ECOHAB 2006: Project Summaries

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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
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
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
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
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
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
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      
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
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”.

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
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.

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.

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
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 resourcesFurthermore, 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 
 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
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.

Last updated: May 15, 2013