “CO2 eutrophication” and warmer water temperatures could provide ideal conditions for the growth of toxic algae over the coming decades.  Preliminary data suggest that HAB species such as raphidophytes may benefit disproportionately under projected year 2100 “greenhouse ocean” conditions, relative to other algal groups such as diatoms. It is imperative that preparations begin for global change-induced increases in damaging toxic bloom events throughout the rest of this century, and beyond. Goals for this project are to evaluate the cumulative impacts of increasing CO2, temperature and nutrients on HAB raphidophytes and dinoflagellates that co-occur in the Delaware Inland Bays (DIB).
Hypotheses and Objectives Hypotheses:  1) Rising CO2 and temperature in concert with increased eutrophication will favor the dominance of raphidophytes and dinoflagellates over competing non-harmful algal species; and 2) These effects will be manifested through changes in gene expression, cell physiology, and ecological dominance. Objectives:  (i) quantitatively assess the effects of increases in CO2, temperature and nutrients on the growth rates and photosynthetic physiology of HAB species, relative to non-HAB species; (ii) evaluate differential expression of critical nutrient and CO2 -regulated genes; and (iii) carry out manipulative experiments with natural algal communities containing HAB species to determine their responses to global change.
Approach: An empirical approach will examine the genetic, physiological, and community-level responses of HAB species from the DIB to changes in CO2, temperature and nutrients. This interdisciplinary investigation will examine global change impacts on expression of carbon- and nutrient-regulated genes (PI Coyne) and cellular nutrient and photosynthetic physiology (PI Warner), as well as holistic determinations of shifts in estuarine algal community structure and HAB dynamics (PI Hutchins). 
Expected Results:  This project will begin to provide definitive answers to the crucial question:  How will HAB events respond to the ever-accelerating pace of anthropogenic global change? Results of this investigation will provide a broad picture of genetic to ecosystem level responses of these HAB groups to a changing world, and supply information that is urgently needed to inform managers and policy makers about future trends in HAB occurrences and impacts.

Investigators: Andrew Juhl, Sonya Dyhrman
EPA Grant Number: R83-3222
Title: Quantifying Grazing on Harmful Algae With a Novel, qPCR-based Technique.
Institutions: Lamont-Doherty Earth Observatory of Columbia University, Palisades, NY and Woods Hole Oceanographic Institution, Woods Hole, MA.
EPA Project Officer: Gina Perovich
Project Period: 03/15/07 – 03/14/10      
Funded by EPA NCER STAR ECOHAB
Description: The proposed, targeted study will develop and apply a novel approach for measuring grazing on harmful algae. Grazing is an important, but poorly constrained, factor in the dynamics of harmful algal blooms (HABs). The new method measures the number of ingested algal cells within grazer gut contents by quantitative polymerase chain reaction (qPCR). Initial development will study grazing by the common copepod, Acartia hudsonica, on the toxic, dinoflagellate, Alexandrium fundyense.
Objectives: The project has three research objectives. 1) Optimize the qPCR assay for quantitative detection of Alexandrium ingested by Acartia. 2) Calibrate and test the qPCR-based measure of grazing rate in laboratory experiments. 3) Use the qPCR-based grazing technique to quantify Acartia grazing rates and their impact on a coastal Alexandrium bloom.
Approach: Preliminary observations show that Alexandrium DNA can be recovered from copepods that have fed on Alexandrium cells. Using similar information as the widely-used gut pigment technique for measuring copepod grazing, the qPCR-based measure of ingested cells will be converted to a specific ingestion rate of A. hudsonica on A. fundyense. Development will begin with laboratory experiments using cultured A. hudsonica and A. fundyense. Degradation of ingested Alexandrium DNA will be determined and ingestion rates of the copepod on Alexandrium will be measured in single- and multi-species prey fields. Once verified in the lab, the qPCR-based method will be used to study the impact of A. hudsonica grazing on an Alexandrium bloom in a coastal bay. During both lab and field work, results of the qPCR-based approach will be compared to copepod ingestion rates determined using the best currently-available method.
Expected Results: The primary advantages of the qPCR-based method over other currently available grazing measures include: in-situ assessment of grazing without incubations, increased sampling resolution, and high sensitivity. Initial application of this new, innovative method will improve understanding of how grazing influences the dynamics of Alexandrium blooms in coastal bays. Increased understanding and quantification of grazing and toxin transfer are important HAB research goals. Ultimately, wide use of the new method could provide high resolution grazing data for improving Alexandrium bloom models. By improving models, the new method will aid forecasting and mitigation of Alexandrium blooms by coastal managers. Once developed, the general method could be used to quantify grazing by many types of zooplankton on many types of phytoplankton. The approach may develop into a tool with widespread application and utility to HAB researchers, monitoring programs, and general oceanographic research.

Investigators: J.H. Paul, D.P. Fries, M. Smith.
Title: Engineering Upgrades and Field Trials of the Autonomous Microbial Genosensor
Institution: University of South Florida
Project Period: 9/1/06-8/31/09
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
Harmful algal blooms can be major catastrophes in terms of economic losses, aquatic organism mortalities, and deleterious impacts on human health. To predict onset of harmful algal blooms, monitor their severity, and to accurately determine their termination, rapid, reliable, and accurate methods are needed to detect HAB species. A major goal is to incorporate rapid and accurate detection methods into ocean observing systems. We have used the ribulose-1,5-biphosphate carboxylase/oxygenase large subunit gene (rbcL) as a molecular tag to detect K. brevis in a prior ECOHAB-funded project. We developed an assay that uses the novel Nucleic Acid Sequence-Based Amplification (NASBA) and molecular beacon technology. NASBA amplification, which is isothermal, is more amenable to field assays and autonomous platforms than PCR, which requires thermal cycling. With prior funding from ONR and NSF, we have incorporated our NASBA-based detection technology into the Autonomous Microbial Genosensor (AMG), the first sensor buoy to perform nucleic acid amplification to detect harmful algae. Based upon our experience with this system we would now like to improve the AMG with several engineering upgrades and embark on a series of field deployments to fully test this system. Our objectives are to: 1. To upgrade the current AMG to a dual channel detection system and other improvements 2. To reduce overall system size and weight by optimizing packaging of the fluidic management system and pressure vessel 3. To build a second AMG unit 4. To determine performance of both units through a series of field deployments. For Objective 1, we will install a second fluorescence channel in the AMG to enable detection of an internal control for quantitation and determination of performance. Alternatively, the second channel can enable detection of a second target species or a different gene (ie. a K. brevis PKS gene). Objective 2 aims to decrease the overall size and weight of the AMG to facilitate easy deployment. Construction of a second AMG (Objective 3) will enable simultaneous deployment and data collection from two sites, which is the main goal of Objective 4. We will manually sample during operation modes of the AMG during field deployments to ensure proper performance, and simultaneous samples will be microscopically counted for K. brevis. The outcome of this research will be an autonomous RNA amplification platform capable of detecting and providing quantitative information on K. brevis populations in near real time. The system will be targeted toward Karenia brevis but with simple modification should be able to target any HAB species. This proposal coincides with the NOAA agency interests described in the RFP: “Development of new methods for measuring HAB cells and toxins, especially those that can be used in observing systems or provide enhanced monitoring capability are especially encouraged”.
http://www.marine.usf.edu/microbiology/genosensor.shtml

Investigators: S. L. Strom and S. Menden-Deuer
Title: Identifying regulatory mechanisms for Heterosigma akashiwo bloom formation: predation interactions with algal behavior and resource use
Institution:            Western Washington University
Project period:  8/15/06 - 8/14/09
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
We propose an experimental investigation into the regulation of Heterosigma akashiwo blooms by protistan predators.  H. akashiwo causes fish kills yearly in coastal waters of the Pacific.  Food web interactions involving H. akashiwo. a raphidophyte that may have multiple modes of toxicity, are poorly understood. Our study focuses on the interactions between H. akashiwo layer-forming behavior, nutrient use, and susceptibility to predation mortality.  Predation and behavioral experiments will utilize heterotrophic protists, the major consumers of phytoplankton in the world’s oceans, and will address both toxicity and predator deterrence as phenomena with different implications for bloom formation and maintenance. This is a novel approach that integrates traditionally separate ‘bottom up’ and ‘top down’ aspects of HAB ecology.  Results will significantly contribute to our understanding of H. akashiwo in coastal food webs, as well as to our knowledge of competitive strategies (layer formation, use of organic nutrient sources, deleterious effects on predators) that are employed by a number of HAB taxa.

Objectives:
1. To determine the relative importance of toxicity versus feeding deterrence in reducing H. akashiwo mortality from protist predators.
2. To investigate the role of H. akashiwo layer formation in deterring predators and, reciprocally, the role of predators in inducing H. akashiwo layer formation.
3. To determine the effect of different nitrogen sources for H. akashiwo growth on toxicity and feeding deterrence of H. akashiwo.
4. To understand how H. akashiwo nitrogen use interacts with H. akashiwo behavior and toxicity to influence predation.

Approach:
We will conduct laboratory experiments with H. akashiwo and heterotrophic protist isolates from the coastal northeast Pacific.  Regional waters and natural blooms of H. akashiwo will be sampled to obtain new isolates of the raphidophyte and of protist predators that both do and do not co-occur with the natural blooms.  Work on layer formation and associated H. akashiwo and protist predator behavior will be conducted in novel spatially structured laboratory environments, using video and motion analysis techniques to quantify individual- and population-level behavioral effects.

Expected Results:
1. An increase in our currently meager knowledge of H. akashiwo toxicity effects on protist predators, potentially the major consumers of this HAB species.
2. Determination of the role of predator deterrence in reducing H. akashiwo mortality.
3. An understanding of the relationship between layer formation by H. akashiwo and the behavior of protist predators.
4. Increased understanding of the potential for organic nutrient use by H. akashiwo, and the effects of algal nutrient source on predation.
5. New understanding of the interactions between resource use and behavior of H. akashiwo and the response of protist predators to this alga.

Investigators: D.M. Anderson, D.J. McGillicuddy, Jr., R. He, B.A. Keafer, C.H.Pilskaln, J. Martin, J. Manning, V.M. Bricelj, J. Deeds, S. Etheridge, S. Hall, J.T. Turner, N.R. Pettigrew, A. Thomas, D.W. Townsend,
Title: GOMTOX: Dynamics of Alexandrium fundyense distributions in the Gulf of Maine: an observational and modeling study of nearshore and offshore shellfish toxicity, vertical toxin flux, and bloom dynamics in a complex shelf sea
Institutions: Woods Hole Oceanographic Insititution, Bigelow Laboratory for Ocean Sciences, Department of Fisheries and Oceans, NOAA/Northeast Fisheries Science Center, National Research Council Canada, Food and Drug Administration, University of Massachusetss, Univeristy of Maine, Stellwagen Bank National Marine Sanctuary
Project Period:  9/1/06 – 8/31/11
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract:
Description: The Gulf of Maine (GoM) and its adjacent southern New England shelf is a vast region with extensive shellfish resources, large portions of which are frequently contaminated with paralytic shellfish poisoning (PSP) toxins produced by the dinoflagellate Alexandrium fundyense. The year 2005 was an historical one for A. fundyense and PSP dynamics in this area, with a bloom that was more severe than any seen in the last thiry years. There are significant challenges to the management of toxic shellfish in this region – in particular the need to document the major transport pathways for A. fundyense, and to develop an understanding of the relationship between blooms and environmental forcings, as well as linkages to toxicity patterns in nearshore and offshore shellfish. An additional challenge is to expand modeling and forecasting capabilities to include the entire region, and to transition these tools to operational, management use.
Objective.  Here we propose GOMTOX – a regional observation and modeling program focused on the GoM and its adjacent New England shelf waters. The overall objective is to establish a comprehensive regional-scale understanding of Alexandrium fundyense dynamics, transport pathways, and associated shellfish toxicity and to use this information and relevant technologies to assist managers, regulators, and industry to fully exploit nearshore and offshore shellfish resources threatened by PSP, with appropriate safeguards for human health.
Approach: GOMTOX will utilize a combination of large-scale survey cruises, autonomous gliders, moored instruments and traps, drifters, satellite imagery and numerical models to: 1) investigate A. fundyense bloom dynamics and the pathways that link this organism to toxicity in both nearshore and offshore shellfish in the Gulf of Maine and southern New England shelf waters; 2) investigate the vertical structure of A. fundyense blooms in the study region, emphasizing the distribution of cells, zooplankton fecal pellets, other vectors for toxin, and their linkage to toxicity in offshore shellfish; 3) assess interannual to interdecadal variability in A. fundyense abundance and PSP toxicity; 4) incorporate field observations into a suite of numerical models for hindcasting and forecasting applications; and 5) synthesize results and disseminate the information and technology, transitioning scientific and management tools to the regulatory community for operational use.
Expected results: At its completion, this program and its predecessors will have produced a comprehensive understanding of the dynamics and forcing mechanisms underlying A. fundyense blooms and the associated toxicity of nearshore and offshore shellfish across a vast and highly complex region. Important hydrographic pathways and branch points will have been identified, and key features and processes characterized. Conceptual models will have been formulated to explain blooms and toxicity throughout the region, and sophisticated numerical models developed and tested that simulate physical, chemical, and biological processes at a highly detailed level over the region. GOMTOX will  thus make significant progress towards an operational bloom forecasting system appropriate for nearshore and offshore shellfish resources.  Furthermore, the information and technology developed by this initiative will contribute greatly to policy decisions concerning the re-opening, development, and management of offshore shellfish industries with potential sustained harvesting value of $50-100 million per year.

Investigators: C. A. Heil, D. Bronk, L.K. Dixon, G. Hitchcock, G. Kirkpatrick, M. Mulholland, J. O’Neil, J.J. Walsh, R. Weisberg 
Title: ECOHAB: Karenia Nutrient Dynamics in the Eastern Gulf of Mexico
Institutions: Fish & Wildlife Research Institution,Virginia Institute of Marine Science, Mote Marine Laboratory, University of Miami - RSMAS, Old Dominion University Research Foundation, University of Maryland, University of South Florida
Project Period: 1 September 2006 – 30 August 2011 
Abstract:
 a) Objectives: The nutrient sources that support and regulate environmentally and economically destructive Karenia brevis blooms in the eastern Gulf of Mexico remain enigmatic.  K. brevis blooms in Florida (FL) are annually predictable, have severe economic and environmental impacts, and are closely monitored and so are an ideal system to examine the complexity of nutrient interactions with harmful algal blooms (HABs) throughout entire bloom cycles (initiation and development, maintenance, and decline). To examine how nutrients regulate K. brevis blooms, the following two hypotheses will be tested: 1) multiple nutrient sources and forms support K. brevis blooms, with the relative contribution of each source depending upon bloom physiological state, bloom environment (e.g., lagoonal, lower estuarine, coastal, offshore), and location along a latitudinal gradient and 2) K. brevis is a mixotroph with a flexible metabolism whose limiting growth factors and metabolic preferences vary with the environment. We propose a workplan that will combine biological, chemical and physical measurements with modeling efforts to examine how K. brevis is able to sustain high biomass blooms in oligotrophic environments for extended periods. 
b) Approach: This proposal brings together a multidisciplinary team with extensive expertise on nutrients, HABs, K. brevis, and the southwest Florida (SWF) environment to identify, quantify and model nutrient inputs and cycling over the entire range of K. brevis bloom stages and environments.  Efforts will combine a retrospective analysis of the 2001 bloom with targeted laboratory studies, comparative field studies across environments and bloom stages, identification and quantification of multiple nutrient sources, measurement of physical flows and three-dimensional coupled biophysical modeling of near and offshore K. brevis blooms and environments.
c) Expected Results and Significance: Effectual HAB management and regulatory interventions are stymied by the lack of an integrated understanding of how nutrients, particularly organic nutrients, regulate blooms temporally and spatially.  The proposed effort, focused on environmentally and economically destructive K. brevis blooms, will provide data necessary to identify regulatory alternatives and will couple results with a public outreach approach individually targeting 1) resource managers and decision makers and 2) stakeholders and the general public via symposiums and workshops, newsletters, public seminars and websites.

Investigators: T. Scheuer, W.A.Catterall, V. Trainer, V.M. Bricelj
Title: Understanding shellfish resistance strategies as a means to predict and manage PSP toxicity
Institution: University of Washington, NOAA Northwest Fisheries Science Center, National Research Council Canada
Project Period:  9/1/06 – 8/31/09
Funded by NOAA NOS NCCOS CSCOR ECOHAB
Abstract
This 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 even lethal effects from harmful algal blooms (HABs) when toxin concentrations are high.  This susceptibility to ingested toxins and thus, ability to accumulate toxins, varies markedly both within and among bivalve species.  The past research of this collaborative group has characterized up to a 50-fold difference in toxin affinity among populations of softshell clams, Mya arenaria, and has shown that a single, conservative mutation in the Na+ channel confers resistance to PSTs.  A key goal of this proposal is to extend this research to more completely characterize the molecular and biochemical basis for the much larger interspecific variation in toxin uptake and sensitivity in bivalves.
The overarching goal of these studies is to understand the factors contributing to shellfish toxicity in the presence of HABs and to reduce their impact by providing tools to predict toxin retention by shellfish.
Specific objectives of this research will be to: 1. characterize the saxitoxin binding region of each of the four functional Na+ channel domains in several shellfish species selected as representative of extremes of nerve sensitivity/resistance to PSTs, 2. Determine the biochemical basis for PSP insensitivity and toxin sequestration in selected bivalve species characterized by prolonged toxin retention of PSTs, 3. determine the molecular  basis for the relative PSP-insensitivity of molluscs compared to vertebrates, 4. develop molecular markers for selection of non-accumulating (nontoxic) bivalve stocks.  Interspecific differences in shellfish susceptibility to toxins will be explored using molecular, biochemical and physiological approaches in clams (Siliqua patula and/or Ensis directus, Spisula solidissima, and Saxidomus giganteus) and mussels (Mytlilus edulis) from historically toxic and non-toxic areas on the Pacific (including Alaska) and Atlantic coasts of N. America. Identification of inter- and intraspecific genetic and biochemical differences will contribute to our fundamental understanding of toxin resistance mechanisms and perhaps open future avenues for detoxification strategies or selective breeding.  Regional characterization of bivalve responses to toxic algae will help to predict the impacts of paralytic shellfish poisoning (PSP) 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.

 

• ECOHAB2005: PROJECT SUMMARIES
  (from the NOAA Oceans and Human Health Initiative Announcement - OHHI 2005)

  • Anderson, D.M., D.L. Erdner, and S.S. Bates (Woods Hole Oceanographic Institution). OHHI 2005: Pseudo-nitzschia: Emerging HAB threat in the Gulf of Maine. 10/1/05-9/30/08. NOAA Award: NA05NOS4781245. Email: danderson@whoi.edu.

    In July, 2003, more than 20 whales, mostly humpbacks, were found dead near Georges Bank. Concurrently, several dead whales were found in nearshore waters of Maine, where 42 unexplained seal mortalities were also reported. Following negative findings for pathogens, attention shifted to algal biotoxins. Domoic acid (DA) was detected in most of the whales that were sampled, with several containing high concentrations. Plankton samples revealed multiple Pseudo-nitzschia species, some known to produce DA. The scientific team concluded that the whales probably died from DA poisoning. As that biotoxin investigation evolved, our lack of knowledge of Pseudo-nitzschia and the lack of research tools for studying this genus and DA in the Gulf of Maine (GOM) became painfully apparent. We knew that multiple Pseudo-nitzschia species occur in the GOM, and that some species are toxic, but we knew nothing of their temporal or spatial distribution or the hydrographic features they are associated with, nor did we have molecular probes and detection and enumeration assays akin to those used by workers studying west coast DA events. Such tools are necessary for field investigations of Pseudo-nitzschia because morphologically similar species can co-occur, with only some being capable of DA production. The west coast probes are not directly applicable within the GOM without testing and probable modification, however, since genetic differences are often sufficient between regions to preclude a direct transfer of this kind of technology. It is clear from the whale deaths and from subsequent examination of toxicity records that DA represents a cryptic or emerging HAB threat in the northeastern U.S. This problem has the potential for serious human health and ecosystem impacts but has not been adequately characterized in the GOM. Little is known about the toxicity and phylogeny of the causative Pseudo-nitzschia species in the region, and none of the GOM coastal states (Maine, New Hampshire, and Massachusetts) routinely monitors for DA in shellfish. Here we propose a field and laboratory study to characterize the species composition, distribution, abundance and toxicity of Pseudo-nitzschia spp. in the GOM. This is the first study of its type in the region, and thus is intended to develop the tools, local expertise and background understanding to respond to outbreaks and provide an assessment of the risk from this group of toxic organisms. A three-year project is proposed. In Year 1, we will establish and characterize Pseudo-nitzschia isolates from the GOM, including: 1) sampling nearshore and offshore waters of the GOM for Pseudo-nitzschia spp.; 2) establishing clonal cultures; 3) identifying the species; and 4) determining their toxicity. In Year 2 we will develop and refine probe sequences and protocols for the molecular quantification of toxic Pseudo-nitzschia species from the GOM, including 1) comparing rRNA gene sequences to existing Pseudo-nitzschia rRNA probes; 2) refining probes to target local species; 3) developing whole-cell and sandwich hybridization assays for toxigenic species; and 4) validating both methods with laboratory and field samples. In Year 3, we will examine the distribution and ecology of Pseudo-nitzschia in the GOM using a) coastal samples collected from phytoplankton monitoring efforts in Maine and Massachusetts; and b) coastal and offshore samples collected during two GOM-wide survey cruises funded through a separate project. Also in this final year we will communicate project findings and transfer technologies to officials responsible for regional shellfish monitoring programs and assess the need for ASP toxin monitoring in coastal shellfish. This information will be of direct interest to the shellfish harvesting industry, to coastal managers responsible for public health and seafood safety, as well as those responsible for ecosystem management, including marine mammal strandings and mortality events.

  • Boyer, G. (State University of New York, Syracuse) and S. Wilhelm (University of Tennessee). OHHI 2005: Identification, characterization and inventory of novel freshwater biotoxins. 10/1/05-9/30/08. NOAA Award: NA05NOS4781251. Email: glboyer@esf.edu

    This proposal seeks to establish facilities and materials to support a major interest of the NOAA Oceans and Human Health Initiative (OHHI) Center at the Great Lakes Environmental Research Laboratory (GLERL). Freshwater biotoxins are markedly understudied relative to those in marine systems: indeed only microcystins produced by Microcystis spp. are currently addressed in any federally funded program on the Laurentian Great Lakes. Recent evidence, however, dictates that biotoxins both new to science (BMAAs) as well as biotoxins new to the Great Lakes (cylindrospermopsin, anatoxin-a) have been present in this system in recent years. Moreover, strains of freshwater plankton (e.g., Planktothrix spp., Anabaena spp.) which were previously thought to be atoxigenic in the Great Lakes have been shown to produce these potent toxins. The investigators propose to establish a line of research that parallels and expands on the current GLERL investigation of Microcystis spp. and microcystins: we will collect and characterize (using biochemical and genetic tools) novel toxigenic cyanobacteria from the Laurentian Great Lakes as well as other freshwater systems, establish molecular and biochemical diagnostics for their presence and the presence of these specific toxins, and generate a series of toxin and cell standards (which are currently unavailable) to be used by GLERL and other researchers. Specific objectives include: (1) To determine the distribution and occurrence of anatoxin-a and cylindro-spermopsin- producing organisms and their toxins in Lakes Erie, (2) To develop an analytical method suitable for determination of the occurrence and spatial distribution of the neurotoxin -N-methylamino-L-alanine (BMAA) in natural samples (3) To identify those organisms responsible for microcystin production in Sandusky Bay. (4) To evaluate the use of rapid high-throughput assays for the detection of cyanobacterial toxins (5) To isolate and genetically characterize toxin-producing strains of Lake Erie cyanobacteria, and (6) To provide both training and reference materials to GLERL and other researchers in terms of both techniques and cell / toxin standards specifically applicable to Great Lakes research. This program will work through the established outreach system at the NOAA GLERL laboratory to inform the public of these emerging health risks, to characterize the biochemistry and ecology of these novel biotoxin producers, and provide toxin and cell-line standards to researchers, systems management and diagnostics labs. Housed at 2 top research institutes, this project will also support the education and training of postdoctoral, graduate and undergraduate personnel whom will be given the unique opportunity to interact with a top government research facility as well as to develop new approaches to understanding the linkages between human health and activities and our indispensable freshwater resources.

  ECOHAB2004: PROJECT SUMMARIES

  • Anderson, D.M. (Woods Hole Oceanographic Institution), C.H. Pilskaln (University of Maine), and B.A. Keafer (Woods Hole Oceanographic Institution). ECOHAB: Alexandrium spp. cyst dynamics in the Gulf of Maine: delivery, deposition, and resuspension. 9/1/04-8/31/07. NOAA Award: NA04NOS4780274. Email: danderson@whoi.edu.

    The predominant harmful algal bloom (HAB) problem in the Gulf of Maine is paralytic shellfish poisoning (PSP), caused by the saxitoxin-producing dinoflagellate Alexandrium fundyense. Blooms of A. fundyense are highly seasonal, consistent with the view that life history transformations between cysts and vegetative cells are major regulatory factors. Another cyst-forming, Alexandrium species blooms in the GOM as well -- A. ostenfeldii. This species is of interest because it produces spirolides, a family of highly potent, fast-acting toxins that are more potent than saxitoxins. The ecology and oceanography of HAB species like A. fundyense and A. ostenfeldii have been relatively well studied, but one aspect of their autecolgy remains poorly understood  their encystment and excystment dynamics. Here we propose a study to focus on several aspects of that dynamic  the processes controlling the delivery, deposition and resuspension of the cysts of these two key Alexandrium species in the GOM. The overall objective is to map Alexandrium spp. cysts over a broad-scale in the bottom sediments and benthic nepheloid layer (BNL) of the GOM, and to relate these distributions to dynamic processes such as resuspension, germination, and encystment fluxes. Specific tasks are to: 1) Develop molecular tools for identification and enumeration of A. fundyense and A. ostenfeldii cysts; 2) Update and expand an existing regional Alexandrium fundyense cyst map to include key cyst accumulation zones in the GOM, as well as the toxigenic A. ostenfeldii; 3) Map the thickness and Alexandrium cyst concentration of the BNL across the study area; 4) Obtain time series data on resuspended cyst abundance within the BNL at two key locations in the GOM; 5) Quantify the time-varying flux of Alexandrium cysts in the upper water column and the time-varying cyst resuspension flux near two cyst deposition zones in the GOM and relate these fluxes to water column samples of Alexandrium vegetative cell abundance and the observed abundance of cysts in bottom sediments; and 6) conduct model runs to assess the relative importance of cyst germination from bottom sediments and the BNL and to assess the role of cells germinating from deep, offshore basins in the bloom dynamics of Alexandrium spp. in the GOM.

    Significance of the proposed study to the overall ECOHAB Program goals: The project will address significant questions related to understanding the sources of HABs and the physical processes that influence their transport and fate. Specific focus on resuspension of cysts as a mechanism by which cysts may be transported from bottom sediments into the overlying water column, or by which newly formed cysts may be maintained in near-bottom waters, will enhance predictive capability of models leading to HAB forecasting. Project results and their incorporation into regional physical modeling efforts will produce an up-to-date and expanded cyst map for the GOM that will include information on regions of resuspended cyst reservoirs.

  • Caron, D.A. (University of Southern California, Los Angeles) and P.E. Miller (University of California, Santa Cruz). ECOHAB: Stimulation of toxic blooms of the diatom Pseudo-nitzschia spp. by urban river discharge into southern California coastal waters. 1/1/05-12/31/07. EPA Award: RD831705. Email: dcaron@usc.edu.

    This is a field-oriented research program to investigate the relationship between freshwater inputs from a highly urbanized region (southern California from the Palos Verdes peninsula to Long Beach) to the growth of Pseudo-nitzschia spp. and the production of domoic acid by members of this diatom genus in the adjacent coastal ocean.

    Objectives/Hypotheses: Freshwater discharge into the Southern California Bight is strongly episodic, highly channelized, and restricted primarily to the winter/early spring. These freshwater inputs contribute substantial amounts of inorganic nutrients, labile organic compounds and trace metals to coastal ecosystems. It is hypothesized that these meteorological events greatly influence phytoplankton dynamics and the formation of harmful algal blooms in these waters. This research program will examine the connection between storm events and Pseudo-nitzschia species success and the production of domoic acid in coastal communities.

    Approach: A sampling grid (up to 45 sites) encompassing three major river discharges will be studied following a major rainfall event in each of two consecutive years. Samples will be collected at 2-4 day intervals for a period of 2-3 weeks following each event. Supplemental samples will be collected bi-weekly throughout the year along a single cross-shelf transect. Remote sensing will be used to guide sampling during the storm events. Plankton abundances will be determined using flow cytometry, FlowCAM and fluorescence microscopy. Pseudo-nitzschia spp. abundance will be determined by microscopy (light, SEM), fluorescent in-situ hybridization, and quantitative real-time PCR. Domoic acid concentrations will be obtained using an immunological method. Physical parameters, nutrient and trace metals will be analyzed. Expected

    Results: This project will discern the patterns of environmental and biological factors stimulating population growth and domoic acid production by Pseudo-nitzschia species in coastal waters of southern California. Key factors leading to blooms of these species and toxicity events will be documented.

  • Coats, D.W., M.R. Sengco (Smithsonian Environmental Research Center) and D.M. Anderson (Woods Hole Oceanographic Institution). ECOHAB: Role of parasitism on HAB dynamics: Amoebophrya sp. ex Alexandrium tamarense. 8/1/05-7/31/08. NOAA Award: NA05NOS4781193. Email: coats@serc.si.edu

    Harmful algal blooms (HABs) develop from the rapid growth and accumulation of certain microalgal species, and cause many deleterious impacts on human health, aquatic organisms, important industries, and the quality of freshwater reservoirs and coastal environments. Globally, HABs have seen a dramatic increase in frequency, magnitude, distribution and impacts in recent years, which has prompted considerable interest in processes that regulate their formation, persistence, and decline. Trophic interactions, like parasitism, may play a significant role in bloom dynamics. Species of Amoebophrya are particularly noteworthy, as they are widely distributed in coastal environments, with infections known for numerous host taxa. Infections prevent reproduction of the host, are short in duration, and invariably result in death of the host, all of which make these parasites likely candidates for controlling host populations. These organisms are even viewed as potentially useful agents for the biological control of HABs.

    This proposal focuses on a recently-isolated dinoflagellate parasite from the genus Amoebophrya and its interaction with its host, Alexandrium tamarense, a bloom-forming, toxic dinoflagellate that impacts U.S. coastal waters. Our first objective is to characterize the parasite in culture to fully establish its identity and relationship to the genus Amoebophrya. This work includes basic parasite morphology/cytology throughout its entire life cycle, and molecular phylogenetic analysis. In conjunction with phylogenetic analysis, our second objective is to develop a series of molecular probes, which are Amoebophrya-specific and specific to our isolate, for later use in field studies. Our third objective is to study host-parasite interactions in the laboratory. This work examines parasite generation time under various conditions: host-parasite ratio, growth phase of host, dinospore age, and environmental conditions (i.e. light, temperature, salinity). The data from this work will be used in modeling the dynamics of host and parasite populations. Our fourth objective is to examine the host range and preference of our isolate. Our fifth objective is to determine the fate of saxitoxins from A. tamarense during and after infection. Lastly, our sixth objective is to study the dynamics of the parasite and A. tamarense (and other dinoflagellate hosts) in the field over time and space to determine the role of parasitism on host populations.

    Overall, this research seeks to further our fundamental understanding of this little-known group of parasitic organisms, and to investigate the role these parasites play in affecting host population dynamics in the laboratory and field. These studies, together with practical and ecological considerations, may allow us to evaluate the strategy of using this parasite as a biological agent to control HABs like those produced by A. tamarense.

  • Dam, H.G., G. McManus and P. Kremer (University of Connecticut). ECOHAB: Linking food web structure, grazer toxin resistance and ecological stoichiometry in understanding harmful algal blooms. 1/1/05-12/31/07. EPA Award: RD831706. Email: hans.dam@uconn.edu.

    Objectives/Hypotheses: The complex dynamics and feedbacks of planktonic food webs determine the formation and fate of harmful algal blooms (HAB), and the trophic transfer of toxins. In principle, both bottom-up forcing (nutrient availability), which constrains the upper limit of plant productivity, and top-down forcing (grazing pressure), which keeps this productivity from reaching its maximum, control HAB. In the simplest case, depletion of top predators and enhanced nutrient supply due to eutrophication can account for the increase of plant production (including HAB) in coastal regions. However such prediction is biased if it ignores three feedback factors seldom considered in tandem in HAB studies: (1) the toxicity of the algae; 2) toxin resistance of grazer populations; and 3) the elemental stoichiometric (C: N: P) imbalance between algae and grazers. Three hypotheses involving these feedbacks will be tested. The first two hypotheses apply to conditions in which the algae are nutrient rich. H1: Trophic cascades are stronger in the presence of toxic algae. H2: trophic cascades are weaker in the presence of toxin-resistant grazer populations. H3: The strength of trophic cascades depends on the interaction of the stoichiometric imbalance of the grazers, the toxicity of the algae and the complexity of the food web.

    Approach: These three hypotheses will be tested in combination of controlled laboratory and mesocosm experiments. Rigorous experimental tests for toxic effect of prey on grazers will be run. Comparative and manipulative trophic cascade studies will also be run with simple food webs consisting of several trophic levels with mixtures of toxic and nontoxic foods, under nutrient replete and depleted conditions, and facing toxin resistant and nonresistant metazoan grazer populations.

    Expected Results: There is an immediate and urgent societal need to understand what factors govern HABs in order to develop effective HAB mitigation strategies. This work will provide some of the required tools to predict under what conditions HAB happen and to what extent the strength of trophic cascades involving toxic algae are modified by toxin-resistant grazers and the elemental composition of grazers and algae. This knowledge is essential for properly designing adequate mitigation plans for toxic algal blooms.

  • Frost, B.W., R.A. Horner (University of Washington, Seattle), C.L. Greengrove, J.E. Gawel, and K. Sian Davies-Vollum (University of Washington, Tacoma). ECOHAB: The relationship between paralytic shellfish toxins and Alexandrium cysts in Puget Sound, Washington. 9/1/04-8/31/07. NOAA Award: NA04NOS4780273. Email: frost@ocean.washington.edu.

    This study addresses the relationship between paralytic shellfish toxins in shellfish based on historical records from the Washington Department of Health and the distribution of cysts and vegetative cells of the dinoflagellates Alexandrium spp. in Puget Sound, Washington. Our hypotheses are 1) cysts of Alexandrium spp. occur throughout Puget Sound; 2) cysts are viable and able to germinate (excyst); 3) cysts are resuspended at constrictions between basins; and 4) PSP is produced by motile cells from germinated cysts. A combination of field surveys and laboratory experiments will be used to test our hypotheses. Surface sediments at 31 sites distributed throughout Puget Sound will be surveyed for cysts. Cysts will be identified, counted, isolated, germinated in culture, and the resulting motile cells tested for PSP production. Water samples collected at the same time will be analyzed for the presence of cysts and motile cells of Alexandrium. Sites where cyst concentrations are high will be reexamined for sediment accumulation and mixing parameters. Sites at constrictions into bays and at sills will be sampled to determine where cyst accumulation, turbulence, and resuspension occur. Longer cores will be examined to determine the chronology of cyst appearance and its relationship to the spread of PSP. Sediments will be analyzed for grain size, metals that might influence the germination of cysts, and 210Pb levels to determine possible age, bioturbation and remixing of surface sediments. Cyst occurrence and viability will be related to physical (temperature, salinity, sediment type) and chemical (oxygen, nutrients, sediment metals) factors that might affect their germination and thus bloom initiation. Sites of seedbeds for potential bloom initiation that need additional monitoring for toxins will be identified, areas of cyst resuspension will be determined, and possible means by which Alexandrium has been spread throughout Puget Sound will be suggested.

  • Hoagland, P., D. Jin, H.L. Kite-Powell, A. Solow (Woods Hole Oceanographic Institution), G. Herrera (Bowdoin College) and B. Keafer (Woods Hole Oceanographic Institution). ECOHAB: Economic impacts of HAB events and the value of scientific predictions. 9/1/04  8/31/06. NOAA Award: NA04NOS4780270. Email: phoagland@whoi.edu.

    The main goal of our proposed research project is to develop a more complete understanding of the ways in which commercial shellfishermen, downstream processors and customers, and government resource managers respond to HAB events in the Gulf of Maine. This understanding will allow us to develop a framework for estimating economic impacts from specific HAB events. A subsidiary but closely related goal is to use this understanding to demonstrate the value of scientific predictions of HAB events. In particular, a thorough understanding of the types, size, and incidence of economic damages will help to clarify the value of public investments in a predictive capacity.

  • Kraeuter, J.N. (Rutgers University), V.M. Bricelj (National Research Council, Canada), E. N. Powell (Rutgers University), E.E. Hofmann, J.M. Klinck (Old Dominion University), J. E. Ward (University of Connecticut), and M.D. Gastrich (New Jersey Department of Environmental Protection). ECOHAB: Effects of Aureococcus anophagefferens brown tides in coastal lagoonal systems: Coupling numerical simulation modeling with field and laboratory studies to determine population effects on the hard clam, Mercenaria mercenaria. 9/1/04-8/31/07. NOAA Award: NA04NOS4780275. Email: kraeuter@hsrl.rutgers.edu.

    Blooms of the brown tide alga Aureococcus anophagefferens have become common in shallow coastal estuaries in the mid-Atlantic US. The Barnegat Bay/Little Egg Harbor system, part of the EPA Estuary Program, has experienced brown tide blooms in 5 of the last 8 years. These blooms inhibit feeding of suspension-feeders and have caused recruitment failure and mass mortalities of commercially important bivalves, including mussels and bay scallops in the region. The hard clam, Mercenaria mercenaria, an ecologically and commercially important species, is documented as sensitive brown tide. Population level effects of short-term bloom events on a long-lived species such M. mercenaria, are difficult to evaluate experimentally. Cumulative sublethal or lethal effects, especially on early life history stages, may not be evident at the population level for many years. In complex systems such as estuaries, it is difficult to isolate population levels effects due to environmental fluctuations and fishing mortality from other factors. Numerical simulation models offer a means to evaluate the relative significance and cumulative effects of these multiple factors. Such a model has been constructed for the hard clam (Hofmann et al., 2003), and preliminary work has incorporated the effects of brown tide, as far as they are known. Initial simulations show that model predictions are sensitive to brown tide effects on early life history stages, for which there is limited information. This additional information could substantially improve the reliability of model predictions.

    This project will combine experimental and modeling efforts to explicitly examine the effects of brown tide on hard clam population dynamics in the Barnegat Bay system. We will experimentally determine effects of brown tide on survival, growth and metamorphic success of hard clam larvae. In addition we will evaluate the size-specific effects of varying concentrations and duration of exposure to Aureococcus on growth and survivorship of hard clam juveniles in the field and in the laboratory. Lastly we will compare the toxicity of various Aureococcus isolates and examine the effects of various concentrations of brown tide in mixed suspensions on feeding activity of adult hard clams using video-endoscopy.

    The results of these experimental manipulations will be used to update and refine the existing hard clam model with a larval submodel larval component that simulates the brown tide effects on rates and processes affecting clam recruitment. The model will then be utilized to evaluate the timing and severity of blooms on the individual, year class and population levels relative to other environmental factor such as seasonal temperature variation, changes in food availability, climate change, and fishing pressure in a typical east coast lagoonal estuary.

  • Larkin, S.L. and C.M. Adams (University of Florida). ECOHAB: Economic effects of HABs on coastal communities and shellfish culture in Florida. 11/1/04-10/31/06. EPA Award: RD83-1707. Email: slarakin@ufl.edu.

    Objectives: (1) To estimate the change in gross revenues to various business sectors of coastal communities affected by HAB (e.g., red tide) events (e.g., test whether changes in restaurant sales are statistically different during periods of red tide and whether the changes are community specific or vary over time);
    (2) To calculate the costs incurred by coastal communities to address the effects of HAB events including planning efforts, contingency plans, beach patrols and cleanup, etc. (which will allow for a test of a minimum community expenditure level); and
    (3) To quantify the effects of HABs and HAB-related harvest regulations on commercial molluscan shellfish operations (which will allow for an evaluation of proposals to alter water quality standards for shellfish harvesting areas). Empirical application will be restricted to Florida for manageability and reduced costs.

    Experimental Approach: Study will use a combination of primary and secondary data, analyzed with econometric techniques and statistical measures. Objective 1 will involve the identification of business sectors impacted by HAB events, such as beachfront lodging and restaurants. A time series of taxable sales will be combined with data on weather (precipitation levels, major storm events, etc.) and HABs (presence and intensity). Municipal and county-level managers and molluscan shellfish (hard clam) culturists will be surveyed following small focus group sessions to identify all HAB-related activities and effects. The managers will be interviewed by telephone to solicit specific information on costs associated with HABs. Culturists will be surveyed by mail to obtain specific information on shellfish losses, harvest closures, cash flow disruptions, etc.). The resulting information will be used to compile a matrix of HAB costs and impacts incurred by coastal communities in Florida.

    Expected Results: The methodologies developed will have broad applicability for investigating the economic effects of HABs. The findings will allow coastal resource managers, local businesses and HAB researchers to better assess the community and business costs resulting from HAB events. This information will provide for a more accurate evaluation of the costs and benefits associated with HAB mitigation, monitoring, regulation, and clean-up efforts. It will also provide data for subsequent analysis of economic impacts.

  • Lin, S. and H. Zhang (University of Connecticut, Groton). ECOHAB: Development of PCR and immunofluorescence methods to measure HAB cell concentration and cell division rate: Karlodinium micrum as a model system. 8/1/05-7/31/08. NOAA Award: NA05NOS4781196. Email: senjie.lin@uconn.edu.

    To monitor dynamics of harmful algal blooms (HABs) and to study potential environmental factors regulating the blooms, it is crucial to simultaneously estimate cell concentration and in situ growth rate (i.e. cell division rate, CDR) at the early stage of bloom development. Although molecular techniques are increasingly used in HAB studies, application of such a powerful technology is still limited to a small number of species, especially in the case of estimation of CDR. A Targeted Individual Study is proposed here to develop a PCR and immunofluorescence (IF) detection system that will allow simultaneous quantification of cell concentration and in situ CDR for Karlodinium micrum. First, extending from the PIs previous research, a dual-gene (mitochondrial cytochrome b-ribosomal RNA) PCR primer set will be developed for quantifying cell concentration (and preliminary survey will be done using existing DNA samples from various estuarine systems). Second, using the proliferating cell nuclear antigen gene (pcna) recently cloned in the PIs laboratory, antibodies will be produced to allow immunofluorecence and estimation of CDR. Meanwhile, empirical correlation between pcna gene expression and growth rate will be attempted as a second way of CDR estimation. Utility of these probes will be assessed using laboratory cultures and field-collected samples spiked with cultured K. micrum. For field application, water samples will be preserved separately for DNA extraction and immunofluorescnece. DNA will be extracted and used in the dual-gene PCR to measure cell concentration. Immunofluorescence of PCNA will be performed and results be applied to a previously established equation for estimation of CDR. In parallel, RNA will be extracted and pcna expression measured using Real-Time quantitative RT-PCR, and CDR will then be estimated using the empirically derived correlation equations. The estimated CDR will be compared with actual CDR measured from the cultures based on cell counts to evaluate the accuracy of the PCR and IF methods. Results from this study will provide specific PCR primers and antibodies and protocols for studying population dynamics and CDR for K. micrum. Results from this study will also provide a framework based on which similar monitoring protocol can be established for other HAB species. In addition, the novel information achieved from this study will be incorporated to courses taught by the PI.

  • Hans W. Paerl, H.W. (University of North Carolina, Chapel Hill), V.J. Paul (Smithsonian Marine Station, Fort Pierce), J.W. Burns (PBS&J), and J.M. ONeil (University of Maryland Center for Environmental Science). ECOHAB: LyngbyaHAB: Ecology of toxic marine cyanobacteria Lyngbya spp. in Florida estuarine and coastal waters. 8/1/05-7/31/08. NOAA Award: NA05NOS4781194. Email: hans_paerl@unc.edu.

    Benthic cyanobacterial (blue-green algal) HABs are becoming more numerous, widespread and persistent in tropical, subtropical and temperate marine embayments, estuaries, and reef environments. Blooms can have many negative impacts, such as directly overgrowing and smothering seagrass, shellfish habitats, and coral reefs. Some nuisance taxa produce toxins and other bioactive metabolites. A genus of particular concern is the filamentous, non-heterocystous nitrogen-(N2) fixing cyanobacteria Lyngbya, species of which (e.g. L. majuscula) are distributed worldwide, especially in the tropical and subtropical oceans. In U.S. coastal waters, Lyngbya blooms have been responsible for fouling large segments of Florida estuaries and bays, the most obvious being Tampa Bay, near-shore reef environments of the Florida Keys, and reefs off of the southeastern coast near Broward County. There are increasing numbers of reports on finfish and shellfish disease and kills as well as human maladies (skin irritations, intoxication) associated with outbreaks of Lyngbya majuscula blooms and other cyanobacterial bloom species. Coastal cyanobacterial blooms are thought to be associated with eutrophication and hydrologic modification of freshwater discharge to marine environments. The following objectives will be carried out in an interdisciplinary effort focused on Tampa Bay, FL (Shell Key): 1) Nutrient additions in in situ bioassays to determine the nutrient(s) that stimulate Lyngbya and other cyanobacterial blooms; 2) Characterize and isolate toxins and other bioactive secondary metabolites produced during bloom and non-bloom events; 3) Develop standards for lyngbyatoxins and aplysiatoxins, 4) Sequence the N2 fixing gene nifH, to examine potential relationships between specific Lyngbya strains, their N2 fixing activity, toxicity, growth and proliferation within Tampa Bay and other Florida Lyngbya populations.

    Parameters we will investigate have been linked to human perturbations of estuarine nearshore environments; hence research products can be evaluated and applied in the context of developing future nutrient and hydrologic management strategies for these habitats and well as risk management plans for humans exposed to Lyngbya toxins. Research results are applicable to subtropical and tropical ecosystems nationally (Hawaii, American Samoa, Guam, Puerto Rico, U.S. Virgin Islands, Gulf of Mexico and S.E. Atlantic) and internationally (e.g., Australia, South Pacific, Caribbean).

  • Parrow, M.W. and J.M. Burkholder (North Carolina State University). ECOHAB: Cell reproduction, the diel DNA cycle, and sexuality in Karlodinium micrum: life cycle processes fundamental to the ecology of a toxigenic estuarine dinoflagellate. 9/1/04-8/31/06. NOAA Award: NA04NOS4780272. Email: mwparrow@unity.ncsu.edu.

    Karlodinium micrum is a toxigenic dinoflagellate that has formed blooms associated with fish kills in temperate estuarine waters and aquaculture facilities in North America, Europe, and Africa. Although this widespread harmful alga has been studied for many years, information on the asexual proliferation and cell cycle of K. micrum is limited, and its life history is virtually unknown: it is unknown whether sexual reproduction occurs, or if sexuality leads to the formation of dormant cysts as a seed population for future blooms, as occurs in many photosynthetic dinoflagellates. Knowledge of these cell cycle and life history features is fundamental to understanding the basic occurrence, ecology, and bloom formation of K. micrum. The proposed research addresses two overall objectives: First, we will examine cell reproduction in multiple strains of K. micrum, and the diel DNA cycle patterns and the intrinsic growth rate of the species from measurements of population-level DNA synthesis. We will test the hypothesis that cell growth, DNA synthesis, and mitosis in K. micrum follow a typical eukaryotic cell cycle pattern (G1, S, G2+M) that can be related to growth conditions and nutritional cell cycle controls. Second, we will examine the sexual life cycle of K. micrum, and the influence of environmental factors on sexuality. We will test the hypotheses that the sexual cycle of K. micrum influences the population dynamics and ecology of the species, and that environmental factors influence sexuality. Cultures of four K. micrum strains will be examined for cell reproduction and sexuality by light and scanning electron microscopy, and for cell DNA content and population DNA distribution by flow cytometry. Cultures in logarithmic and stationary growth will be profiled over time to measure the rates and patterns of DNA cell cycle progression and restrictions, and intrinsic growth rates for the species will be calculated from cell cycle data. The sexual life cycle of K. micrum will be examined with supporting cell DNA measurements, and the expression of sexuality will be studied in relation to population growth phase and different nutrient (N, P), temperature, and light regimes. This research will advance understanding of the basic cell and life cycle processes that fundamentally influence the ecology of K. micrum. It directly addresses primary needs identified by the ECOHAB program to characterize harmful algal species, reproduction, life stages, and environmental controls on population distribution and abundance.

  • Place, A.R., J. Adolf, and T. Bachvaroff (University of Maryland Biotechnology Institute). ECOHAB: Consequences and causes of variable toxicity in Karlodinium micrum  a cosmopolitan ichthyotoxic dinoflagellate. 9/1/04-8/31/07. NOAA Award: NA04NOS4780276. Email: place@umbi.umd.edu.

    For decades, high densities of the dinoflagellate Karlodinium micrum have been associated with aquatic faunal mortalities worldwide. Recently we have described several toxic compounds (karlotoxins, KmTx) from K. micrum, both in the laboratory and in the field, with hemolytic, ichthyotoxic, and cytotoxic properties which may explain some of the observations associated with high densities of this organism. Our research has revealed substantial variability in toxin yields for different isolates (ca. 0.1 - 1 pg cell^-1). Moreover, samples collected during fish kills have contained 10-100 fold this amount on a per cell equivalent (10-12 pg cell^-1). We find that a geographic strain variation exists in the toxin produced among K. micrum populations from Southeastern estuaries of the United States. All K. micrum isolates and samples from the Chesapeake Bay contained KmTx1 while all strains from North Carolina to Florida contained KmTx2. Cellular toxicity occurs through non-selective permeabilization of plasma membranes, leading to osmotic cell lysis. Susceptibility to karlotoxins is determined by membrane sterol composition, which also appears to underlie K. micrum's immunity from its own toxins. It is our fundamental premise that K. micrum populations have an extensive variability in toxin production, both in terms of amount and type, and that karlotoxins are primarily produced to aid in prey capture. The proposed work will pose the following questions:
    

    • Do environmental conditions and/or genotype modulate toxin production? (Project #1)
      
    • Does genetic strain variation exist within and between populations of K. micrum, and does this genetic variation vary during bloom events? (Project #2)
      
    • Do differences in the structure of KmTx1 and KmTx2 correlate with biological activity (Project #3)

  • Sarnelle, O., S. Hamilton, J. Rose, S. Peacor (Michigan State University), and H. Vanderploeg (NOAA Great Lakes Environmental Research Laboratory). ECOHAB: Complex interactions between harmful phytoplankton and grazers: variation in zebra mussel effects across nutrient gradients. 1/1/05-12/31/07. EPA Award: RD831708 and NOAA support to GLERL. Email: sarnelle@msu.edu.

    Filamentous and colonial cyanobacteria are the most important taxa causing harmful phytoplankton blooms in freshwater. A long-standing tenet in lake ecology is that summer blooms of harmful cyanobacteria are a characteristic response to nutrient enrichment and a symptom of eutrophication. However, recent events in the Great Lakes region suggest that the invasion of the zebra mussel (Dreissena polymorpha) is altering well-established functional relationships between nutrient loading and cyanobacterial dominance via the promotion of Microcystis aeruginosa, a toxic species of cyanobacteria, in low-nutrient lakes. We examine the interaction between D. polymorpha and M. aeruginosa with large-scale field manipulations of mussel density and nutrients coupled to research aimed at elucidating underlying mechanisms.
    Objectives/Hypotheses:
    1) experimentally determine whether the effect of D. polymorpha on M. aeruginosa changes direction across a broad gradient of phosphorus loading;
    2) identify thresholds in P loading at which the D. polymorpha effect changes direction;
    3) understand the mechanisms underlying the complex interaction between D. polymorpha and M. aeruginosa, with the explicit goal of predicting the consequences of changes in nutrient loading on harmful phytoplankton abundance in invaded habitats;
    4) determine the degree to which experimental results from inland lakes are relevant to the interaction between D. polymorpha and M. aeruginosa in the western basin of Lake Erie; and
    5) determine the extent to which D. polymorpha promotion of M. aeruginosa translates into increased levels of cyanobacterial toxin levels in the Great Lakes.
    Approach: The centerpiece of the project is a set of three enclosure/mesocosm field manipulations that test the interactive effects of phosphorus availability and Dreissena on M. aeruginosa biomass and dominance. These factorial experiments will be conducted in Gull Lake and Lake Erie. The mechanistic component will include the development of a new theory of herbivore-phytoplankton interactions that can explain negative, positive and neutral effects of an herbivore on the abundance of a harmful phytoplankton species, quantification of selective grazing and per capita nutrient excretion by zebra mussels under widely varying environmental conditions, genetic characterizations of M. aeruginosa via HIP-PCR, and monitoring of cyanobacterial toxin production.
    Expected Results: We will determine whether the effect of D. polymorpha on M. aeruginosa changes direction across a broad gradient of phosphorus loading, and if so seek to identify critical loading thresholds that can be applied in the management of invaded habitats. We expect to achieve a better general understanding of the mechanisms underlying herbivore-nutrient-phytoplankton interactions. Measurements of toxin levels will directly quantify a potentially critical threat to public health, and when coupled to genetic characterizations of Microcystis, should further our ability to predict when and where toxic blooms are likely to occur in Dreissena-infested habitats. Our research may begin to shed light on the question of why Microcystis aeruginosa appears to be uniquely responsive to Dreissena invasion relative to other phytoplankton species.

  • Schultz, I.R., D. Woodruff, and A.D. Skillman (Battelle Pacific Northwest Division). ECOHAB: Domoic acid kinetics and trophic transfer in shellfish: an integrated laboratory and estuarine mesocosm study. 1/1/05-12/31/07. EPA Award: RD31703. Email: irv.schultz@pnl.gov.

    We hypothesize that physiologically based pharmacokinetic models mathematically analogous to the type developed in vertebrates can be adapted for marine invertebrates based on the known physiology of decapod crustaceans and bivalve mollusks. These models will be used to predict the uptake and disposition of the marine algal toxin domoic acid in Dungeness crabs (Cancer magister), Pacific razor clams (Siliqua patula) and blue mussels (Mytilus edulis). Validation of individual kinetic model predictions and trophic transfer of domoic acid will be achieved through a combination of focused laboratory experiments and the use of large-scale estuarine mesocosms containing razor clams and Dungeness crabs.
    Approach: We have successfully applied recent improvements in analytical detection of domoic acid to study the excretion of the toxin in shellfish after intravascular injection and repetitive hemolymph removal. This technique will be used in conjunction with controlled laboratory feeding studies to develop a detailed data set on the uptake, tissue distribution and elimination of domoic acid in shellfish. Physiologically based kinetic models will be developed and specifically parameterized for crabs and bivalves using a combination of recently published and experimentally determined values for the cardiovascular and gastrointestinal / digestive systems of shellfish. Validation of model predictions will initially be performed from indoor laboratory studies and then from the results of a large scale estuarine mesocosm containing razor clams and crabs. In the mesocosm study, 500 clams verified to contain domoic acid will be collected from contaminated Washington State coastal sites. The clams will be added to the mesocosm along with adult Dungeness crabs (previously unexposed to domoic acid), which will feed on the clams. Individual clams and crabs will be repetitively monitored for hemolymph concentrations during the study. Crabs will also be intravascularly injected with 15N-labeled domoic acid to allow simultaneous determination of elimination and uptake of domoic acid. Expected Results: The validated models of domoic acid kinetics in shellfish will provide researchers and risk assessors a useful tool for exploration of mechanisms controlling selective retention of domoic acid by certain shellfish and allow more accurate predictions of depuration times to below permissible limits. When used in conjunction with forecasting models of Pseudo-nitzschia blooms, the predicted levels of domoic acid in shellfish can be better estimated along with the potential economic consequences of recreational and commercial shellfish closures.

  • Shumway, S.E. (University of Connecticut, Storrs). ECOHAB: Assessment of the potential for introduction of harmful algal bloom (HAB) species via shellfish transport. 1/1/05-12/31/07. EPA Award: RD831704. Email: sshumway@uconnvm.uconn.edu.

    Published records and other data clearly indicate that cells and cysts of many HAB species can pass intact and viable through the digestive tracts of bivalve molluscs. These cells and cysts are capable of establishing new cell cultures under laboratory conditions. Shellfish are routinely transplanted between areas during normal aquaculture, shellfishing, and shellfish restoration practices. While the potential role of bivalves as vectors of HAB species has been recognized by many authors, their actual role in this process has not been examined. We propose a three-year study to assess potential pathways of introduction and consequences of shellfish transfer (commercial or personal) on HAB distribution, i.e. to determine the risk of transferring toxic algal cells/cysts during transport of live bivalves between sites, and to establish and evaluate mechanisms to minimize these risks using best management practices (BMPs). In addition to establishing means of mitigation and control, we have also included a strong outreach education component because we believe that education will play a major role in stemming or slowing the transfer of HAB species by way of shellfish movement.

    Objectives: Our major objectives are to: (1) determine which algal species pass intact and viable through the digestive tract of commercially important bivalve molluscs; (2) determine the extent to which washing and purging shellfish intended for transfer can slow or eradicate the potential transfer of HABs; (3) determine when bivalves are safe to transport following exposure to HAB species; (4) assess field populations in areas of known HAB outbreaks for the presence of viable cells/cysts in resident shellfish populations during non-bloom periods; and (5) provide information to the user groups through presentations, management agencies, trade publications, pamphlets, and web pages.
    Approach: We will address these questions using standard laboratory techniques and well-established experimental protocols to determine rates of uptake, retention, and elimination of toxic cells/cysts, as well as excystment and release from fecal strands and the subsequent culture of viable cells. In collaboration with industry we will design, apply, and evaluate management strategies and BMPs.

    Expected Results: This research will be valuable to aquaculturists, watermen, processors, and managers for public health, and will assist in both habitat management and preservation of habitat integrity. Understanding the vectors for transfer of HAB species is critical to responsible environmental stewardship. This study specifically addresses special emphasis areas 1 and 4(b)prevention and mitigation strategies, and the sources, fates, and consequences of HABs in food webs and fisheries. Results from this study will ensure that the user groups are provided the most current information available, in a useable format, to control and mitigate impacts of HABs on public health, shellfish aquaculture, and the environment.

  • Thomas, A. and H. Xue (University of Maine). ECOHAB: Oceanographic links to Alexandrium-imposed toxicity in the Gulf of Maine. 9/1/04-8/31/07. NOAA Award: NA04NOS4780271. Email: thomas@maine.edu.

    We propose a data mining project based on remote sensing, numerical modeling and statistical analyses that will identify and quantify links between coastal toxicity caused by Alexandrium in the Gulf of Maine and oceanic variability. The Maine Department of Marine Resources (DMR) monitors toxin levels in multiple species of shellfish at over 300 Maine coastal sites throughout non-winter months. The same sites and/or species are not necessarily sampled each time. Nevertheless, over 25 years of these data provide an unparalleled documentation of HAB variability along the Maine coast and our best window into the interannual variability of Alexandrium dynamics. Annual in situ surveys of potentially relevant oceanographic characteristics to compare with the DMR record are not available. Three sources of data do provide systematic and consistent metrics of oceanographic variability for extensive, overlapping time periods. 1) Satellite data. Our sea surface temperature (SST) image database provides 4-5 images/day, 1985 - today. These data define our study period (1985-2006), allowing analysis of interannual variability in SST and surface thermal patterns indicative of circulation as time/space series within each year. A shorter timeseries (1997-2004) of daily SeaWiFS multispectral data supplement the SST data. 2) Model fields. We will reconstruct major 3D hydrographic structure and circulation in each study year using our Gulf of Maine numerical model. Based on the 3D Princeton Ocean Model, our hindcasts will use meteorological forcing from the NCEP Eta reanalysis, river discharge, assimilation of daily satellite SST fields and climatological open ocean boundary conditions. 3) River discharge and meteorological records in each study year provide coincident ancillary data. The overarching hypothesis that structures our investigation is: Interannual differences in the location, timing and magnitude of toxicity events along the Maine coast are associated with interannual variability in Gulf of Maine oceanographic patterns. We will statistically isolate and quantify dominant patterns in the multidimensional and gappy toxicity record. A suite of metrics indicative of oceanographic structure and forcing will be extracted from the image time series, numerical model output fields and ancillary data (e.g. dominant time/space SST variability, location/strength of specific frontal zones, timing/location of patterns indicative of specific circulation, pycnocline depth, timing of stratification, cross-shelf salinity structure, coherence of alongshore currents, etc.). We then use correlation functions and multivariate statistical tools to identify and quantify those characteristics of Gulf of Maine oceanography most consistently linked to toxicity events. We propose an iterative system of analysis, basing initial approaches on previous ECOHAB results and earlier analyses of subsets of the toxicity record, modifying our metrics as we learn which environmental parameters vary most closely with toxicity timeseries. Our results 1) simplify dominant patterns of variability in the 22+ year toxicity record 2) identify those Gulf of Maine environmental characteristics most closely linked to toxic events 3) deliver a system of easily monitored HAB ocean indices to managers and 4) point to the most promising oceanic features upon which to focus future, more physiologically, based research.

  • Van Alstyne, K.L. (Western Washington University) and T.A. Nelson (Seattle Pacific University). ECOHAB: Harmful ulvoid macroalgal blooms in Washington State: Distribution, environmental effects, and toxin production. 8/1/05-7/31/08. NOAA Award: NA05NOS4781192. Email: kathy.vanalstyne@wwu.edu.

    Blooms of harmful ulvoid green macroalgae occur regularly in Washington (WA) coastal waters and are typically composed of Ulva spp. and Ulvaria obscura. Anecdotal information from aquaculturists suggests they are increasing in magnitude. Their harmful effects towards seagrasses, fish, and invertebrates are generally thought to result from smothering or anoxia. However, recent studies have isolated toxins from bloom-forming algae and found them to be detrimental towards macroalgae, microalgae, and invertebrates. Anecdotal evidence suggests that toxin release by ulvoid blooms may be having community-level impacts.
    Objectives: The goals of our research are to: (1) determine when and where ulvoid blooms occur in the Puget Sound/ Northwest Straits region, (2) determine the physical, chemical, and biological factors associated with blooms, (3) determine the importance of nutrients, light, and herbivores in controlling the growth of ulvoid algae, and (4) examine toxin production by bloom forming algae and the impacts of toxins on co-occurring plants and animals.
    Approach: We will assess spatial and temporal changes in bloom occurrences by analyzing underwater videos taken by the WA Department of Natural Resources from 2000-2008. We will measure environmental factors associated with bloom formation by monitoring sites where blooms typically have and have not occurred in the past. Manipulative field experiments will be used to determine the effects of light and nutrients on the growth and physiology of bloom forming algae. Field measurements will be used to measure grazing rates on algae and bioassay guided fractionation methods will be used to isolate algal toxins. Toxicological assays will be used to assess the effects of toxins on marine plants and invertebrates.
    Expected Results: This research will advance our understanding of the causes, controls, and impacts of green algal blooms in WA, and is likely to be broadly applicable to macroalgal blooms in other locations. Our research will provide a better understanding of spatial variability in harmful macroalgal blooms, of the mechanisms that initiate bloom formation and contribute to their persistence, and of the importance of toxins in mediating the effects of these blooms on surrounding communities.

  ECOHAB2003: PROJECT SUMMARIES

  • Edwards, K.A. (U.Washington). ECOHAB: Satellite analysis of the physical forcing of algal blooms in the Pacific Northwest coastal ocean. 9/1/03-8/31/06. NASA Award. Email: edwards@apl.washington.edu

    This project seeks to identify and monitor physical conditions which favor harmful algal blooms (HABs) in Pacific Northwest coastal waters. This goal will be served by integration and analysis of satellite datasets relevant to the physical oceanography of this upwelling region, including some which have not been used to study its dynamics. The proposed work aims to directly support the currently funded project 'ECOHAB PNW: Ecology and Oceanography of Toxic Pseudo-nitzschia in the Pacific Northwest Coastal Ocean' (B. Hickey and V. Trainer, Lead PIs), in two ways. First, derived satellite products will be developed for use in initialization, forcing, and validation of the biophysical models of ECOHAB PNW. These products, based primarily on QuikSCAT winds and AMSR temperatures, are an improvement on commonly used NCEP products. Second, a satellite-based environmental index will be developed to summarize physical conditions associated with the growth and transport of toxic PN to coastal razor clam beaches. The index will be based on the results and hypotheses of ECOHAB PNW and aims to both validate these hypotheses and to provide a valuable monitoring tool for coastal fisheries managers and researchers. The regular availability and resolution of the chosen satellite datasets are valuable in the study of spatially variable phenomena, harmful algal blooms, which evolve in time. This satellite exploration of the physical conditions favorable to Pseudo-nitzschia blooms will augment the moored and shipboard observations of ECOHAB PNW.

  • Ferry, J.L. (U. South Carolina) and P. M. Moeller (NOAA). ECOHAB: Chemical degradation pathways for the natural attenuation of marine biotoxins. 9/1/03-8/31/06. EPA Award: RD83-1042. Email: ferry@mail.chem.sc.edu

    Objectives/Hypotheses: During harmful algal bloom events, toxins are dispersed into the food web through planktonic, detrital, or solution pathways. We hypothesize that this transfer is occurring against a continuous backdrop of chemical reactions that can act to attenuate the chemical signature of the bloom in the water column, including direct and indirect photooxidation, adsorption onto suspended solids, and hydrolysis. Photoactive suspended solids may also engage in photocatalyzed oxidation or reduction of the toxin.

    Approach: We will test this hypothesis by exposing solutions of several purified toxins to a matrix of different possible oxidizing conditions, including illumination in the presence of photosensitizers (Fe oxides, colloidal and crystalline; NO3-; varying levels of dissolved organic matter) under several different water quality conditions (varying salinity, pH, total carbonate, and suspended clays or colloidal silica). Dissolved organic matter (DOM) may also provide a hyrdophobic microenvironment for toxins to partition into, so we will measure the partitioning constant (Koc) for these toxins into dissolved organic matter as well, with particular emphasis placed on measuring how DOM and Koc vary with water quality and affect adsorption on suspended solids.

    Expected Results: The overarching goal of the proposal is explore the fundamental fate and transport processes that govern the abiotic processing of marine toxins. The specific objectives of the study are to a) build a library of multivariate models for describing the half-life of a given toxin as a function of light intensity, suspended solids, and water quality during a bloom, b) to identify degradation products, for further toxicity evaluation or use as chemical markers of abiotic degradation in the field, and c) build databases of Koc with respect to water quality. We believe this knowledge will be critical for predicting the impact of a harmful bloom event, and also that it will yield valuable insight into the possible ecological function of marine toxins based on new understanding of their persistence in the environment.

  • Franks, P.J.S. (SIO) and Farooq Azam (SIO). ECOHAB: Dynamics and mechanisms of HAB dinoflagellate mortality by algicidal bacteria. 10/1/03-9/30/06. NOAA Award NA17RJ1231. Email: pfranks@ucsd.edu

    Our primary objective is to quantify the ability of algae-killing bacteria belonging to the genus Cytophaga to influence the population dynamics of the red-tide forming dinoflagellate Lingulodinium polyedrum. We will initially combine controlled microcosm experiments manipulating cultures of axenic phytoplankton, bacteria, and DOC, with simple models to determine the critical dynamics and mechanisms that govern this algal-bacterial interaction. We will follow these with experiments utilizing mixed bacterial cultures together with L. polyedrum to explore how competition or inhibition among bacteria may influence algicidal bacterial growth, and therefore their effect on their algal target. Finally we will use mesocosm experiments with L. polyedrum bloom water to determine if the same dynamics that occur in laboratory experiments can be induced in the natural environment. The specific questions to be answered are: 1. Do the growth and ectoenzyme activities of algae-killing bacteria influence Lingulodinium polyedrum populations under conditions that mimic the natural DOC concentrations of a dinoflagellate bloom? 2. Do bacterial competition and inhibition affect the ability of algae-killing bacteria to cause algal mortality and to subsequently cause the decline of a red tide? 3. Can the results from our controlled laboratory experiments apply to a complex community of viruses, bacteria, phytoplankton, and protozoa, such as a mesocosm taken from a red tide? The results from our study will: 1. Clarify the potential role of algicidal Cytophaga bacteria in the termination of phytoplankton blooms. Past studies have independently demonstrated that many algicidal bacteria isolates belong to the genus Cytophaga and that Cytophaga spp. increase in numbers toward the end of L. polyedrum blooms. This study will determine if the increase of Cytophaga spp. can affect L. polyedrum bloom duration. 2. Determine whether biological control of HABs with algicidal bacteria is feasible. While no published account of biological control of algal blooms with bacteria exists, such a control strategy may one day be attempted. Prior knowledge of the conditions necessary for successful algal mortality is crucial before such efforts are proposed. This study will determine the effects of DOC concentration and competition with other bacteria on the ability of algicidal bacteria to effectively cause algal mortality. 3. Increase the understanding of bacterial-algal interactions in marine ecosystems. While algicidal bacteria actively cause algal mortality in laboratory cultures, the mechanism and significance of this phenomenon in marine systems is unknown. This study will determine if algicidal bacteria can cause algal mortality under environmentally relevant conditions.

  • Hutchins, D.A, S.C. Cary, and K.J. Coyne (U. Del.), and M. Doblin (ODU). ECOHAB: Investigation of toxic Raphidophyte population dynamics using molecular and physiological tools. 9/1/03-8/31/06. EPA Award RD83-1041. Email: dahutch@udel.edu

    Project Summary: In 2000, an abrupt and unprecedented bloom of the toxic Raphidophyte Chattonella verruculosa reached densities as high as 107 cells/L in the Delaware Inland Bays (DIB), causing massive mortality of marine life. Extensive monitoring revealed the presence of Raphidophytes throughout the bays and blooms have occurred several times since their discovery. Initially thought to consist of unialgal blooms of Chattonella, it has since become clear that Raphidophyte blooms in the bays are instead made up of a consortium of four Raphidophyte species. The abundance of each species in the blooms varies, suggesting that there are species-specific responses to the environment and that inter-specific interactions between Raphidophytes have variable outcomes, or both. The effects of environmental and physical factors, as well as biotic interactions, on the dominance and succession of mixed Raphidophyte blooms are currently unknown. Objectives: The goals of this project are (1) to gain a better understanding of the effects of environmental perturbations and grazing pressure on Raphidophyte community dynamics; (2) to identify environmental factors that stimulate the growth of Raphidophytes relative to other algal taxa; and (3) to investigate the potential of Raphidophyte cyst distributions as an indicator of seasonal bloom "hot spots". We will investigate the following hypotheses: H1: The relative abundance of species within Raphidophyte assemblages is controlled by physical and chemical conditions, such as light, nutrient concentrations and nutrient ratios. H2:The relative abundance of species within Raphidophyte assemblages is controlled by inter-specific interactions, such as differential grazing rates. H3:The abundance of Raphidophytes as a group relative to other algal taxa is affected by bottom-up controls, especially P enrichment. H4: Local strains of Raphidophyte species are grazed at similar rates as other community members.H5: Resident populations of Raphidophyte cysts in Delaware Inland Bays sediments can be used as a predictive determinant for seasonal blooms. Experimental Approach: Sensitive molecular techniques, HPLC pigment analysis and microscopic methods will be used to assess the relative abundance of the four Raphidophyte species vs. other major algal taxa in the DIB. Raphidophyte assemblages at several key sites in the DIB will be routinely monitored along with environmental parameters such as nutrients, light, temperature and salinity. Sediment samples will also be collected periodically throughout the course of the investigation to determine the distribution and relative abundance of Raphidophyte cyst populations. Laboratory investigations will evaluate the effect of bottom-up (nutrients and light) and top-down (grazing) controls on the four Raphidophyte species individually, in mixed assemblages and in natural populations. Expected Results: The proposed investigation addresses fundamental questions of Raphidophyte physiology and population dynamics. We will (1) determine physiological requirements and tolerances to nutrients and light for the four Raphidophyte species in culture; (2) evaluate the effect of grazing on Raphidophyte species and assemblages; (3) evaluate the effect of environmental factors on Raphidophyte population dynamics in natural assemblages; (4) identify resident Raphidophyte cyst populations in natural sediments and correlate the distribution and relative abundance of Raphidophyte cysts to seasonal blooms.

  • Landsberg, J., K. Steidinger, L. Flewelling, B.Richardson (FMRI), S. Hall (USFDA), G. Doucette (NOAA). ECOHAB: Pyrodinium bahamense: a new saxitoxin threat in the USA. 09/01/03- 08/31/06. NOAA Award NA03NOS4780196. Email: jan.landsberg@fwc.state.fl. us

    From 1 January to June 2002, 14 cases of Puffer Fish Poisoning (PFP) were reported in Florida, three cases in New Jersey, and two cases in Virginia. All illnesses were linked to puffer fish originating from the Indian River Lagoon on Florida's east coast. State and federal officials issued health advisories and banned puffer fish harvesting in the Indian River Lagoon. PFP is usually caused by ingestion of tetrodotoxins that can cause fatal human poisonings similar to Paralytic Shellfish Poisoning (PSP) due to saxitoxins. Unexpectedly, saxitoxins, not tetrodotoxins, were confirmed in the Indian River Lagoon in southern puffer fish, in mollusks (below regulatory limits), and in the dinoflagellate Pyrodinium bahamense. The 2002 PFP cases are the first to: a) confirm saxitoxin poisoning associated with the consumption of puffer fish originating from the United States, b) confirm saxitoxins in marine waters in Florida, and c) implicate the dinoflagellate P. bahamense as a new source of saxitoxins in the United States.

    The goal of our proposal is to confirm that P. bahamense is an emerging HAB threat to human health and natural resources in Florida, with a potential to impact other states. Objectives include: (1) examine the morphology, genetic variability, and toxicity of P. bahamense varieties from Florida in comparison to other regions; (2) determine the geographical distribution, concentrations, and tissue localization of saxitoxin, and derivative toxins, in puffer fish and bivalves in Pyrodinium hot spots and control sites; (3) determine the origin of saxitoxins in P. bahamense (microalgae versus bacteria); (4) verify the transfer of saxitoxins from P. bahamense to bivalves and puffer fish.

    Approach: To achieve these objectives we propose to: (1) examine P. bahamense from live field samples, cultures, and preserved material from different geographic regions using light, scanning, and transmission electron microscopy; (2) culture multiple strains of P. bahamense to high biomass for morphological examination, genetic analyses, toxin characterization, microbiology, and animal exposure studies; (3) conduct genetic analyses on P. bahamense from live field samples, cultures, and preserved material using PCR, gel electrophoresis, and recombinant technology; (4) test fish, shellfish, and Pyrodinium from different geographical locations in Florida for saxitoxins and tetrodotoxins using mouse bioassay, ELISA, or other rapid screening methods; (5) characterize the toxin profiles of saxitoxins in animal tissues and Pyrodinium using HPLC and LC-MS; (6) identify, characterize, and isolate bacterial communities and strains from puffer fish species and tissues, and Pyrodinium samples from a variety of sources, using Biolog and DGGE; (7) identify and culture common bacterial strains associated with Pyrodinium and puffer fish and screen for saxitoxin and tetrodotoxin production; (8) expose target shellfish to saxitoxins from Pyrodinium and Alexandrium cultures, expose non-toxic puffer fish species from Florida and from non-toxic control areas to experimental toxic shellfish, and expose non-toxic puffer fish to Pyrodinium and Alexandrium cultures in controlled environmental wet laboratory facilities; (9) verify and characterize toxin transfer to puffer fish from dinoflagellates via shellfish or directly from dinoflagellates.

    Expected Results: The sudden appearance of saxitoxins at potentially lethal concentrations in an area previously unknown to have such toxins, signals a new and unprecedented public health and natural resource problem for Florida and, potentially for other states. The public has been advised to refrain from eating puffer fish from the Indian River Lagoon, and the harvesting ban that was imposed on 25 April 2002 remains in effect. With saxitoxins confirmed in a number of animals, as well as in P. bahamense, a more widespread public health risk from not only PFP but also PSP warrants continuous monitoring activities, increased surveillance, and enhanced research to confirm and manage the source of the toxins. This lethal toxin also has the potential to cause significant economic and ecological resource impacts, posing a significant threat to natural resources, fisheries, and endangered species. Results from our research will: 1) confirm the origin and source of toxins; 2) determine the distribution of toxic P. bahamense in Florida and confirm its identity with other toxic strains and varieties; 3) provide data on the distribution of saxitoxins within selected aquatic biota in Florida; 4) will assist in the development of appropriate management and mitigation plans for the protection of public and natural resources health.

  • Lohrenz, S. E. (University of Southern Mississippi), G. J. Kirkpatrick (Mote Marine Laboratory), O. M. E. Schofield (Rutgers). ECOHAB: Optical Detection and Assessment of the Harmful Alga, Karenia brevis. 7/1/2003-9/30/2006. ONR Award N00014-03-1-0896 and NASA Award NNG04GA02G. E-mail: steven.Lohrenz@usm.edu.

    The primary goal of this project is to refine and evaluate optical approaches to detect and monitor bloom events of the red tide species, Karenia brevis. The traditional means of detecting and monitoring K. brevis blooms are slow, labor intensive, and spatially limited, relying primarily on shipboard sample collection and direct microscopic observations. New capabilities provided by in situ and remote optical sensors promise to enhance the detection and monitoring effort. However, these new capabilities require detailed knowledge of bloom optics to achieve their full potential. Recent work has provided evidence that K. brevis blooms exhibit unique spectral signatures in absorption, backscattering, and remote sensing reflectance. Data collected in conjunction with the Florida ECOHAB program, as well as new observations acquired during this proposed research, will be used to evaluate optical algorithms for detection and monitoring of red tide events. A multi-tiered approach is proposed, including laboratory studies of optical properties of cultures of K. brevis, a hierarchical modeling effort to simulate the contribution of K. brevis populations to bulk optical properties measured in the field, and an evaluation of algorithms to retrieve optical properties from remote sensing reflectance. Laboratory measurements conducted as part of prior-funded Florida ECOHAB research have provided preliminary information about absorption and scattering properties of K. brevis populations. Further study is needed to examine how inherent optical properties vary at the organismal level in response to different conditions of nutrient and light availability, and changes in population size distributions related to growth and cell division. The laboratory studies will be used to establish a database of optical properties of K. brevis under different environmental conditions. This database will be essential for the next phase of our study, that is, to simulate the influence of natural populations of K. brevis on inherent and apparent optical properties and identify key optical indices for discerning blooms of K. brevis. Prior ECOHAB-funded work has demonstrated the utility of Mie Theory to simulate variations in inherent optical properties using abundance and size distribution data from natural populations of K. brevis. Coupling this approach with radiative transfer modeling, we propose to examine the sensitivity of bulk optical properties and remote sensing reflectance to changes in distributions (e.g., vertical migration) and abundance of K. brevis populations in relationship to other optical constituents (e.g., other phytoplankton, colored dissolved organic matter, sediment, bottom effects). Finally, in collaboration with other investigators, we will examine the utility of inversion methods to retrieve phytoplankton absorption from reflectance, and subsequently evaluate this with regard to optical criteria for detection of K. brevis. An anticipated product of this effort will be the development of improved optical approaches for detection of K. brevis that may be applied to observations made using moored or ship-deployed instrumentation, autonomous vehicles, or satellite or aircraft remote sensing.

  • Mitchell, B. G., M. Kahru, and C. Hewes (UCSD/SIO). ECOHAB: Role of mycosporine amino acids in UV photoecology of harmful dinoflagellates. 9/1/03 ­ 8/31/06. NASA Award. Email: gmitchell@ucsd.edu

    An important goal of the ECOHAB and MERHAB programs is to improve early detection of harmful algal bloom (HAB) formation and to predict growth of the species of concern. Early detection and monitoring of HABs requires automated monitoring methods to discriminate harmful species within a mixed-species phytoplankton assemblage before the HAB dominates, and to assess the physiological acclimation state needed for accurate growth models. In general, the cellular composition of light absorbing pigments (chlorophylls, accessory caroteniods) is fundamentally related to the growth physiology and acclimation state of phytoplankton. UV-absorbing mycosporine amino acids (MAAs) have been widely reported in bloom forming dinoflagellates as well as other phytoplankton taxa and, like pigments absorbing in the visible, are expected to vary by species and within a species in response to factors that regulate growth.

    We propose to collaborate with field programs at the Florida Marine Research Institute and the Mote Marine Laboratory that are focused on recurring dinoflagellate blooms on the west Florida shelf. Our work will characterize the UV spectral properties of harmful blooms and quantify MAAs produced by the phytoplankton communities of interest. The role of MAAs in screening harmful UV radiation (UVR) will be quantified. Changes in the relative abundance of MAAs for natural communities will be studies with respect to light and nutrients to test the hypothesis that dinoflagellates dynamically vary their MAA composition in response to these environmental controls. Spectral shifts in UV absorption associated with changes in the MAA composition will be explored as a potential tool for characterization of both the taxonomic composition and the physiological acclimation of the population. Spectral microphotometry in the visible region for single cells will allow estimates of taxon-specific cellular chlorophyll-a (chla) to carbon (C) ratios, a fundamental parameter commonly found in many models of phytoplankton growth. The combined information on MAAs, UV absorption spectra, and cellular quotas of C:chla will be used to improve early detection, assessment of physiological status, and growth models of harmful phytoplankton species.

  • Pierce, R.H. (Mote), R.W. Dickey (FDA), J.L.C. Wright (UNC), and K.A. Steidinger (FMRI). ECOHAB: Structure, toxicity, and persistence of brevetoxins and of brevetoxin-conjugates responsible for neurotoxic shellfish poisoning. 9/1/03 ­ 8/31/06. NOAA Award NA03NOS4780197. Email: rich@mote.org

    a) Hypothesis/Objectives The hypotheses to be tested are: 1) Neurotoxic Shellfish Poisoning (NSP) results not only from accumulation of algal toxins (brevetoxins) but also from brevetoxin-conjugates produced by shellfish; 2) Different molluscan species produce different brevetoxin-conjugates that are retained in the shellfish to different degrees; 3) Brevetoxins and brevetoxin-conjugates are transferred to natural predators where they are metabolized to new conjugated forms. The objectives are: 1) To identify the major NSP constituents (both parent brevetoxins and brevetoxin-conjugates) in NSP-contaminated clams and oysters and establish the toxicity of each constituent; 2) To determine the relative rates of accumulation and persistence of NSP toxins in clams and oysters exposed to the same natural bloom; and 3) To identify and characterize the brevetoxin - conjugates in whelks preying on NSP - contaminated clams. b) Experimental Approach Two approaches will be used for obtaining NSP-contaminated shellfish: 1) Archived tissue from NSP-contaminated shellfish (collected and archived at -80ºC from red tide blooms that have occurred from 2001 to 2003 in Sarasota Bay, Florida) will be used for identification of major NSP toxins (using LC/MS/MS and NMR) and for establishing the toxicity of each constituent by mouse bioassay; 2) Routine collection and analysis of NSP constituents in clams, oysters and whelks from a common site along the Florida Gulf coast during and following annual red tide blooms. Pilot studies were performed with shellfish collected from the Sarasota Bay site during and following the September through December, 2001 K. brevis bloom. The presence of K. brevis cells and brevetoxins was verified in the water. Clams (Merceneria merceneria), oysters (Crassostrea virginica) and whelks (Busycon sp.) were collected periodically from a common site during and following the bloom. Tissue from the NSP-contaminated shellfish was archived at -80^oC. The toxicity of this tissue from exposed shellfish was determined by mouse bioassay and the presence of toxins and previously reported metabolites (Plakas et al., 2002) was determined by liquid chromatography-mass spectroscopy (LC-MS) (Pierce et al., 2003). This archived tissue and additional collections will be used for extraction, separation and identification of the NSP constituents, using LC-MS to guide the fractionation process and final structural identification by NMR analyses (Crouch et al., 1995; Plakas et al., 2002). Once the identity of NSP constituents has been established, several dozen oysters and clams from current red tide exposures will be collected for isolation and purification of NSP constituents. Toxicity will be determined by mouse bioassay as well as by receptor-binding assay, or by N2a neuroblastoma cell assay. Additionally, comparisons of LC-MS and enzyme-linked imunosorbent assay (ELISA) will be obtained to assess these methods for routine detection of NSP-contaminated shellfish (Baden et al., 1995; Dickey et al., 1999; Pierce and Kirkpatrick, 2001; Naar, et al., 2002; Plakas et al., 2002). c) Significance Results of this study will establish and verify the identity, toxicity, persistence and trophic transfer of brevetoxins and brevetoxin-conjugates in clams, oysters and whelks. This information is critical for assessing public health risk from NSP and for management of NSP events. Results will address Agency interests of public health, valuable sustainable fisheries, manage