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, management of coastal resources, characterization and detection of HAB toxins, and ecological fate and transport of HAB toxins.

  • Van Dolah, F. M. (NOAA), G. R. DiTullio (U. Charleston, SC). ECOHAB: Physiological and genomic approaches to understanding responses of the Florida red tide dinoflagellate, Karenia brevis, to iron limitation and oxidative stress. 9/1/03 -8/31/06. NOAA Award NA03NOS4780198 (to U. Charleston). Email: Fran.Vandolah@noaa.gov

    A goal of the ECOHAB Program is to provide scientifically sound approaches to the management of harmful algal blooms. For HABs such as the Karenia brevis, which occur as a natural component of an ecosystem, rather than as a result of coastal eutrophication, the most viable management tool may be a predictive model suitable for forecasting bloom occurrence and landfall. The accuracy of such predictive models is dependent upon the precision of biotic and abiotic processes that are incorporated into them. Thus insight into the physiological responses of a HAB species to environmental parameters is critical. This proposal addresses two gaps in our knowledge of K. brevis physiology critical to understanding the initiation and growth phases of K. brevis bloom development: (1) the response of K. brevis to Fe in bloom initiation, (2) and the mechanisms by which K. brevis deals with oxidative stress in the growth phase of bloom formation.

    K. brevis blooms generally initiate in waters replete in P, but depleted in N and Fe. A major hypothesis resulting from ECOHAB Florida proposes that input of Fe into the Gulf of Mexico by Saharan dust events triggers the initiation of K. brevis blooms, indirectly, by supporting blooms of the Fe-dependent N2 fixing cyanobacterium, Trichodesmium erythraeum, which in turn provide sufficient N to support blooms of K. brevis. Yet a 40 year time series of T. erythraeum and K. brevis abundances on the west Florida coast does not uniformly support the occurrence of T. erythraeum blooms prior to the onset of K. brevis blooms. Early research into K. brevis nutrient requirements also suggested a link between Fe and K. brevis blooms, but Fe metabolism in K. brevis has not been studied in detail. Phytoplankton respond to Fe limitation through genetic regulation that alters the profile of Fe requiring proteins. One such protein is the Fe-S protein ferredoxin (Fd), critical for photosythetic electron transport, which is replaced by flavodoxin (Flv) under Fe limited conditions. The ratio of Fd to Flv has thus been widely used to as a biomarker of Fe limitation in microalgae. We have isolated cDNAs for Fd and Flv from K. brevis, and propose to utilize these probes, in conjunction with classical physiological approaches, to characterize the responses of K. brevis to Fe. The results of this work will provide critical input, currently lacking, for predictive models for K. brevis blooms currently under development.

    During bloom growth, K. brevis undergoes cell division with an average rate of 0.3 div/day, well below its theoretical maximum of 1/day dictated by circadian control of its cell cycle. K. brevis is positively phototactic, resulting in dense surface populations that are exposed to surface light intensities of 1800 _E m-2s-1. Therefore, significant energy is spent on coping with oxidative stress, which may contribute to its low rate of growth. The known mechanisms by which K. brevis accommodates oxidative stress include the induction of HSPs and antioxidant systems and photoadaptive responses including changes in pigment profiles and protein expression levels. In addition, we have recently identified the proposed antioxidant DMSP and DMSP lyase in K. brevis. Here we will investigate the role of DMSP and its metabolites in the responses of K. brevis to oxidative stress. In the proposed project, we will first employ classical physiological approaches to define the responses of K. brevis to Fe limitation and oxidative stress, and will then investigate the genetic regulation of such responses using a cDNA microarray that we will develop from cDNA libraries to K. brevis under both replete and stress conditions. The proposed genomic approach will reveal novel insight into the physiological mechanisms that permit K. brevis to proliferate under seemingly hostile conditions, and may provide tools by which to characterize the physiological status of naturally occurring blooms.

  • Villareal, T. A. (UT Austin), S. L. Morton and P. Moeller (NOAA). ECOHAB: Toxin content in the ciguatera-causing dinoflagellate Gambierdiscus toxicus. 9/1/03 ­ 8/31/06. NOAA Award NA03NOS4780238. Email: tracy@utmsi.utexas.edu

    Ciguatera is the leading form of seafood intoxication in the world and accounts for nearly all HAB-related medical expenses in the U.S., a cost estimated at >$20 million annually. The disease results from toxins produced by the benthic dinoflagellate Gambierdiscus toxicus that are bio-magnified up the food web. While there is evidence that anthropogenic activity such as construction or eutrophication increases ciguatera outbreaks, the factors controlling toxin content are poorly known. It is unclear whether ciguatera outbreaks are related to some combination of increased toxin content in existing G. toxicus populations, population replacement by more toxic strains, or complex issues in coral reef food web dynamics. Despite the obvious need to quantify toxin content in G. toxicus, there are only scattered papers with results that are now suspect.

    This data void stems from a lack of standards and methodology for quantitative ciguatoxin analysis and from the use of batch cultures rather than continuous cultures to examine nutrient-toxin relationships. Analytical capability is a fundamental problem since both the mouse bioassay and the receptor binding assay (toxicity rather than toxin content) appear compromised by maitoxin contamination and matrix effects specific to G. toxicus extracts, respectively. The NOAA Marine Biotoxin Laboratory now has CTX 1C standards and the analytical capability to measure ciguatoxin in G. toxicus. Our combined research program focuses on how nutrient stress and source alters ciguatoxin content in G. toxicus and uses continuous culture to rigorously define physiological state. We will screen clonal isolations in batch cultures to define light and temperature optima, as well as the gross effects of nutrient source (inorganic versus organic forms) on toxin content. Using this information, we will induce varying degrees of N and P limitation in cyclostats (continuous cultures on a light:dark cycle) under varying light and temperature using both inorganic and organic sources, determine toxin content, and measure a series of physiological and chemical indices that quantify the physiological state of the organism. Cyclostats are a critical component since they reflect more accurately the low nutrient system these dinoflagellates occur in, allow creation of defined physiological states, and avoid the pitfalls of the boom-and-crash batch cultures. We will use clonal isolations from both the Pacific and Caribbean to resolve key issues of toxin content and toxin identity as well as to determine if responses to nutrient stress and/or source are generic in G. toxicus. This collaboration exploits the combination of analytical tools and knowledge of microalgal physiology/ecology available in our labs. This study will provide a quantitative basis for assessing the impacts of eutrophication and modified nutrient supply on ciguatera and provide a basis for future fieldwork.

  ECOHAB2002: PROJECT SUMMARIES

  • Anderson, D.M., M. R. Sengco (WHOI), R. Pierce/J. Culter, (Mote Marine Laboratory), R.M. Greene/ M. Lewis (EPA Gulf Ecology Division), M. Bricelj (Institute for Marine Biosciences, NRC Canada). ECOHAB: Control of harmful algal blooms using clays: Phase II. 9/1/02-8/31/05. NOAA/COP. E-mail: danderson@whoi.edu

    Harmful algal blooms (HABs) pose a serious and recurrent threat to marine ecosystems, fisheries, human health, and coastal aesthetics worldwide. These phenomena are caused by growth and accumulation of microscopic algae, some of which produce potent toxins. The significant public health, economic, and ecosystem impacts of HABs suggest that these phenomena would be legitimate targets for direct control or mitigation efforts. Nevertheless, there has been relatively little research on HAB control strategies in the U.S. The objective of this renewal proposal is to continue our investigations of the feasibility of a promising control strategy: the removal of cells from the water column through co-flocculation with clays. The approach relies on the ability of certain clays to scavenge particles, including algal cells, from seawater, carrying them to bottom sediments where they may be buried and decomposed. Clay-mitigation has the potential to be environmentally benign and cost-effective. In this renewal, our sole focus will be on the Florida red tide organism, Karenia brevis, and specific tasks will be to: 1) determine cell removal efficiency and cell viability in flow; 2) determine the fate of toxins within cells and bound directly to clays (with and without flocculants) following treatment and sedimentation; 3) determine the capacity of clay to remove dissolved toxins in seawater; 4) determine the bioavailability and toxicity of sedimented clay/cell flocs on a benthic deposit feeder; 5) assess the impacts of clay on a range of bivalve species with different feeding strategies; 6) conduct mesocosm studies to characterize cell and toxin removal in natural bloom populations, as well as benthic impacts of sedimented clay and toxin on natural communities; 7) develop and test strategies for dispersing and tracking a plume of clay and flocculated cells in a natural system; 8) conduct a pilot-scale treatment of a bloom in a well-defined (small) area and evaluate transport, deposition, removal efficiency of cells and toxin, overall effectiveness and biological and chemical impacts. The research proposed here will fill important gaps in the scientific knowledge needed for a critical evaluation of the utility and suitability of clay flocculation in HAB management. The concept remains promising for control of certain types of HABs, but additional experimental work is needed before this strategy can be considered for use at any significant scale on natural bloom populations.

  • Campbell, L. (TAMU) and John R. Gold (TAMU). ECOHAB: Linking population and physiological diversity in a toxin-producing dinoflagellate. 9/1/02-8/31/05. EPA Award R830413. Email: lcampbell@ocean.tamu.edu

    The overall goal of this research is to understand the dynamics of blooms of the toxic dinoflagellate Karenia brevis in the Gulf of Mexico. Blooms of K. brevis along the Texas coast are increasing in frequency, with the number of red tide events reported during the 1990's alone equaling the total for the previous four decades. The source population and specific factors influencing bloom initiation and intensity are poorly understood, particularly in the western Gulf. Preliminary results from our laboratory demonstrate genetic diversity within isolates of K. brevis from Texas waters. This genetic diversity may serve as a repository from which populations bloom in response to appropriate environmental conditions. This emphasizes the need for new, hypervariable genetic markers that can be utilized in population- and species-level studies of harmful algal blooms. Objectives of this project include: (1) Optimizing a suite of hypervariable, nuclear-encoded DNA markers (microsatellites) that have been developed to characterize genetic diversity among isolates of K. brevis; (2) Establishing clonal cultures of K. brevis during the onset, bloom, and decline of a red tide event in order to assess genetic and physiological variability within a bloom; and (3) Testing the following null hypotheses: (a) spatial/temporal samples from a single bloom are genetically homogeneous; and (b) geographic isolates of K. brevis from the northern Gulf are genetically homogeneous.

    Approach: A suite of microsatellite markers will be employed as tools to link diversity and structure of isolates of K. brevis with the physiological and ecological bases of bloom formation. This is the first broad-scale application of microsatellites in studies of toxic dinoflagellates. For each clonal isolate established during the course of a bloom event, allele distributions at approximately 10-15 microsatellite loci will form the basis for tests of temporal (genetic) homogeneity. Physiological characterization of unique clones will consist of determining growth rates and cellular brevetoxin levels at three light irradiances and five salinities in a factorial design. Data analysis primarily will include tests of spatial and temporal homogeneity (including molecular analysis of variance or AMOVA) of allele (haplotype) distributions (frequencies). Estimates of haplotype (nucleon) diversity and intrapopulational nucleotide diversity will also be generated. Neighbor-joining of genetic distance matrices will be used as a means to assess genetic and evolutionary relationships among spatial and temporal samples.

    Expected results: A database for dinoflagellate microsatellite alleles will be initiated for the Gulf. Initial results will assess population-genetic structure and elucidate levels of genetic variation and diversity within blooms as they develop. Ultimately, results will provide profiles of genetic and ecological diversity on appropriate spatial and temporal scales to test rigorously hypotheses regarding various environmental variables and how they affect and influence bloom formation and population structure of species of Karenia. The work will be critical to interpretation of dynamics of field populations and in models used to predict occurrences of harmful algal blooms.

  • Dyhrman, S. (WHOI) and D.M. Anderson (WHOI). ECOHAB: The development of a single-cell field diagnostic for nitrogen limitation in harmful algae. 11/18/02-11/17/05. EPA Award R83-0415. Email: sdyhrman@whoi.edu

    This project proposes the development of an assay to identify cell-specific acetylglucosaminidase activity using the substrate ELF-NAG (ELF 97 N'-acetylglucosaminide). Preliminary data indicates this enzyme is nitrogen-regulated in Alexandrium and that this assay could be an important new tool for identifying and monitoring nitrogen nutrition in field populations of harmful algae. In short, this work addresses gaps in our knowledge of nutritional physiology in harmful species and in our ability to identify the nutritional factors that regulate bloom dynamics.

    Objectives: The research proposed here will test and further refine an assay for the N¹-acetylglucosaminidase activity using the ELF-NAG substrate. Specific objectives are to: 1) Test for ELF-NAG labeling in a suite of HAB species. 2) Pursue a method for quantifying ELF-NAG labeling on a single-cell basis in a model Alexandrium species. 3) Relate the regulation of the ELF-NAG labeling to the physiological condition of the Alexandrium cells. 4) Further characterize the physiological function of the N'-acetylglucosaminidase enzyme. 5) Develop the assay for use on field populations of Alexandrium fundyense. 6) Test the assay on field populations of the toxic dinoflagellate A. fundyense during nutrient addition experiments in limno-corrals.

    Approach: The research will be performed with a suite of harmful algae in the laboratory to identify the regulation of N'-acetylglucosaminidase activity. More detailed studies will focus on Alexandrium to refine a single-cell assay for N'-acetylglucosaminidase activity that can be used in field populations.

    Expected Results: This research will advance our knowledge of nitrogen assimilation and nitrogen stress responses in harmful algae. We anticipate the development of a well-tested method for identifying single-cell N'-acetylglucosaminidase activity in different genera. As this enzyme appears to be indicative of nitrogen stress in Alexandrium, such an assay could be used to identify nitrogen stress in field populations.

  • Forward, R.B. Jr. (Duke) and PA. Tester (NOAA/NOS). ECOHAB: Effects of the toxic dinoflagellate Karenia brevis on copepod survival, feeding, swimming behavior, and photobehaviors involved in diel vertical migration. 9/1/02-8/31/05. NOAA/COP. Email: rforward@duke.edu

    Objectives
    1. Determine lethal levels of Karenia brevis cells and brevetoxin for the 3 copepod species by calculating the LD₅₀ for ingested brevetoxin and the LC₅₀ for both external exposure to cells and to dissolved brevetoxin. 2. Measure grazing rates of 3 copepod species (Acartia tonsa, Centropages typicus, and Temora turbinata) upon exposure to sublethal concentrations of K. brevis. 3. Determine sublethal concentrations of K. brevis cells and brevetoxin that alter aspects of swimming and behavioral responses to light involved in diel vertical migration by the 3 copepod species.

    Approach
    The effects of the toxic dinoflagellate Karenia brevis on 3 species of copepod (Acartia tonsa, Centropages typicus, and Temora turbinata) will be compared to effects of a non-toxic bloom forming dinoflagellate (Heterocapsa triquetra), as well as to purified brevetoxins, in mortality, grazing, and behavioral experiments. In order to differentiate lethal and sublethal levels of toxin exposure, a 24 hr LD₅₀ for brevetoxin ingestion will be calculated for each copepod species using brevetoxin body burdens as measured by capillary electrophoresis. Additionally, a 24 hr LC₅₀ for exposure to K. brevis cell and purified brevetoxins will be calculated for each species. Grazing experiments will determine whether copepods feed upon K. brevis at sublethal concentrations when no other food choices are available, and will estimate rates of ingestion. Sublethal effects of exposure to K. brevis cells and brevetoxin on copepod behavior will be assessed by determining the effects of different sublethal concentrations on copepod swimming and photoresponses involved in diel vertical migration (DVM). Behavior will be monitored in an apparatus that mimics the underwater angular light distribution, recorded with a video system and quantified with a motion analysis system.

    Expected Results
    Working under the premise that normal behavioral responses (swimming and DVM) of copepods aid in regulating exposure to K. brevis, results from this study will provide predictions for the contribution of copepods to the dynamics of harmful algal blooms and the transfer of algal biotoxins to higher trophic levels within marine food webs. Specifically: 1. Mortality studies will predict whether tolerance to K. brevis and brevetoxin varies among copepod species, and accordingly whether species may differentially co-exist with K. brevis during persistent blooms that vary considerably in concentration over large spatial scales. Comparing mortality and brevetoxin body burdens of copepods in K. brevis cell treatments with those in purified brevetoxin treatments will indicate whether these 2 potential routes of exposure (internal exposure to ingested cells and external exposure to dissolved brevetoxin) are equally toxic. 2. Grazing studies will test whether copepods feed upon sublethal concentrations of K. brevis cells when no other food choices are available: a common situation during the persistent natural monospecific K. brevis blooms that occur annually on the west Florida shelf. This information will help to quantify the overall impact that copepod grazing may have on the time course of bloom formation and decline, and will provide valuable additional evidence regarding species-specific differences in grazing rates on K. brevis. 3. Copepod swimming behavior and photoresponses involved in DVM determine the position of these organisms in the water column, and in turn their vulnerability to visual predators such as fish, as well as exposure to vertically stratified dinoflagellate layers during blooms. Alterations in copepod behavior induced by the ingestion of K. brevis cells or aqueous exposure to brevetoxins would suggest that during blooms, the transfer of carbon in copepod tissues, and of brevetoxin incorporated into these tissues, to higher trophic levels may be altered.

  • Giner, J.L. (SUNY) and G.H. Wikfors (NMFS). ECOHAB: Harmful algal bloom sterols and their effects on shellfish and crustaceans. 09/01/02 ­ 08/31/05. NOAA/COP. Email: jlginer@syr.edu

    The goal of these studies is to provide the knowledge base necessary to develop ways of mitigating the detrimental effects of HABs on marine invertebrates. Many harmful algae such as the "red tide" and "brown tide" organisms, contain unusual sterols - we have found unusual sterols in Aureococcus anophagefferens, Aureoumbra lagunensis, Gymnodinium breve, and Pfiesteria piscicida. We propose that these sterols interfere with the sterol nutrition of invertebrates such as arthropods and mollusks which, unlike vertebrates, rely entirely on dietary sources for the cholesterol needed for their cellular membranes and for the biosynthesis of hormones. By interfering with the growth and reproduction of invertebrates, HAB sterols have detrimental consequences both for fisheries and for the zooplanktonic grazers that might otherwise control an algal bloom. Because this is a relatively unexplored area, we propose to systematically investigate the effects of HAB sterols on selected mollusks and crustaceans using feeding experiments. Experiments will also be carried out to reverse the harmful effects of HAB sterols by dietary supplementation with beneficial sterols.

    The proposed research will lead to a better understanding of the sterol nutritional requirements of economically and ecologically important invertebrate species, and will contribute to the understanding of issues such as bloom initiation and fisheries recruitment. This work is expected to lead to HAB mitigation strategies, such as sterol supplementation in aquacultural settings, or to maintain and promote grazer populations during bloom events. The information gained in these studies will also be important for the utilization of HAB sterols as food chain and environmental biomarkers, and as biomarkers for HAB paleochronology.

  • Gobler, C.J. (Southampton College, LIU), D.A. Caron (USC), and D.J. Lonsdale (SUNY). ECOHAB: Impact of microbial consumers and nutrients on the abundance of Aureococcus anophagefferens during New York brown tides. 9/01/02 to 8/31/05. NOAA/COP. Email: cgobler@southampton.liu.edu

    Within Long Island's estuaries, there exists a great disparity in the frequency with which brown tides of the pelagophyte, Aureococcus anophagefferens occur, as several bays experience blooms on an annual basis, while others typically remain bloom-free. The sporadic natural of these bloom events is most evident along Long Island's south shore estuaries, such as Great South Bay (GSB), where brown tides recur annually within its western extents, but occur only sporadically in the eastern portion of the bay. Although dissolved organic nitrogen appears to play a role in stimulating the growth of A. anophagefferens in Long Island estuaries, the intrinsic growth rates of A. anophagefferens seem to be similarly nitrogen-replete throughout GSB. In contrast, our preliminary data indicates that net population growth, and thus bloom dynamics, within the estuary are significantly affected by relative (cross-estuary) differences in grazing pressure by zooplankton populations. We hypothesize that such differences in brown tide specific grazing rates may be a function of protistan species composition. The goal of this project, therefore, will be to investigate the role of protistan grazing on A. anophagefferens in Long Island estuaries with a specific aim of elucidating factors which may contribute to reduced grazing pressure on A. anophagefferens relative to other phytoplankton. Our secondary goal will be to ascertain how nutrients sources (benthic porewater and compounds of varying C:N ratios) may interact with reduced grazing pressure to yield higher net growth rates for the brown tide alga. We will conduct field experiments within Long Island's south shore estuaries to quantify microzooplankton grazing rates on brown tide and other phytoplankton, while simultaneously characterizing the composition of the protistan grazing communities. We will conduct experiments to differentiate the potential impact of various size planktonic consumers (meso-, micro-, nano-) on brown tide and other phytoplankton populations. During both types of experiments, we will investigate the impact of naturally occurring nutrient sources (e.g. benthic porewater), as well as individual organic and inorganic nutrients, on brown tide growth rates in the presence and absence of various grazer populations. This project will, therefore, determine the composition of protistan communities which are capable of controlling brown tide blooms, as well as communities which tend to avoid Aureococcus cells. Moreover, we will determine the importance of nutrient supply to Aureococcus net growth rates relative to grazing removal by planktonic consumers. Finally, this project will determine whether differences in grazing rates on Aureococcus are due to direct changes in protistan communities or due to a cascading effect within the pelagic food web.

  • Heil, C.A. (USF). ECOHAB: Humic acid utilization by the HAB dinoflagellates Karenia brevis and Alexandrium tamarense: application of a new radioisotopic technique. 9/1/02-8/31/05. NOAA/COP. Email: cheil@seas.marine.usf.edu

    The environmental history of blooms of the HAB dinoflagellates Karenia brevis and Alexandrium tamarense suggest that terrestrially derived, high molecular weight, dissolved organic material (i.e. humic acids) may be important during the initiation and maintenance bloom stages of blooms of each species. Examination of the bioavailability of humic acids to phytoplankton has been hindered by the chemical complexity of humic material and the lack of a method to radiolabel this material for uptake studies. I have developed and tested a novel technique of labeling humic material by which ¹²⁵Iodine is attached to extracted humic and fulvic material by a lactoperioxidase labeling technique. The resulting ¹²⁵I-labeled humic substances have been used to successfully examine the uptake of this material by phytoplankton under a variety of environmental conditions in preliminary experiments. This study proposes to isolate humic acids from the rivers which impact Alexandrium tamarense blooms in the Gulf of Maine and Karenia brevis blooms on the west Florida shelf, to utilize this labeling methodology to provide ¹²⁵I-labeled humic acids from these rivers, and to use these ¹²⁵I-labeled humic acids in uptake experiments with these two species examining their bioavailability and the conditions under which these species potentially utilize humic acids. In conjunction with ¹²⁵I uptake experiments, uptake of ¹⁴C-labelled humic 'model' compounds (synthesized by a peroxidase-initiated radial polymerization of a mixture of phenolic compounds, peptides, amino acids and carbohydrates labeled with ¹⁴C in either proteinaceous or aromatic component) will be used to provide information on the fate of specific components of humic acids within cells.

  • Hickey, B.M. (U. Washington), V. Trainer (NMFS), W. Cochlan (SFSU), M. Foreman, A. Pena, R. Thomson (Dept. Fisheries & Oceans, Canada), E. Lessard (U. Washington), M. Wells and L. Connell (U. Maine), and C. Trick (U. Western Ontario). ECOHAB PNW: Ecology and oceanography of toxic Pseudo-nitzschia in the Pacific Northwest coastal ocean. 9/1/02-8/31/07. NOAA/COP and NSF. Email: bhickey@u.washington.edu

    This project will study the physiology, toxicology, ecology and oceanography of toxic Pseudo-nitzschia species off the Pacific Northwest coast, a region in which both macro-nutrient supply and current patterns are primarily controlled by seasonal coastal upwelling processes. Recent studies suggest that the seasonal Juan de Fuca eddy, a nutrient rich retentive feature off the Washington coast serves as a ³bioreactor² for the growth of phytoplankton, including diatoms of the genus Pseudo-nitzschia (PN). Existing ship of opportunity data are consistent with the working hypothesis that the seasonal Juan de Fuca eddy is an initiation site for toxic PN that impact the Washington coast and that upwelling sites adjacent to the coast are less likely to develop toxicity.

    The long term project goal is to develop a mechanistic basis for forecasting toxic PN bloom development here and in other similar coastal regions in Eastern Boundary upwelling systems. Specific study objectives are: 1) To determine the physical/biological/chemical factors that make the Juan de Fuca eddy region more viable for growth and sustenance of toxic PN than the nearshore upwelling zone; 2) To determine the combination of environmental factors that regulate the production, accumulation, and/or release of DA from PN cells in the field; 3) To determine possible transport pathways between DA initiation sites and shellfish beds on the nearby coast.

    The objectives will be met with an integrated suite of field and laboratory studies on two 21 day cruises per year, moored bio/chem/physical sensors as well as circulation and biophysical modeling in a study area that includes both the eddy and also a typical coastal upwelling region. The key factors responsible for high cell densities of toxigenic PN spp. and the variable levels of cell toxicity will be investigated with on-deck incubation studies and comprehensive in situ measurements including macronutrients, micronutrients (Fe, Cu), bacteria and grazing abundance as well as photosynthetic radiation, stratification and velocity shear. Aging of blooms will be studied by following drogued patches of water both from the eddy and from a nearshore upwelling region. Toxification of coastal shellfish will be determined using beach sampling sites maintained by the Olympic Region HAB program. A coupled biophysical model of the region enhanced with assimilated survey data will be used to examine the potential for bloom generation in offshore eddy and nearshore upwelling regions (e.g., stratification, nutrient sources, strength and timing) as well as to assess transport pathways of toxic PN to the coast under a variety of environmental and physiological conditions.

  • Kirkpatrick, G. (Mote Marine Lab.), O. Schofield (Rutgers U.), M. Moline (Calif. Polytechnic State U.), and D. Webb (Webb Res. Corp.). ECOHAB: Distributed detection and adaptive 3-D mapping of harmful algal blooms incorporating autonomous underwater vehicles. 9/1/02-8/31/04. NOAA/COP. Email: gkirkpat@mote.org

    This project will utilize autonomous underwater vehicles (AUV) equipped with optical phytoplankton discriminators (OPD) to evaluate two hypotheses concerning the development of Karenia brevis blooms. These two hypotheses involve biological, chemical and physical interactions that are unobservable with current technologies. The first hypothesis involves the initial development of a Trichodesmium sp. bloom offshore of west Florida over the continental shelf supported by the deposition of iron-rich Saharan dust. Organic nitrogen compounds released by Trichodesmium then stimulate growth of K. brevis. The second hypothesis invokes the biophysical interaction between the vertical swimming behavior of K. brevis and the convergence of water masses at density fronts associated with freshwater plumes around coastal inlets.

    Slocum Gliders, equipped with OPDs, will be used to investigate the Trichodesmium-K. brevis hypothesis. These deployments will take advantage of the long endurance and range capability of the glider coupled with the ability to discriminate Trichodesmium from K. brevis and other phytoplankton classes. A science crew onboard the R/V Suncoaster will collect the necessary biological, optical and hydrographic information to verify the detection and to characterize the transport of the phytoplankton.

    A REMUS/OPD system, with its higher maneuverability, but reduced endurance, will be well suited to investigating the accumulation of K. brevis in and around frontal boundaries associated with coastal inlets. Verification measurements of the phytoplankton community, marine optics and hydrography will be obtained from the small boats supporting the REMUS/OPD system.

  • Lapointe, B.E. (HBOI) and C.S. Yentsch (Plankton Research and Instruments). ECOHAB: Physiology and ecology of macroalgal blooms on coral reefs off southeast Florida. 10/14/02-10/13/04. EPA Award R83-0414. Email: lapointe@HBOI.edu

    Objectives/Hypotheses: Over the past several decades, macroalgal blooms have degraded the biodiversity and growth of coral reef ecosystems experiencing anthropogenic nutrient enrichment. This problem has reached a critical stage in southeast Florida where blooms of Codium isthmocladum in the early 1990¹s were followed by a succession of Caulerpa spp. between 1998 and 2001 on fringing reefs in 20 to 50 m depths. Preliminary evidence supports the hypothesis that the decade-long succession of macroalgal blooms is linked to increasing land-based discharges of ammonium derived from sewage via groundwaters and ocean outfalls. To date, little is known of the seasonal patterns in growth and photosynthesis of Caulerpa spp., the relative importance of upwelled nitrate versus ammonium as a nitrogen source, or the potential for herbivores to control these blooms. This project will provide a two-year study of the physiology and ecology of Codium and Caulerpa spp. with the objectives of measuring seasonal patterns in benthic cover, photosynthesis, dark respiration, optical properties, tissue C:N:P ratios and _ 15N values, uptake of NH4+ and NO3- under different combinations of irradiance and temperature, and the potential for herbivores to control the blooms.

    Approach: To achieve these objectives, we propose to: 1) use underwater digital video to quantify seasonal growth patterns (as % cover of reef surface) of the target species on two fringing reefs, 2) measure seasonal changes in tissue C:N:P and _ 15N of the target species, 3) measure seasonal changes in spectral absorption, reflectance, dark respiration, and photosynthesis of the target species with and without ammonium enrichment, 4) use controlled, laboratory experiments to determine the effects and interactions of temperature and irradiance on uptake of NH4+ and NO3- by the target species, and 5) conduct controlled grazing experiments in both the lab and field to quantify the potential for generalist and specialist herbivores to control standing crops as a function of the C:N ratio.

    Expected Results: Achieving these objectives will advance our understanding of how physical, chemical, and biological factors interact to initiate, sustain, and terminate macroalgal blooms on coral reefs in southeast Florida. This research will be useful to the scientific community and resource managers not only in south Florida but worldwide by demonstrating the physiological and ecological bases for the formation and termination of harmful macroalgal blooms on coral reefs.

  • Lefebvre, K, N.Scholz, V.Trainer (NMFS). ECOHAB: Effects of algal toxin exposure in early life history stages of fish. 10/1/02-9/30/05. NOAA/COP. Email: Kathi.Lefebvre@noaa.gov

    It is well established that the toxins produced during harmful algal blooms (HABs) can kill fish. However, the chronic effects of sublethal toxin exposures are poorly understood. Potential impacts on the embryos and larvae of marine free-spawning fish are a major concern because these sensitive developmental stages may be unable to avoid the dissolved toxins that algal cells release into the surrounding water during HABs. Moreover, where blooms of multiple HAB species overlap in space and time, they can produce mixtures of different algal toxins in the marine environment. Little is known about the interactive effects (antagonistic, additive, or synergistic) of these mixtures on early development in fish. Algal toxins, alone or in mixtures, may have chronic effects on developing fish that negatively impact recruitment, survival, or reproductive success at later life history stages. Fish populations that are at risk include northern anchovies (Engraulis mordax) and Pacific herring (Clupea pallasi), species that are vital to marine food webs and commercial fisheries. The overall goal of the proposed research is to define the specific sublethal effects of algal toxins, alone or in mixtures, on early development in these economically and ecologically important marine fish species.

    To this end, we propose the novel use of a biomedical model system, the zebrafish (Danio rerio), to evaluate the sublethal effects of two algal toxins (domoic acid and saxitoxin), on fish embryos and larvae. Zebrafish are a National Institute of Health-approved experimental system for studying fundamental mechanisms of development and developmental toxicity in vertebrates. Using this well-defined and easily manipulated system, we will conduct rapid and sensitive phenotypic screens to determine the impacts of algal toxins on the anatomy, physiology, and behavior of developing fish. Our aim is to identify sublethal indicators of developmental toxicity that can be clearly related to the health or performance of fish at later life history stages. Phenotypic screens in zebrafish will be used to define a discrete set of toxicological endpoints that are specific for the two toxins (or their mixtures). These endpoints, or pathways of developmental toxicity, will then be validated in northern anchovies and Pacific herring. The proposed research will therefore proceed in two stages. The first stage (Years 1 & 2) will use the zebrafish model to identify sensitive and specific indicators of algal toxin-induced injury in fish embryos and larvae. The second stage (Year 3) will use these markers to establish sublethal toxicological thresholds for domoic acid and saxitoxin in the marine species of concern.

    This research will provide empirical data that is critically needed to address the chronic effects of HABs on the viability, fecundity, and recruitment of key fish species in the marine environment. The results will provide considerable insight into the relationships between HABs and the productivity of marine fish populations. The findings will also have immediate predictive benefits for natural resource managers and provide an improved scientific basis for mitigation strategies that are designed to protect and maintain sustainable fisheries. Finally, the proposed research will establish zebrafish as a new experimental tool for resolving the chronic impacts of other algal toxins on the health of a wide variety of marine fish species.

  • Lewitus, A.J. (Belle W. Baruch Inst.) and A. Ringwood2 (SC Dept. Natural Res.). ECOHAB: Widespread Kryptoperidinium blooms in South Carolina estuaries: improving detection, understanding why they form, and assessing their impact on shellfish. 9/1/02-8/31/05. NOAA/COP. Email: Lewitusa@mrd.dnr.state.sc.us< /a>

    Since their discovery in 1998, dense Kryptoperidinium blooms have been observed in several SC estuaries from Georgetown to Hilton Head (over 100 miles apart). These blooms have recently been shown to cause physiological stress to oysters. Given their widespread distribution and potential to adversely affect shellfish, the ecological and economic impacts of these newly observed blooms may be considerable.

    This proposed study seeks to advance understanding of the identity of the bloom organism(s), the factors driving bloom dynamics, and potential bloom impacts on shellfish health. Our objectives are to: (1) determine the taxonomy of the Kryptoperidinium clade, and develop molecular tools to enhance their identification and detection (Hyp: Genetic markers will reveal geographic delineation of Kryptoperidinium bloom species), (2) determine the ecophysiological factors that regulate bloom formation, maintenance, and decline (Hyp: Direct uptake of high molecular weight DOM by Kryptoperidinium sp. stimulates bloom formation and supports bloom maintenance), and (3) determine the physiological stress responses of oysters to the SC blooms (Hyp: The physiological functioning of oysters is compromised by exposure to Kryptoperidinium blooms).

    Objective 1 will be accomplished using SEM, epifluorescence microscopy, pigment profiles, histone analyses, multiple gene sequencing, and ³FISH² probes. For Objective 2, we will use tracer and bioassay experiments, focusing on the roles of nutrients, DOM, bacteria, and grazing on bloom regulation. For Objective 3, indicators of physiological stress will be measured in bioassays using: 1) native oysters from bloom locations, and hatchery-reared oysters 2) placed in cages in situ and 3) exposed to bloom samples in the laboratory.

    We will target 3 research needs of ECOHAB; HAB cell characterization/detection; the influence of human and natural factors on HAB initiation, distribution, and accumulation; and the consequences of HABs on food webs and fisheries. We expect to define the taxonomy of the Kryptoperidinium clade, which is fundamental to the identification/distinction of bloom species. By developing PCR- and FISH-based probes, we will establish routine protocols for Kryptoperidinium sp. detection and quantification. The nutrient bioassays and tracer experiments will allow identification of the conditions that favor bloom formation and maintenance, and in conjunction with information from an ongoing statewide monitoring program, will be used to forecast areas prone to Kryptoperidinium blooms, and develop hypotheses on the potential influence of anthropogenic nutrient loading. Oyster bioassays will generate new information on the mechanisms and extent of harmful effects from these blooms. The enhanced predictive capabilities for bloom regulation and impact on shellfish will generate novel and valuable criteria for coastal management and mitigation strategies.

  • Lin, S. (UCONN). ECOHAB: Use of dual-gene PCR and immunofluorescence to investigate spatial and seasonal distribution of Pfiesteria and P. shumwayae in Long Island Sound and adjacent waters. 9/1/02-8/31/04. NOAA/COP. Email: slin@uconnvm.uconn.edu.

    Pfiesteria piscicida and P. shumwayae are potentially toxic heterotrophic dinoflagellates and important grazers in estuarine ecosystems. Geographic and seasonal distribution of these organisms and mechanisms by which the distribution is regulated are not well understood in general and have not been investigated for estuaries in New England. In this study, spatial and temporal distribution of these species will be investigated for Long Island Sound (LIS) which features a nutrient gradient along the east-west axis, Chesapeake Bay, which has been impacted by Pfiesteria spp, Narragansett Bay, and Boston Harbor. For P. piscicida, a recently established dual-gene PCR protocol and immunofluorescence will be employed. For P. shumwayae, a similar dual-gene PCR technique will be established and used to detect this species and quantify its cell concentration. This study is designed to test the following hypotheses: 1) Pfiesteria piscicida and P. shumwayae may be present widely in coastal waters and estuaries without toxic outbreaks; 2) Eutrophicated condition in western LIS may be more favorable for P. piscicida and P. shumwayae to occur and thrive than the relatively pristine eastern LIS; 3) Seasonal fluctuation and spatial variation of abundance of P. piscicida and P. shumwayae biomass is correlated with variation in chlorophyll a and nutrient concentrations; 4) Spatial limits of Pfiesteria spp. distribution may in part be related to salinity.

    Results from this study will be useful for understanding the potential linkage between P. piscicida/P. shumwayae and fish kills and factors regulating spatial and temporal distribution of P. piscicida. The results will also provide valuable information on the potential role of this organism in estuarine ecosystems. Conclusions made from this study will provide reference for making management decisions for the coastal and estuarine environments. Furthermore, the methodology, after validation through inter-comparison in this study, will be a useful tool for monitoring P. piscicida and P. shumwayae in estuaries in the US and in other countries.

  • McGillicuddy, Jr., D.J., D.M. Anderson, A. Solow (WHOI), D.R. Lynch (Dartmouth College), and D. Townsend (U. Maine). ECOHAB: Predictive models of the toxic dinoflagellate Alexandrium fundyense in the Gulf of Maine: quantitative evaluation, refinement, and transition to operational mode for coastal management. 1/1/03-12/31/05. NOAA/COP. Email: dmcgillicuddy@whoi.edu

    Coupled physical-biological models of Alexandrium fundyense in the Gulf of Maine have matured to the point that it is now feasible to assess their suitability and potential value in an operational context. Our strategy in this undertaking consists of three main elements: (1) evaluation of predictive skill in a hindcast mode using data from the three field years of the ECOHAB-GOM program (1998, 2000, and 2001); (2) improvement of the models in light of what is learned in that evaluation; and (3) formulation of a plan for transition of the models to operational use. Of particular importance in the latter activity is design of an observational network needed to drive the models in order to achieve a specified level of accuracy.

    Results from the ECOHAB-GOM program have shown that the dynamics of Alexandrium fundyense blooms are regional in scope, spanning the waters from the Bay of Fundy down into Massachusetts and Cape Cod Bays. Thus, a gulf-wide modeling approach is necessary. Furthermore, given the large domain of interest and complex hydrodynamics characteristic of this region, data assimilation is an essential element of skillful prediction. Our hindcasts will be based on hydrodynamic simulations constructed using the Dartmouth suite of models and associated assimilation methodologies. Velocity measurements from ECOHAB-GOM shipboard ADCP surveys and moored instrumentation will serve as the primary constraint on the hindcast circulation. Biological measurements will be assimilated into the ³forward² coupled problem via initial conditions. We will also explore the inverse problem of inferring the biological sources and sinks necessary to account for observed changes in Alexandrium fundyense abundance given the underlying circulation.

    Our work on coupled physical/biological dynamical models will be complemented by research on much simpler empirical models. Statistical linkage of shellfish toxicity with various environmental indices (e.g. meterological forcing, hydrography, NAO) will provide the basis for empirically-driven predictions. Both statistical and dynamical modeling activities will contribute to better understanding of the system, and thereby improve our ability to forecast changes. Progress toward transition of this suite of models to an operational framework will be fostered through collaborations with agency representatives, management personnel, applied modelers, and observational network operators. Yearly workshops will facilitate communication amongst the interested parties. The final product of this larger working group is expected to be a detailed implementation plan for a system to carry out operational forecasting of Alexandrium fundyense in the Gulf of Maine, including the identification of possible academic, public, or private institutions where the operational model might be housed.

  • Plumley, F.G. (U. Alaska). ECOHAB: A molecular genetic system for identification of the saxitoxin genes. 9/1/02-8/31/05. NOAA/COP. Email: fffgp@uaf.edu

    The biochemistry and molecular biology of HAB toxin synthesis is not well known. The focus of this proposal is the molecular biology of saxitoxin, the etiological agent of paralytic shellfish poisoning (PSP). The specific objectives of this proposal are to clone and identify the genes involved in the synthesis of saxitoxin, the ³saxitoxin genes.²

    Metagenomics is the primary experimental approach to be used for this work. Amplified Fragment Length Polymorphism (AFLP, a modified differential display technique) is the backup strategy. Briefly, metagenomics is the cloning of large DNA fragments using artificial bacterial chromosomes (e.g., pBAC) as vectors and the expression of the cloned genes in a host bacterium. The host will be E. coli K12, as mandated by NIH requirements for recombinant DNA involving manipulation of genes that encode toxins. The large DNA fragments containing the ³saxitoxin genes² will be obtained from toxic Aphanizomenon flos-aquae and Anabaena circinalis. Saxitoxin-producing E. coli colonies will be screened via HPLC. The backup method, AFLP, will be used to identify differentially expressed mRNAs recovered from Aphanizomenon flos-aquae grown either with or without urea as an exogenous nitrogen (N) source. Recent work has shown that addition of urea to the culture medium completely suppresses saxitoxin accumulation in this cyanobacterium.

    Identification of the ³saxitoxin genes² is the expected result of this project. Success with the metagenomics approach will provide a molecular genetic system that can be conveniently manipulated using standard molecular protocols to determine the function of each gene. Long-term goals include identification of DNA probes that can be used to differentiate toxic and non-toxic algae, and to determine at the molecular level how environmental growth conditions affect expression of the ³saxitoxin genes,² and hence, toxicity.

    Many outstanding questions related to saxitoxins that are congruent with ECOHAB objectives can best be addressed through study of saxitoxins at the genetic and/or molecular levels. This proposal address three research themes (1, 4, & 5) highlighted in the solicitation: … Gene probes as tools for environmental monitoring programs 1. Characterization and detection of HAB cells 4. Enhancing predictive and early warning capability for the occurrence of HABs … Gene probes as tools in laboratory/ field studies to understand physiological ecology 5. Enhancing predictive and early warning capability for the occurrence and impact of HABs

  • Shull, D.H. (Gordon College). ECOHAB: Ecology of benthic deposit feeders and toxic dinoflagellates. 9/1/02-8/31/04. NOAA/COP. Email: dshull@gordon.edu

    Deposit-feeding soft-bottom benthos are exposed to and consume toxic dynoflagellate cells and cysts in marine sediments. Dinoflagellate cells and cysts can be abundant in marine sediments, especially following dinoflagellate blooms. Because harmful algal blooms of certain species of dinoflagellates may be seeded by germination of cysts, consumption of cysts by deposit feeders may have important consequences for both the dinoflagellates and for the benthos. Deposit feeding results in the transport of cysts within the sediment, which can alter rates of germination. Digestive processes in deposit-feeder guts can potentially degrade cysts, decreasing their viability. This consumption could also result in the accumulation of these toxins in deposit feeders and introduce toxins into the benthic food web.

    The interaction between deposit feeders and the resting stages of dinoflagellates represents a gap in our understanding of dinoflagellate ecology. The primary objective of this research is to determine the effects of deposit-feeding benthos on the transport and germination of cysts, focusing on three species of dinoflagellates, Alexandrium tamarense, Gymnodinium catenatum, and Scrippsiella cf lachrymosa. A second objective is to determine whether deposit feeders accumulate toxins by ingesting toxic species. I propose to examine the effects of deposit feeder ingestion on cyst viability and germination with a series of laboratory and field experiments. The effects of deposit feeder digestion on cysts will be assessed by exposing cysts to deposit-feeder digestive fluids in vitro. This work will be followed with experiments using live deposit feeders in the laboratory to assess the effects of both deposit feeding and burial on cyst germination. Measurements of saxitoxin concentrations in digestive fluids and tissues will assess the importance of digestive processes in saxitoxin accumulation by deposit feeders.

    The effects of deposit feeders on the transport of buried cysts will be addressed by the application of a mechanistic model of cyst transport within the sediment. The model will be used to predict changes in cyst profiles due to bioturbation by deposit feeders. Field studies will be conducted in Salt Pond on Cape Cod and in at a site in the western Gulf of Maine to compare model-predicted cyst profiles with measured profiles. Differences in modeled and measured cyst profiles will be used to assess the importance of excystment and other processes in determining the distribution, transport, and loss rates of dinoflagellate cysts in the benthos.

    Expected results of this study will fill several gaps in our understanding of dinoflagellate and deposit-feeder ecology. We will assess whether deposit feeders accumulate saxitoxin. We will determine whether deposit feeders are a source of cyst mortality in the benthos and whether they alter rates of germination in the field. Finally, the mathematical model of cyst transport and germination in the sediment may help in forecasting harmful algal blooms seeded by cyst germination and may better constrain the initial and boundary conditions for coupled physical-biological HAB models.

  • Smith, C.M. (UH), C. Hunter (Waikiki Aquarium), J. Harrigan (HI Dept. Health), R.T. Nishimoto (HI Div. Aq. Res.), F. Sansone (UH), and G. Tribble (USGS). ECOHAB: Nuisance macroalgal blooms in coastal Maui: assessment and integration of physical factors and biological processes. 02/01/03-01/31/07. NOAA/COP. Email: celia@hawaii.edu

    The primary goal of the proposed study is to determine the causes of nuisance algal blooms on coastal West Maui through an inter-disciplinary approach and utilizing innovative technologies. Objectives will be to a) characterize a nuisance algal bloom from a biological perspective focusing on distribution, abundance, ecology, and physiological status of the target species, b) quantify unique environmental and water quality parameters within the bloom area, c) identify sources and quantities of nutrient input to the reef ecosystem, d) examine details of algal species biology and physiology to identify specific factors driving rapid growth rates, and f) develop a comprehensive model that includes a variety of physical and biological parameter to predict growth and persistence of nuisance algae. The limitations of past research on Maui algal blooms resulted from a lack of integration of efforts into a cohesive program that could provide links between the various factors in a complex scenario. Through recent advances in 3-dimensional groundwater modeling, linking land-based nutrients to oceanic inputs using tracers, and conducting algal physiological experiments in situ, we have the opportunity and the tools to make these important links in ways not possible five years ago. By assembling a team of researchers from a variety of disciplines (geology, geophysics, hydrology, chemistry, biology, and physiology), including State of Hawaii resource managers, and employing new and innovative research tools, we have perhaps for the first time the resources needed to answer these difficult questions.

  • Valiela, I. (Boston U.) ECOHAB: Controls of harmful macroalgal blooms: experimental and comparative studies. 9/1/02-8/31/05. NOAA/COP. Email: valiela@bu.edu

    Macroalgal blooms are increasing along the shorelines of the US and the world. They create major disruptions in natural communities, as well as alter water quality, and eliminate important valuable coastal habitats. These blooms impair human uses of the affected coastal areas and reduce commercially important fin- and shelfish stocks.

    To synthesize the information that will be needed to manage or mitigate macroalgal blooms, we propose a comprehensive definition of the relative role of the controls of blooms of green, red, and brown macroalgae. The controls suggested in the literature include increased nutrient supply, lowered grazing pressure, successful recruitment, and changes in physical factors. We will examine the relative importance of nutrient (N and P) supply and grazer pressure by in situ enrichment and caging treatments; the importance of recruitment mechanisms will be assayed by records of reproductive performance of the algae in the experiments and by experimental installation of settlement tiles in the various sites, so as to measure success in settlement of immature stages of macroalgae, and survival and recruitment to adult fronds. We will also measure 15N of the macroalgae to explicitly link supply of nitrogen (ambient or experimental addition) to the response of the macroalgae in the experiments and field surveys.

    The work will be done in intensive sites along the Massachusetts coast (Cape Cod and Nahant), where we can follow time courses of the responses to experiments across the seasons. We will run the same experiments in comparative sites (Jobos Bay, Puerto Rico; Tijuana Estuary, California, Venice Lagoon, Italy, and Maasholm Bay, Germany), selected to span a wide variety of sites with well-established green, red, and brown macroalgal blooms, and sites where there is substantial ancillary information. To test the relative importance of differences in physical conditions (temperature, light, salinity, and water flow), we will run the experiments in selected places within and among the intensive and comparative sites, so as to span a range of physical and latitudinal conditions. The data from this varied array of sites ought to furnish guideposts to develop a comprehensive view of the relative role of the major presumed controls of macroalgal blooms under a wide variety of conditions.

    To synthesize the various data sets, we will use recent advances in stage-structured matrix models. With the models (and inputs from our cage and tile experiments) we will estimate demographic statistics and will do simulations in which we predict the occurrence of blooms of green, red, and brown macroalgae under a variety of circumstances. These syntheses can then form a basis for management practices.

  ECOHAB2001: PROJECT SUMMARIES

  • Anderson, D.M. (WHOI). ECOHAB: Toxin composition variability as an indicator of nutritional status of Alexandrium field populations. 9/1/01-8/31/04. NOAA/COP. E-mail: danderson@whoi.edu.

    Harmful algal blooms (HABs) are a serious economic and public health problem throughout the world. In the U.S., the most widespread HAB problem is paralytic shellfish poisoning (PSP), a potentially fatal neurological disorder caused by human ingestion of shellfish that accumulate saxitoxins produced by dinoflagellates in the genus Alexandrium. PSP closures affect thousands of miles of U.S. coastline and numerous fisheries resources annually.

    Understanding the processes that lead to the growth, accumulation, and negative impacts of toxic dinoflagellates is a fundamental goal of HAB research, and an essential element of prevention, control, and mitigation (PCM) strategies. In particular, the factors that regulate dinoflagellate toxicity need to be elucidated, as these directly affect the nature and quantity of toxins that enter the food chain. For PSP toxins, this requires knowledge of not only the abundance of the toxic cells, but of their total toxicity (toxin content) and the chemical nature of that toxicity (toxin composition), since more than 20 saxitoxins exist, with widely varying potencies. Furthermore, efforts to model or predict toxicity require a detailed parameterization of the manner in which these toxicity characteristics are affected by environmental factors such as temperature and nutrients. There have been studies of Alexandrium toxicity as it is affected by environmental factors, but that database proved inadequate when it was recently used to interpret field observations during a major Alexandrium bloom in the Gulf of Maine. In particular, some of the patterns that were expected based on the laboratory data were not observed, such as a decrease in toxin content or a significant increase in C toxins as the cells became nutrient limited, both of which had been considered robust indicators of nitrogen limitation. Part of the problem in extrapolating laboratory culture studies to field populations is that the former did not use Alexandrium isolates representative of the region under study, nor has a "generic" response or diagnostic indicator been identified that applies to all Alexandrium populations under all conditions. In addition, the experimental procedures did not adequately simulate conditions in the natural environment, especially the transition to sexuality and cyst formation that occurs under nutrient limitation. The overall goal of the project proposed here is to use semi-continuous cultures and field-deployed mesocosms to characterize and parameterize the manner in which Alexandrium populations vary in toxicity in response to environmental and nutritional fluctuations. Specific project objectives are to: 1) Establish a set of Alexandrium isolates that represent the extremes of regional and temporal population variability for the Gulf of Maine in particular, and for globally distributed Alexandrium, in general; 2) Document the extent to which toxin content and composition vary in these isolates under different nutritional conditions using semi-continuous cultures; 3) Determine the extent to which nutrient-driven toxicity variations are modulated by temperature; 4) Study the effects of sexual induction and cyst formation on patterns of toxin accumulation; 5) Identify robust indicators of nutrient limitation, considering trends in toxin content and a variety of toxin composition ratios; 6) Evaluate the validity of these nutritional indicators using mesocosm deployments and controlled nutrient additions at sites of natural Alexandrium blooms; 7) Use the observed patterns of toxin composition variability to infer saxitoxin biosynthetic and interconversion pathways and mechanisms and identify generic responses to nutrient limitation; and 8) Incorporate nutrient and toxin variability into existing physical/biological coupled models for Alexandrium in the Gulf of Maine.

    The information to be gained from this study will add much to our ability to understand, model and predict PSP outbreaks caused by Alexandrium, and to assess the extent to which natural populations are nutrient limited using an indicator which is unique to this group of saxitoxin-producing organisms.

  • Buskey, E.J. (UT-Austin). ECOHAB: The role of zooplankton grazing in harmful algal bloom (HAB) dynamics and trophic transfer of toxins. 10/01/01-09/30/04. NOAA/COP. Email: buskey@utmsi.zo.utexas.edu

    The objective of this proposal is to better understand the potential role of zooplankton grazers in the initiation and maintenance of harmful algal blooms, and to better define their role in the transfer of toxins through the food chain where they can potentially be ingested by humans or endangered wildlife species. This project will focus on the toxic dinoflagellate Gymnodinium breve which causes extensive fish kills, human health risk and economic loss along the Gulf of Mexico coast. Protozoan grazers have rapid growth rates and are the most likely grazers on harmful algal species to be capable of controlling blooms during their initial phase. Metazoan planktonic grazers such as copepods may also graze on harmful species, but their longer generation times make them poor candidates for controlling blooms. However, since some copepods tend to avoid consuming toxic phytoplankton species, they may switch to alternate prey, including protozoan grazers of harmful algal species, which may indirectly aid the formation or maintenance of blooms. A series of laboratory experiments is proposed to measure the grazing and growth rate of zooplankton grazers on harmful algal blooms and the impact of metazoan planktonic grazers on the interaction between protozoa and harmful algae species, to determine the role of planktonic grazers in harmful algal bloom dynamics and as vectors to transfer toxins to higher trophic levels. Although several studies have shown that some species of harmful algae are not acutely toxic to zooplankton, there has been less study of sublethal effects of toxins on zooplankton. If these toxins, many of which act as neurotoxins, affect the behavior of zooplankton, this might make them more likely prey for visual predators. Changes in behavior could both make the zooplankton more conspicuous to their predators and might also reduce the efficacy of their escape behaviors. In this study we propose to use video computer motion analysis techniques to see if toxins produce abnormal swimming behavior in zooplankton that ingest them. We will also use high speed video to carefully examine the kinetics of copepod escape behaviors to see if zooplankton that ingest toxic algae have impaired escape responses, making them more likely prey and increasing the chances of biological magnification of toxins. If the opportunity presents itself, we also propose to sample zooplankton during an outbreak of red tide along the Texas coast to examine feeding behavior of zooplankton under natural conditions and to test for copepod growth and toxin content within bloom areas.

  • Connell, L. (UMaine), V. Trainer (NOAA/NWFSC), and V.M. Bricelj (IMB). ECOHAB: A molecular basis for differential susceptibility and accumulation of PSP toxins in commercial bivalves. 10/1/01-09/30/04. NOAA/COP. Email: laurie.connell@umit.maine.edu

    The proposed multidisciplinary research collaboration will characterize the complex mechanism underlying bivalve susceptibility to paralytic shellfish toxins (PSTs) and species-specific toxin accumulation. In mammals, PSTs affect nerve function via specific block of the voltage-sensitive Na+ channel. Bivalves, however, clearly have adaptations that permit them to tolerate toxins in their algal food. Specifically, "insensitive" bivalve species are known to harbor, without apparent harm, high concentrations of PSTs, while more "sensitive" species attain relatively low toxin levels and can suffer sublethal or lethal effects from harmful algal blooms (HABs). This susceptibility to ingested toxins and thus, ability to accumulate toxins, varies markedly both within and among bivalve species.

    Although many examples of resistant and sensitive Na+ channels have been described in nature, little is known about the genetic basis of these different subtypes in molluscan shellfish. For example rat and human cardiac Na+ channels are resistant to toxins, whereas their skeletal muscle Na+ channels are sensitive. Additionally, natural resistance to pesticides has developed in the Na+ channel gene of some insect populations. Our current ECOHAB research has characterized up to 50-fold differences in toxin affinity among populations of Mya arenaria, providing evidence for the presence of a Na+ channel mutation that confers resistance to PSP. A key goal of this proposal is to more completely characterize the basis (at the molecular level) for inter- and intraspecific variation in toxin uptake and sensitivity in bivalves. Specific objectives of this research will be to characterize toxin tolerance (and thus also toxin accumulation) at the level of (1) the Na+ channel gene, with specific focus on the known sites of PST action; (2) the isolated nerve; (3) behavior (burrowing); and (4) feeding physiology and toxin uptake. Interspecies differences will shed light on protective mechanisms used by various shellfish to resist the effects of toxins using both short-term behavioral and long-term genetic strategies, as well as on the fate of toxins through the ecosystem.

    This research will help to develop genetic markers for identification of bivalves that have natural mutations in the Na+ channel gene. These will be related to the organisms¹ ability to tolerate (and thus concentrate) toxin. Interspecific differences in shellfish susceptibility to toxins will be explored in oysters (Crassostrea gigas, C. virginica), and clams (Mya arenaria, Siliqua patula, and Saxidomus giganteus) from historically toxic and non-toxic areas on the Pacific (including Alaska) and Atlantic coasts of N. America. Genetic markers will allow selection of non-accumulating bivalve stocks in a given region, thereby reducing harvest losses. Identification of inter- and intraspecific genetic differences will contribute to our fundamental understanding of toxin resistance mechanisms and perhaps open future avenues for selective breeding. Regional characterization of bivalve responses to toxic algae will help to predict the impacts of paralytic shellfish poisoning (PSP) at the level of primary consumers over a wide geographical range. Understanding of the relationship of specific toxin vectors to the intensity and frequency of HABs in a given area, will contribute to improved management of commercially important shellfisheries and assessment of human health risks associated with HABs.

  • Dam, H.G. (UCONN-Avery Point) ECOHAB: Ecological and evolutionary consequences of the spreading of Alexandrium to grazers, and implications for bloom formation and maintenance. 08/01/01-07/31/04. NOAA/COP. Email: hans.dam@uconn.edu

    Harmful algal blooms (HABs), which pose a potentially serious threat to public health and to fisheries, have been increasing in frequency and duration worldwide. However, the ecological and evolutionary consequences of the spreading of HABs to grazers, the ramifying effects on food web structure and function, and on the transfer of toxins are not well understood. The toxic dinoflagellate Alexandrium spp. has been spreading from the north (Bay of Fundy, Canada) to the south (New Jersey, USA). The frequency and toxicity of Alexandrium blooms decrease from north to south. Because of the longer time to which grazers in the northern waters have been exposed to the putative selection pressure represented by toxic Alexandrium, one may hypothesize that these grazers have developed adaptations to better cope with this toxic alga. Indeed, preliminary studies in our laboratory analogous to transplant experiments are consistent with this hypothesis of local adaptation. That is, females of the copepod Acartia hudsonica from southern populations (NJ) naïve to toxic Alexandrium fed a toxic strain of Alexandrium (from ME) display significantly reduced ingestion and egg production rates relative to females from northern populations (ME) historically exposed to toxic Alexandrium. Our general goals are to understand the mechanisms responsible for these biogeographic differences, to evaluate the Darwinian fitness consequences of toxic Alexandrium to naïve and historically exposed populations of copepods, and to test for rapid copepod evolution to toxic Alexandrium. The specific goals are: 1) To determine whether physiological incapacitation by Alexandrium is responsible for the observed difference in ingestion and egg production rates between naïve and historically exposed populations of copepods. 2) To examine acclimation to toxic Alexandrium in the naïve and historically exposed populations of copepods. 3) To examine the effects of toxic Alexandrium on life history traits and population fitness of historically naïve and historically exposed populations of copepods. 4) To examine whether toxic Alexandrium can induce rapid evolution in historically naïve populations of copepods.

    To address goal 1, ingestion and egg production rates will be compared for naïve and exposed populations fed toxic Alexandrium in short term experiments (hours). For goal 2, physiological acclimation by copepods will be examined by comparing copepod rate processes through length of time (1-15 d) of exposure to toxic Alexandrium. For goal 3, changes in copepod fitness will be examined by rearing the population from egg through adult on diets with and without toxic Alexandrium, and determining age-specific survivorship and fecundity, and the finite population growth rate as a function of diet. For goal 4, genetic adaptation of copepods will be tested with selection experiments with a naïve population exposed to a diet containing toxic Alexandrium and determining changes in fitness over generations (1-12).

    The biogeographic context of this study through its tests of local adaptation of grazers to a toxic phytoplankter will provide a significantly improved understanding of the consequences of HABs to a key group of planktonic consumers, and hence in foodwebs and fisheries. In essence, our study will provide the first detailed understanding of how a key group of grazers responds ecologically and evolutionary to the spreading of HABs. In addition, results of this study will be essential in testing the long-held assumption that toxic algae bloom because of failure of grazing control. If indeed local adaptation of grazers take place, then the grazing-failure hypothesis will require substantial revision. This knowledge is essential to the understanding of HAB formation and maintenance and to predictive models of HABs.

  • Doucette, G.J. (NOAA/CCEHBR). ECOHAB: Algicidal bacteria targeting Gymnodinium breve: population dynamics & killing activity. 09/01/01-08/31/03. NOAA/COP. E-mail: greg.doucette@noaa.gov

    Blooms of the toxic dinoflagellate Gymnodinium breve occur in coastal waters throughout much of the Gulf of Mexico, but are especially problematic along the Florida coast where these annual events last at least 3-4 months and exact a large toll on the regional economy with losses estimated at about $20 million per episode. As a result, there is a growing interest in exploring strategies for the prevention, control, and/or mitigation of G. breve blooms. Understanding the population dynamics of harmful algal blooms (HABs) and elucidating the factors controlling transitions between bloom phases are critical for effectively dealing with HABs such as Florida¹s red tides, and ultimately minimizing their socio-economic impacts. Thus, a logical approach for efforts aimed at identifying potential strategies for managing G. breve blooms and their negative consequences is to acquire knowledge of natural mechanisms involved in regulating population growth of this species.

    Several reports of algicidal bacteria in high concentrations at the onset of HAB termination have led to the suggestion that such microbes may directly or indirectly lead to the decline of blooms. The issue of bacterial involvement in terminating algal blooms has only recently been addressed in relation to HABs occurring in U.S. waters. In a previous ECOHAB project, our group initiated a search for algicidal bacteria active against G. breve in waters of the west Florida shelf. This effort led to the first discovery of algicidal bacteria targeting a HAB species resident along the U.S. coast. In fact, two bacterial strains lethal to G. breve were isolated, classified phylogenetically using 16S rDNA sequences, and their killing activity partially characterized. Moreover, we have used the sequence data to develop taxon-specific rRNA probes, which have been used as tools to begin examining the population dynamics of these algicidal bacteria. Additionally, study of the killing activity revealed that both algicidal strains released a dissolved bioactive compound(s) into the growth medium, causing the death and lysis of G. breve.

    We have developed a conceptual model for the population dynamics of algicidal bacteria over the course of a G. breve bloom event based on our previous findings, and propose to test these ideas in laboratory cultures, microcosms, and field populations with the work described herein. A principle hypothesis is that algicidal bacteria targeting G. breve, initially a background component of the ambient bacterial community, increase in both absolute and relative abundance over the course of a bloom. Taxon-specific rRNA probes and other molecular tools (e.g., DGGE) employed in our previous work will provide the means to track populations of these algicidal bacteria within mixed microbial assemblages in laboratory cultures and microcosms, as well as samples taken from G. breve blooms, and thus to better assess their involvement in promoting bloom decline. The other focus of this project will be identification of the bacterially produced algicidal compound(s) and elucidation of its mode of action, thereby permitting development of an assay for the detection and measurement of this activity.

    These complimentary lines of investigation involving both the organism and its biologically active compound(s) will yield a better understanding of the role played by algicidal bacteria in terminating G. breve blooms, as well as further insights into the potential use of these bacteria as part of an effective management strategy for HABs and their frequently devastating impacts.

  • Durbin, E.G. (URI), G.J. Teegarden (Bowdoin College), R. Campbell (URI), and A.D. Cembella (Institute for Marine Biosciences, NS). ECOHAB: The role of zooplankton grazers in harmful algal bloom dynamics. 09/01/01­08/31/04. NOAA/COP. Email: edurbin@gso.uri.edu

    Harmful algal blooms threaten public health and economic activities (fisheries, aquaculture, and tourism) in coastal environments. The biological interactions of harmful species and their competitors and predators are significant and may determine bloom dynamics and ecosystem impacts. Results from our recent ECOHAB study suggest that grazer response to harmful algal blooms will vary with spatial and temporal changes in grazer communities and prey field assemblages. Based on these results, we propose a study of the role of grazing by zooplankton in the population dynamics of Alexandrium, and the transfer and fate of toxins in marine food webs. Field and laboratory experiments will test these principal hypotheses: 1) Grazer response to toxic Alexandrium in natural assemblages is dependent on Alexandrium concentration and abundance relative to co-occurring phytoplankton (e.g. stage of bloom development). 2) Grazing pressure on Alexandrium depends on the composition and abundance of the co-occurring phytoplankton community. 3) Grazing pressure on Alexandrium depends on the composition, abundance, and selective feeding ecology of the grazing community. 4) Significant losses of both toxin and carbon from ingested Alexandrium cells result from destructive rejection of toxic cells, with implications for toxin transfer and secondary production. Laboratory experiments will: 1) Examine the feeding behavior and selectivity of dominant zooplankton from the Gulf of Maine in gradients of Alexandrium cell concentrations and alternate prey. 2) Investigate the acute and chronic effects of toxic Alexandrium exposure on grazing behavior, toxin assimilation, detoxification, growth, and reproduction. Field studies in the Grand Manan Basin, an upstream source region of Alexandrium for the Gulf of Maine, will use fine-scale drifter studies to: 1) Determine the feeding behavior of zooplankton in this region of higher abundance of Alexandrium. 2) Investigate the role of grazing by zooplankton in controlling Alexandrium blooms. 3) Determine fine-scale distribution of zooplankton, potential co-existence of zooplankton and layers of Alexandrium, and the temporal dynamics of zooplankton feeding processes in layers, using process studies in distinct water masses. 4) Describe the accumulation of toxin in zooplankton tissues and determine whether toxin accumulation in zooplankton poses risks to higher trophic levels. The results will address several program objectives. Characterization of grazer response over a continuum of bloom conditions will provide basic information on the significant role of grazers in the regulation (or lack thereof) of harmful algal blooms. A grazer deterrent effect (which would promote bloom formation), apparent under some but not all conditions, is a hypothesized ecophysiological role for phycotoxin production, and our proposed study will directly address the validity of this hypothesis. Zooplankton grazers can act as vectors of toxin in food webs; the proposed study will provide quantitative assessment of this threat to ecosystems over a variety of bloom conditions. The results will expand on our recent ECOHAB study to provide quantitative estimates of grazing impact over the range of Alexandrium bloom conditions, currently lacking, but necessary for accurate modeling of harmful bloom dynamics.

  • Kamykowski, D. (NCSU) and G. S. Janowitz (NCSU). ECOHAB: Laboratory and numerical modeling studies of Gymnodinum breve behavior to aid in predicting natural bloom events. 09/01/01-08/31/04. NOAA/COP. Email: dan_kamykowski@ncsu.edu

    Abbreviated versions of the hypotheses to be tested are: 1) populations of Gymnodinium breve can be reliably quantized (all cells divide together every 3rd day) by isolating the cells that vertically migrate to form a population aggregate in the mid-afternoon surface layer into a separate culture; 2) in nutrient replete water columns, unequal cell division by Heterocapsa illdefina and G. breve parent cells yields a high variance of biochemical content among daughter cells at all depths immediately after cell division; 3) all G. breve cells in a nutrient replete water column have similar generation times irrespective of the depth occupied; 4) G. breve cells that grow in a water column where nitrogen is vertically patchy undertake diel vertical migrations influenced by the vertical location of the nitrogen source; 5) the toxin content of aggregating and non-aggregating G. breve cells differs because of unequal allocation at division and/or because of different, behaviorally determined light/nutrient exposure in the water column; and, 6) environmentally and behaviorally realistic physical-biological models are required for accurate HAB prediction. These hypotheses will be tested using proven experiment protocols applied to 225 L cultures of G. breve and will be extended to natural populations by refining our evolving physical-biological numerical models.

    Predicting when and where different dinoflagellate species significantly contribute to HABs presently is compromised by the incomplete information on how cells use their motility. This information is difficult to obtain with field populations because of unknown population history associated with patchy events in time and space that occur in a dynamic fluid environment. The proposed laboratory work, under conditions where the population history is controlled and the same population is continuously available, will help define the biological context within which cell behavior influences vertical position in the water column and, thus, cell growth/division rates and horizontal transport.

  • Kudela, R.M. (UCSC), Smith G.J. (SJSU) and Armbrust, V.E. (UW). ECOHAB: Development of Molecular and Biochemical Signatures for the Detection of Toxin Production In Pseudo-nitzchia spp. Under Nutrient Stress. 10/15/01-10/15/04. E-mail: kudela@cats.ucsc.edu

    The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used to interrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested:

    H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia.
    H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development.
    H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer.

    Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.

  • Maranda, L. (URI), S.L. Morton (NOAA), and P.E. Hargraves (URI). ECOHAB: Diarrhetic toxins and Prorocentrum lima in New England coastal waters. 09/01/01-08/31/04. NOAA/COP. Email: lmaranda@gso.uri.edu

    The overall goal of this proposal is to determine the potential for diarrhetic toxins to contaminate shellfish resources along the coast of New England. This research will contribute to a better understanding of the population dynamics of the toxin producer, Prorocentrum lima, within the epibiotic communities of wild and cultured shellfish and examine the relationship between the abundance of P. lima and shellfish contamination with diarrhetic toxins. Our specific objectives are: 1) to determine the seasonal distribution of P. lima associated with wild and cultured shellfish, 2) to elucidate the quantitative relationships between P. lima and the epibiotic community fouling cultured shellfish, and 3) to determine the toxin load and composition of the epibiotic community, and of wild and cultured shellfish over time. Three hypotheses will be tested: 1) P. lima from New England coastal waters is toxigenic with respect to production of okadaic acid and derivative active compounds, 2) P. lima is more abundant within the epibiotic community associated with suspended shellfish rather than with wild shellfish, and 3) Shellfish grown in suspension culture become contaminated with DSP toxins at a faster rate and at a higher level than wild shellfish.

    The seasonal distribution of P. lima and epibiota will be assessed at eight sites along the New England coast where P. lima is either known or suspected to occur; four sampling stations will be located at shellfish aquaculture facilities and four will be in coves open to wild shellfish harvesting when in season. Toxin analysis will be performed on epibiota < 90 µm) and on the digestive glands of shellfish at given locations. Toxin will be reported in terms of protein phosphatase inhibition activity and toxin composition.

    We know P. lima is widespread in New England coastal waters. This presence signals the potential for diarrhetic shellfish poisoning. We know that low levels of okadaic acid-like activity have been detected in blue mussels along the coast of Maine. We now need to find out whether and to which extent this DSP potential can be realized and under what conditions. This proposal attempts to fill some of these gaps (first area of emphasis of the ECOHAB solicitation), especially in light of changes in aquaculture practices from on-bottom to suspension mode (specific topic #2), creating conditions favorable for the growth of P. lima. We expect to provide results which will allow us to verify whether the pattern observed in eastern Canadian waters may repeat and affect shellfish and public health in the U. S. Northeast.

  • Mulholland, M.R. (ODU) and E. Minor (ODU). ECOHAB: Nutritional factors promoting the growth and dominance of Aureococcus anophagefferens in coastal waterways. 09/01/01 ­ 08/31/03. NOAA/COP. Email: mmulholl@odu.edu

    Dissolved organic material (DOM) has been implicated as a causative agent promoting the growth of harmful algal species and initiating blooms in the inland waterways of the Eastern United States. In particular, the Brown Tide chrysophyte, Aureococcus anophagefferens, occurs at bloom densities in the inland waterways of NY, DE and MD. It is likely that its geographical range extends into VA. These areas tend to be shallow and have restricted flushing and therefore are impacted by nutrient inputs from terrestrial runoff, groundwater inputs and sediment resuspension. The objectives of this project are to: identify compounds and compound classes that stimulate the growth of A. anophagefferens, determine possible sources of DOM and its importance to the nutrition of this species, assess how competition for DOM and nutrients affects growth of A. anophagefferens relative to co-occurring taxa, and determine the nutrient conditions that promote blooms.

    To meet our project goals, we have selected field sites that have similar physical attributes (e.g., circulation and morphology) but different densities of A. anophagefferens. We will conduct intensive process studies at a site in Chincoteague Bay, where A. anophagefferens is known to occur at high densities, and at more southerly sites that are less urbanized, have different sources of DOM and have not previously experienced high abundances of this species (e.g., Hog Island Bay), but may be vulnerable to blooms if there are changes in nutrient conditions. These sites have the advantage of being near Norfolk and the Virginia Institute of Marine Science¹s Eastern Shore Laboratory (ESL) in Wachapreague, VA. In addition, there are long-term monitoring data available for both sites. At each site, and one near to Wachapreague, we will employ a variety of new techniques to characterize DOM, identify and measure the primary pathways of C and N cycling and determine the competitive interactions that affect the cycling of DOM and its use by mixotrophs, such as A. anophagefferens. In addition, we will conduct outdoor mesocosm experiments at ESL using natural populations. This will enable us to determine the effects of longer-term (14 - 21 days) nutrient enrichments on competition for DOM and nutrients and competition among co-occurring species. This work will answer basic questions regarding the extent to which organic N and C supports the growth of A. anophagefferens, the nutrient conditions that stimulate blooms and mixotrophy, how competition for DOM varies depending on its source and composition, and differences among areas that experience blooms and those that do not.

    Paul, J.H. (USF). ECOHAB: Quantitative detection of transcriptionally active carbon fixation genes in the Florida red tide organism, Karenia brevis. 09/01/01-08/31/04. NOAA/COP. Email: jpaul@seas.marine.usf.edu

    By conservative estimates, harmful algal blooms cost the U.S. over $50 million/year. In the Gulf of Mexico, K. brevis has caused economic loss and massive marine animal (including mammal) mortalities for over a century. Methods for rapid detection of blooms and monitoring of specific K. brevis strains are greatly needed. We have recently cloned and sequenced a portion of the K. brevis carbon fixation gene, rbcL. As a coding gene (in contrast to rRNA genes) we hypothesize that there might be enough genetic diversity present in rbcL to differentiate K. brevis strains. Additionally, as a functional gene which is expressed in viable cells, we can detect its expression (as mRNA) as an indicator of viable (transcriptionally active) cells. Our overall objective is to understand the function of this gene in this organism, its regulation, its organization within the plastid genome, and its ability to serve as a marker for K. brevis detection in the field. Our specific objectives are: 1) To design Real Time PCR probes for detection, quantitation, and differentiation of Karenia strains based upon rbcL sequence data 2) To assess the performance of the probes with Karenia strains in cultures and in field samples and 3) To determine the molecular regulation of the carbon fixation genes in K. brevis. For the first objective, we will sequence strains provided by Karen Steidinger of the Florida Marine Research Laboratory and design probes based upon sequence alignments. For the second objective, we will test probes against these strains and natural seawater samples containing bloom and non-bloom levels of K. brevis. For the third objective, carbon fixation and rbcL transcript abundance will be determined in diel studies in cultures to understand regulation of the carbon fixation operon. Collectively these studies will yield a functional approach to the issue of carbon fixation in K. brevis, as well as provide probes for the specific detection of active strains in the field.

  • Frances M. Van Dolah, F.M. (NOAA/CCEHBR). ECOHAB: Cellular mechanisms mediating bloom longevity and bloom termination in a toxic dinoflagellate, Gymnodinium breve. 09/01/01-08/31/04. NOAA/COP. Email: fran.vandolah@noaa.gov

    Development of effective management strategies for addressing the occurrence and impacts of harmful algal blooms is dependent upon adequate insight into the mechanisms that control bloom initiation, growth and termination. Dinoflagellates are the major group of microalgae responsible for the production of toxins that impact human and environmental health. Among these, the Florida red tide dinoflagellate, Gymnodinium breve, is among the most notorious. G. breve blooms occur almost annually off the west coast of Florida, where they cause extensive fish kills, marine mammal mortalities, and human illness due to both respiratory irritiation and neurotoxic shellfish poisoning. Blooms of G. breve initiate offshore, and become a threat to coastal ecosystems and humans only when carried inshore by prevailing wind and oceanographic conditions. Once in coastal waters, G. breve cells may experience dramatic changes in environment, resulting in temperature, salinity, pH, light, turbulence, and oxidative stresses. As a consequence, blooms may either rapidly die out, or they may adapt and persist for many months within coastal embayments, where their impacts on the coastal populations of marine animals and coastal economies may be devastating.

    A goal of the ECOHAB Program is to develop scientifically sound approaches to prevention, control and mitigation of HABs. The processes that result alternatively in cellular adaptation and bloom persistence, or cell death and bloom termination, may be suitable targets for developing predictive indicators or control strategies applicable to limited, sensitive areas. Analogous to cancer research, we must understand the cellular processes driving the consequences of this "decision point" in the fate of a bloom, before we can potentially modulate its outcome. In this project, we propose to elucidate cellular mechanisms in G. breve that mediate adaptation of G. breve cells to environmental stressors, and mechanisms that mediate G. breve cell death.

    All eukaryotic cells possess stress proteins that are activated in response to denaturing cellular stress, including heat shock proteins (sHsps), molecular chaperones (Hsp60, Hsp70), and antioxidant systems (e.g., GSH, SOD). In the current project we propose to identify stress proteins present in G. breve and characterize their expression in response to relevant environmental stressors likely to be encountered by coastal blooms. Failure of cells to adapt to such stressors results in cell death. The pathways present in dinoflagellates that trigger cell death are largely uninvestigated. Programmed cell death (PCD) is a highly conserved mechanism by which eukaryotic cells selectively die. Although previously thought to have evolved in multicellular organisms, recent evidence indicates the presence of at least certain components of this pathway in unicellular protists. We propose to determine the presence of the PCD in G. breve, evaluate its activity during the growth, stationary phase, decline phases of laboratory cultures, and determine the triggers for its activation.

    Following characterization of stress responses and cell death pathways in laboratory investigations, we will evaluate their expression in growth vs termination phase blooms of G. breve on the west coast of Florida. This proposal represents the first attempt to define cellular mechanisms responsible for bloom termination in a toxic dinoflagellate. The results of this project will yield molecular biomarkers of physiological status in naturally occurring blooms and may identify novel targets for control measures. Although this work will be carried out in G. breve, we anticipate that insight gained in this species, and tools developed for this species, will be broadly applicable to other toxic dinoflagellate species.

  • Vogelbein, W.K. (VIMS), L.W. Haas (VIMS), K.S. Reece (VIMS), and J.D. Shields (VIMS). ECOHAB: Life history and pathogenicity of Pfiesteria shumwayae. 07/01/01-06/30/04. NOAA/COP. Email: wolf@vims.edu

    The toxic ambush-predator dinoflagellate, Pfiesteria piscicida, is considered a serious emerging fish and human health problem in some Mid-Atlantic U.S. estuaries. Adverse health effects are thought to be caused by potent exotoxins, the production of which appears intimately tied to life cycle transformations and specific environmental cues that regulate them. The life history of P. piscicida is complex and reported to consist of 24 stages including flagellated, amoeboid and cyst forms, several of which are reportedly toxic. However, many aspects of the life history and factors that influence toxigenicity remain poorly documented and controversial. Recently a second species, soon to be described and named Pfiesteria shumwayae, was discovered. Little published information is available on this undescribed species other than that it is believed to be similar to P. piscicida in "toxigenicity" and complex life history. We recently developed a 96 hr larval fish bioassay that provides, for the first time, a sound experimental approach to critically investigate life history and toxigenicity issues of Pfiesteria spp. under rigorously controlled laboratory conditions. This bioassay is the center piece of our proposed studies. Preliminary tests of the assay using our "pathogenic" P. shumwayae cultures, suggest that this species differs substantially from P. piscicida in life cycle complexity and "toxigenicity". We propose several refinements to the larval fish bioassay that will allow critical investigation of the role of environmental cues in life stage transformations and toxigenicity of P. shumwayae including: development and application of "gnotobiotic" fish, exposure studies in sterile membrane-compartmentalized culture flasks, one- and two-cell exposures with sterilized fish tissues and application of our recently-developed, highly specific suite of molecular probes. The objectives of the proposed project are to conduct laboratory and field studies to: (1) document the life cycle of P. shumwayae, (2) determine the biotic and abiotic environmental cues that regulate life cycle transformations and toxigenicity, (3) identify which of the life history stages are pathogenic or "toxigenic", and (4) examine the role of filter feeding organisms (e.g., menhaden and oysters) in the regulating life history events and expression of toxigenicity in this new dinoflagellate. Our approach is to examine different life cycle stages using flow cytometry for ploidy, lectin biomarkers for strain or stage specificity, histopathology, SEM and TEM for toxigenicity and life stage studies, and labeled molecular probes to verify participation of the various observed life forms (e.g., amoebae) in the P. shumwayae life cycle. We have observed P. shumwayae and other PCOs in the alimentary tracts of fishes from the laboratory and the field. Thus, do menhaden and oysters serve as vectors in the life cycle of Pfiesteria spp., or does passage through the alimentary tract of these filtering organisms induce life stage transformations directly? Can we develop them as biomonitors of environmental outbreaks of Pfiesteria spp.? We outline feasibility studies using in situ hybridization methodology and real-time PCR technology to identify and quantify life cycle stages in the tissues of these model filter-feeders and their possible important role in modulating stage transformations and toxigenicity.

  ECOHAB1999: PROJECT SUMMARIES

  • Anderson, D.M. (WHOI) and J. Rensel (Rensel Associates). ECOHAB: Mitigation of fish-killing algal blooms using clay. 09/01/99- 08/31/01. National Sea Grant Office. Email: danderson@whoi.edu.

    Harmful Algal Blooms (HABs) are a serious and growing threat to coastal waters throughout the U.S. and the world. One of the more significant HAB impacts is the mass mortality of aquaculture fish, leading to substantial economic losses on fish farmers and their insurers. Fish farmers have a few techniques for reducing the impacts of HABs, but none are fully reliable or effective; and several involve serious risk for loss of the fish and facilities. The overall objective of this project will be to field test a combination of strategies - clay treatment (to remove cells from the water column) and perimeter skirting (to retain treated water and prevent recontamination). Neither of these technologies is unique individually, but they have never been applied in combination in the manner we envision. This will also be the first concerted bloom control or removal effort attempted in the U.S. for fish mariculture. Preliminary laboratory experiments indicate there are specific types of clays that effectively remove several HAB species at relatively low application rates. As part of this proposal, more work will be conducted to optimize the types of clay needed for Heterosigma removal, and to establish clay loadings and application protocols best suited for fish farms. The project will then conduct a series of pilot-scale clay treatments inside experimental fishnet pens operated by an active net-pen aquaculture facility. This facility has an NPDES permit allowing for water column and benthic impact zones and we will strive to retain all effects within that zone and not exceed Washington State water quality standards for turbidity (i.e., no greater than 5 NTU above background). Effects of the clay treatment on the fish, the plankton community, the water quality, and the benthic community will all be assessed. The information to be collected in this manner is essential if this approach to HAB mitigation is to be used on a larger scale at fish farms or other environments.

  • Haab, T. C. ( ECU), J. Whitehead (ECU), D. Lipton (UM), J. Kirkley (VIMS), and G. Parsons (UDE). ECOHAB: The economic effects of Pfiesteria in the mid-Atlantic region. 09/01/99- 08/31/01. National Sea Grant Office. Email: HAABT@MAIL.ECU.EDU.

    While significant amounts of research are currently being conducted to assess the biological, ecological and environmental effects of Pfiesteria piscicida and other harmful algal blooms (HABs), very little work has been conducted to look at the economic impacts or lost benefits due to Pfiesteria outbreaks or HABs. To begin to assess the economic impacts of Pfiesteria and HABs, we propose a 2-year Mid-Atlantic (North Carolina, Virginia, Maryland and Delaware) study of seafood consumption combining both revealed and contingent behavior questions. The study will consist of two surveys, analysis, and a final report. The surveys (to be administered by the East Carolina University Survey Research Laboratory) will consist of two parts. The first will be a combined phone-mail survey of the Mid-Atlantic region focusing on current seafood consumption patterns and reactions to harmful algal blooms, Pfiesteria outbreaks, and education materials. The second part of the survey will consist of in-person interview surveys in North Carolina. The survey instrument will be similar to the phone-mail survey but will allow for more visual material. This will also allow us to test for differences or similarities between survey formats. By combining revealed behavior regarding seafood consumption with contingent behavior regarding future seafood consumption plans under a variety of realistic hypothetical outbreak and policy scenarios the proposed study will measure the economic effects of a Pfiesteria outbreak on the Mid-Atlantic seafood market.

    Questions to be addressed include: What are the current seafood consumption patterns in the Mid-Atlantic region? What is the effect of a HAB/Pfiesteria outbreak on seafood consumption in the Mid-Atlantic region? What are the economic welfare effects of an outbreak? What are the potential economic impacts of an outbreak (as opposed to welfare losses)? What are the regional similarities or differences in response to outbreaks (Albemarle/Pamlico Sound estuary system versus Chesapeake system)? Do respondents respond differently when shown the risk or counter-information in person rather than through the mail or over the phone? Are contingent behavior surveys capable of identifying substitution effects.

    The project represents a case study of the economic impacts of Pfiesteria on the Mid-Atlantic seafood industry as well as a methodological project to understand the conveyance of risk information to consumers. As such, the project will benefit the National Sea Grant College Program goal of evaluating the cost-effectiveness of potential management actions relating to Pfiesteria and HABs.

  • Hoagland, P. (WHOI), D. Jin (WHOI), and H. Kite-Powell (WHOI). ECOHAB: A framework for estimating the economic impacts of Harmful Algal Blooms (HABs) in the United States. 09/01/99- 08/31/01. National Sea Grant Office. Email: hoagland@whoi.edu.

    We plan to develop a methodological framework for estimating the economic impacts of HABs on local, regional, and national scales. The proposed study is designed to achieve the following objectives: develop a framework for the economic impact analysis of HABs using an I-O modeling approach; develop detailed case studies using the framework (specific locations and types of damages); develop local, state, and national level I-O models for the case studies, generate economic impact estimates, and calculate multipliers; summarize the impact at the local, state, and national levels, and present a national overview of economic impacts HABs; extend the analysis of Anderson et al. (1998) to estimate the economic impacts of HABs during 1993-1998, resulting in a 12 year window for estimating the average annual impacts of HABs; and conduct sensitivity analyses of key factors, e.g., individual and cumulative impacts: human health, fisheries, and recreation.

    This study employs an input-output (I-O) methodology to estimate, on a consistent basis, the regional and national economic impacts of HABs. The I-O methodology was developed to characterize the nature of interactions among the sectors of an economy. Application of the I-O methodology will result in estimates of the scale of economic impacts and the distribution of those impacts across economic sectors at relatively low cost relative to other valuation methods. Application of I-O analysis will help to identify geographic locations where further work should be conducted using more intensive and more costly methods of economic valuation to estimate the economic damages associated with HABs and to scale the appropriate policy responses and mitigation actions.

    There is a current need to develop estimates of the economic impacts and distributional effects of marine natural hazards such as HABs. This kind of information is needed to begin to think carefully about appropriate policy responses to such hazards. To date, very little work has been done to account for the economic impacts of HABs in the United States and to report them on a consistent basis. Aggregate impacts average about $42 million per year, which is on the scale of a single HAB event with major economic impacts. When a HAB event resulting in major economic impacts is added to the annual average, impacts could approach or even exceed $100 million. The results of our study will allow public and private decision-makers to gain a better perspective on the scale of the economic impacts and employment effects associated with HABs and the distribution of impacts and employment effects across the sectors of the relevant economies. These decision-makers will be able to use the estimates to begin to assess the risks of HAB events at the local, state, and national levels and to scale appropriate policy responses to deal with those risks.

  • Kana, T.M. ( HPEL), H. MacIntyre ( HPEL), J. Cornwell ( HPEL), and M.. Lomas (HPEL). ECOHAB: Benthic-pelagic coupling and LI Brown Tide. 09/01/99-8/31/02. NOAA/COP & NYS Sea Grant. Email: kana@hpl.umces.edu.

    The proposed research is on the controls of the initiation and maintenance of Long Island (New York) Brown Tide events. The central focus of the work is on the role of sediment and benthos as mediators of nutrient exchange with the water column. It is hypothesized that the release of dissolved organic nitrogen (DON) from the benthos plays a significant role in the selection of Aureococcus anophagefferens in Long Island bays. The release of DON relative to dissolved inorganic nitrogen (DIN) can be affected by multiple factors including the movement of groundwater, decomposition of recently sedimented organic matter, resuspension events, and light limitation. Current hypotheses regarding the control of Brown Tide by DON/DIN ratios and known controls of sediment nutrient exchange suggest that sediment processes may help explain regional differences in the occurrence of Brown Tides across the Long Island bays. A coupled benthic-pelagic model is presented as a framework for studying the role of sediments in Brown Tide behavior.

    The proposed research will involve intensive sampling of several habitats in the Peconic Bay region where Brown Tide exhibits different annual behaviors. Measurements of sediment nutrient exchange, assimilation of nitrogen nutrients by the phytoplankton assemblage and the distribution of light through the water column will provide the necessary data to evaluate differences in benthic-pelagic coupling that relate to the incidence of A. anophagefferens blooms.

    In addition, the proposed research will include a continuation of physiological experiments on A. anophagefferens. New technology developed in the PI¹s laboratory will allow accurate bioenergetic measurements of A. anophagefferens growth and photosynthesis under diverse organic nutrient conditions. The results of this work will be used to interpret the ability of A. anophagefferens to utilize organic matter in the field.

  • Kudela, R.M. (SFSU) and W. Cochlan (SFSU). ECOHAB: Regulation of P. australis by C, N, Si interactions. 09/01/99-8/31/02. NOAA/COP. Email: kura@mbari.org.

    The recognition that harmful algal blooms (HABs) are becoming increasingly prevalent in coastal waters and that one such HAB genus, Pseudo-nitzschia, is a common bloom-forming diatom found in the United States, has demonstrated our limited understanding of the ecophysiology of this toxigenic diatom, and more generally of the factors which control coastal productivity. Historically, it has been assumed that the growth of coastal phytoplankton (diatoms in particular) is limited by the relative availability of nitrogen. Recently, this idea has been increasingly questioned, with recognition of the potentially important role(s) of other controlling factors, such as the availability of silicate (an obligate requirement for siliceous organisms), other trace elements, and the complex physiological responses associated with diatom bloom formation. Several models which explicitly test and attempt to quantify the role of nitrogen, silicon, light, and the complex physiological responses to these factors are now available. Although another group is currently examining the production of DA in cultured Pseudo-nitzschia, no one has attempted to integrate these various factors into a coherent ecological framework. We propose to examine the ecophysiological characteristics under which the Pseudo-nitzschia genus becomes dominant in coastal waters and initiates domoic acid (DA) production.

    The goal of the proposed research is to examine the interactions of multiple nutrients (C, N, Si) and light using continuous and batch culturing methods in the laboratory, and to test these interactions in the field. We have the unique ability to utilize both stable and radio-isotope tracers (15N, 14C, 32Si, 32P) for these experiments, and are proposing to integrate both field and laboratory measurements. This approach to understanding DA production sets us apart from the other research groups in Monterey Bay, and provides the ability to assess the overall dynamics of this system. The following two hypotheses will be tested as part of this project. H1. Silicate limitation has modulating effects on the rate of nitrogen uptake and cell growth, normalized to carbon, nitrogen, or pigments in natural diatom assemblages. H2. Significant expression of DA by diatoms (i.e. toxic outbreaks) in the field are rare because nitrogen or light limitation typically occurs before silicate or phosphorous limitation in diatom-dominated coastal upwelling regimes. The primary product of this proposed research is the development of more detailed knowledge of the interactive role(s) of these factors in the development and regulation of HABs in particular, and coastal production in general; by doing so, we can provide the basis for future successful management and modeling efforts.

    By capitalizing on the extensive background in research of multiple light and nutrient interactions in Monterey Bay, the extraordinary oceanographic and laboratory resources available at MBARI and SFSU, and the continuing commitment by MBARI and colleagues at UCSC to the development of a DA research program, there is an unprecedented opportunity to better understand the regulation of potentially toxic diatom blooms. Related but fundamentally different efforts at MBARI by Dr. Chris Scholin (development of a real-time monitoring capability using genetic probes), and at the University of California Santa Cruz by Drs. Mark Wells and Dave Garrison (influence of trace-metal availability on DA production in P. australis), provide the opportunity to develop a synergistic research program in Monterey Bay. We will also coordinate our efforts with other groups who are interested in DA dynamics by participating in the regional bi-monthly meetings held at UCSC. Clearly, if we are to understand how and why toxic blooms of Pseudo-nitzschia occur in areas such as Monterey Bay, we must first understand the fundamental controlling factors regulating new and primary production by diatoms. This proposal will address both of these questions.

  • Lonsdale, D. (SUNY at Stony Brook), D.A. Caron (WHOI), and R. Cerrato (SUNY at Stony Brook). ECOHAB: Causes and prevention of Long Island Brown Tides. 09/01/99-8/31/02. NOAA/COP & NYS Sea Grant. Email: dcaron@whoi.edu.

    The research project is an experimental study of brown tides caused by outbreaks of the pelagophyte Aureococcus anophagefferens in Long Island coastal waters. This project entails the manipulation of natural water samples enclosed in mesocosms on-site in the Peconic Bay estuary system (Coecles Harbor, Shelter Island). The research we propose is a direct result of, and continued effort on, our studies to understand the factors leading to outbreaks, persistence, decline and (potentially) prevention or mitigation of brown tides. During the past three years, we have established an experimental mesocosm system in which we have been able to consistently shift phytoplankton community structure to dominance by A. anophagefferens. We propose to exploit this experimental system, and our results to date, to address three related aspects of the ecology of this harmful algal bloom species. First, we propose a study to examine changes in plankton community structure that take place as A. anophagefferens increases in relative and absolute abundance within a natural plankton assemblage, and the effects that perturbations to the pelagic food web have on success or failure of the brown tide alga. We hypothesize that selective grazing by microbial consumers in the plankton provide A. anophagefferens with a competitive advantage relative to other phytoplankton, and thus bring about a shift in the assemblage to dominance by the brown tide alga. This work will entail planktonic food web manipulations and microbial population measurements, as well as experimental determinations of growth and mortality of A. anophagefferens and co-occurring phytoplankton. Second, we propose a set of experiments to examine the effect of the chemical form of growth-limiting nutrients and the rate of nutrient loading as factors affecting brown tide initiation and the magnitude of blooms. We hypothesize that nutrient loading rate, but not the form of the nutrient per se, can affect the relative abundance of A. anophagefferens in the phytoplankton assemblage, but that total nutrient loading dictates bloom magnitude. We will experimentally investigate the effect of a number of specific nutrients that have been implicated in the stimulation of brown tide outbreaks. Third, we will employ our mesocosms to investigate how filter-feeding bivalves affect planktonic food web structure, and how their activities affect the absolute and relative abundance of A. anophagefferens.

  • Shields, J.D. (VIMS), W. Vogelbein (VIMS), L. Haas (VIMS), H. Kator (VIMS), and V. Blazer (USGS). ECOHAB: Pfiesteria or fungus? Etiology of lesions in menhaden. 09/01/99-08/31/02. EPA. Email: jeff@vims.edu.

    Menhaden, Brevoortia tyrannus, develop ulcerous skin lesions that have been attributed to exposure to Pfiesteria piscicida toxins. The characteristic lesions present as deep penetrating circular ulcers with intense granulomatous inflammation. The presence of these lesions in conjunction with counts of presumptive Pfiesteria-like cells in water samples are the primary criteria for river closures in Maryland and Virginia due to local, recent Pfiesteria activity. There is, however, controversial evidence that the lesions are caused by an oomycete fungus, Aphanomyces sp., either as a primary or secondary invader. The fungus is almost always associated with the lesions, with hyphae penetrating the tissues and organs of the infected fish.

    Biological and environmental stressors appear central to the etiology of ulcers on menhaden. We hypothesize that the development of the disease requires some initiating stressor(s) that erode, damage or penetrate the epidermis and expose the underlying dermis. Recent experiments with striped bass and tilapia exposed to sublethal levels of P. piscicida indicate that fish experience an initial loss of the epidermis. This may provide the portal of entry for propagules of Aphanomyces to attach and invade the dermis and internal tissues of the fish. The resulting granulomatous ulcer is subsequently colonized by secondary invaders (bacteria, saprophytic fungi, etc.), that further influence the pathogenesis of the lesion. Hence, we suggest that the syndrome is most likely a multifactorial disease that arises from the interplay between fish, fungus, and stressors such as Pfiesteria, hypoxia, pH, or pollution.

    The goal of the project is to identify the interrelationships between menhaden, Pfiesteria, and Aphanomyces, and the environmental conditions that may modulate or contribute to the epizootics of ulcers on the fish. The objectives are to conduct controlled laboratory exposure studies with menhaden to (1) identify the causal agents responsible for the ulcerous lesions, and (2) identify contributory environmental and biological conditions that are required for the development and progression of the lesions.

  • M. Sieracki (BLOS). ECOHAB: The effects of microbial food web dynamics on the initiation of Brown Tide blooms. 09/01/99- 08/31/02. NOAA/COP & NYS Sea Grant. Email: msieracki@bigelow.org.

    Since its first, dramatic appearance in 1985, a great deal of research has been done on the brown tide-forming phytoplankton, Aureococcus anophagefferens. As a result, our understanding of its biology and the ecology of the Long Island Bays where it occurs have grown considerably. Aureococcus possesses an unusual ability to use organic nitrogen and carbon, which may allow it to thrive in the turbid, low inorganic nutrient conditions that prevail in the summer in these embayments. However, a variety of other picoalgae, including the typically dominant Synechococcus may also be capable of effectively utilizing the same environment. It is critical to determine what events are controlling the composition of the spring, pre-bloom populations in the impacted areas. To address these issues, we propose here to examine the growth and grazing of Aureococcus within the context of the microbial plankton community. Our experimental manipulations of grazing pressure and organic nutrients during the initiation phase of the bloom will show us how the organism gains a foothold in the "picoalgal niche" that opens at this time of the year.

    Our sampling period will bracket the important "hinge point" period, beginning in late April and ending in late May, when a fundamental shift to small cells occurs. We will sample in an area where Aureococcus biomass is accumulating and it is becoming a dominant component of the population, to compare with our previously collected profiles of "non-bloom" populations. We believe that the picoalgal niche is typically occupied by Synechococcus, and that Synechococcus must be selectively removed or reduced to open the niche to Aureococcus. We also believe that a unique bacterial consortia co-occurs with Aureococcus, and may play a crucial role. Thus, our focus will be the picoplankton community, including phototrophic and heterotrophic components. A reduction in Synechococcus populations may be associated with a bloom of a highly size- selective protozoan grazer.

    We will use a combination of in situ observations of microbial community structure and experiments on natural populations to evaluate some of the possible controlling factors. Dilution experiments will be conducted on the evolving microbial community throughout the bloom initiation period to estimate selective grazing rates on different components of the phytoplankton community. With Synechococcus populations diminished, we hypothesize that Aureococcus outcompetes other co-occurring eukaryotic picoalgae through its ability to utilize dissolved organic carbon and nitrogen. Its association and possible symbiosis with a unique bacterial consortia may also be a selective advantage.

    An understanding of these processes at the early stages of a bloom is critical to the ultimate goal of increasing abilities to predict and mitigate blooms of this harmful organism.

  • Silver, M..W. (UCSC), G. Doucette (NOAA), and R. Tjeerdema (UCD). ECOHAB: Domoic acid in a coastal food web. 09/01/99-8/31/02. NOAA/COP. Email: msilver@cats.ucsc.edu.

    In US coastal waters, domoic acid (DA), a toxin produced by several common species of the diatom Pseudo-nitzschia, has caused marine bird and mammal deaths and contaminated seafood products, resulting in human health problems, fishery closures and economic losses. Relatively little is known about DA transfer through food webs, and toxin detection is poor using the present sentinel species. Our proposal is concerned with examining this transfer process in a coastal site frequently subject to blooms of the toxigenic diatoms. This study will take place in Monterey Bay, a Pacific coastal site with seasonally abundant populations of DA-producing Pseudo-nitzschia, to determine the chronology of toxin passage into key food web intermediaries, both benthic and pelagic. We hypothesize that DA in the nearshore benthos reaches consumers via plankton and various detrital pathways, as reflected in the toxicity of the sand crab Emerita analoga. In the offshore region, we hypothesize that DA reaches key intermediaries, including planktivorous fish (anchovies and sardines), squid and krill, which route toxins to a wide variety of higher vertebrates and cause mortalities such as those observed in Monterey Bay in 1991 and 1998. We will test the hypothesis that benthic Emerita and pelagic anchovies serve as better sentinel species for inshore and offshore DA levels, respectively, than does the currently-used intertidal mussel, Mytilus californianus. The field study uses weekly or biweekly sampling in the nearshore region for DA-producing diatoms, sentinel benthic species (E. analoga and M. californianus), and benthic sediments, with intensified collections when toxigenic species are abundant. In the offshore region, we compare the pattern of DA in krill and commercially landed anchovies, sardines, and market squid, intensifying our offshore sampling during HAB events, with the pattern of DA in plankton populations offshore being measured by collaborators. We test the hypothesis that DA concentrations decline with increasing trophic level, which has important fishery consequences. NOAA/NOSS Marine Biotoxins Program will provide measurements of DA in some of the key organisms studied (sand crabs and krill), whereas the UCSC team will measure toxins in the others (fish, squid, mussels, sediments).

    These results will provide a better understanding of the pattern of toxin transmission through marine food webs, both benthic and pelagic. The research also will lead to recommendations for more effective biomonitoring for governmental agencies concerned with seafood safety and for environmental agencies concerned with protecting vulnerable marine vertebrates, including endangered species of seabirds and mammals that forage on the potentially DA-contaminated species studied here.

  • Wells, M. (UCSC) and R. Wessling (UCSC ). ECOHAB: A new chemosenser for domoic acid based on molecular imprinting. 09/01/99-8/31/01. NOAA/COP. Email: mwells@cats.ucsc.edu.

    We propose to develop a novel, highly sensitive analytical method for determining DA in tissue, plankton, and seawater. Our approach builds upon recent advances in the areas of molecular imprinting of resins and in the ability to fluorescently probe chemical environments. Briefly, we will develop a chemosensor for DA by cross-linking a polymer matrix around the analyte (DA), creating micro-cavities, or imprints for DA within the resin structure. The "template" DA is then washed from the resin leaving the imprints vacant. The imprints will contain binding sites specifically situated for DA. The resulting sterochemistry of the imprint will impart very high specificity for DA over other low molecular weight substrates, overcoming one of the two major analytical hurdles.

    Detection of DA within the imprints, the second major analytical hurdle, we be accomplished by incorporating fluorescently-active molecules that are influenced by the presence of DA. We will experiment with two different approaches; fluorescent binding sites for DA which fluoresce upon association with DA, or molecules embedded within the walls of imprints which fluoresce upon the expulsion of aqueous media from the cavity by DA.

    The combination of imprinting and fluorescence detection has been demonstrated in other fields to give very high selectivity and sensitivity. Based on equivalent sensitivity, it should be possible to detect the presence of DA in seawater containing as few as 1 cell ml-1. This optically-probed chemosensor will be ideally suited for simple, robust detection units that can be used on vessels, skiffs, or the beach. Ultimately, it should be possible to deploy molecular-imprinted chemosensors on mooring-based, satellite-reporting platforms in regions susceptible to toxigenic diatom blooms to provide an effective early warning coastal network system.

  ECOHAB1998: PROJECT SUMMARIES

  • Andersen, R.A. (Bigelow Laboratory for Ocean Sciences). "ECOHAB: Culturing and cryopreserving Pfiesteria and Pfiesteria-like organisms." 10/01/98-10/14/01, EPA Award R826793.

    Certain aspects of research on Pfiesteria and Pfiesteria-like algae may be enhanced by the availability of culture isolates. In particular, laboratory experimentation would benefit from the availability of cultures to investigate the autecology and toxicology of these dinoflagellates as well to investigate interactions of Pfiesteria and Pfiesteria-like with other organisms. Presumably, laboratory results will have implications for understanding blooms, predicting occurrences, and minimizing deleterious effects. The proposed research is to first establish and maintain cultures of Pfiesteria and Pfiesteria-like organisms. Second, basic culturing experiments are proposed to determine optimal growth conditions and to establish the range of tolerance for temperature and salinities. Pfiesteria and Pfiesteria-like organisms appear to be obligate phagotrophs, but another common dinoflagellate phagotroph, Oxyrrhis marina, has been found capable of growing on a defined medium. Thus, the third aspect of the proposed research is to develop a defined organic medium that will support the growth of Pfiesteria and Pfiesteria- like organisms. Finally, most microscopic organisms in culture gradually "evolve" over time, sometimes changing in their growth characteristics, toxicity, and ability to exhibit alternate life stages. Cryopreservation offers relief from these undesirable attributes of "evolving" culture strains. The fourth aspect of this research is to establish cryopreservation protocols and to maintain cryopreserved cultures for culture strains of Pfiesteria and Pfiesteria-like organisms.

    Lead PI: R.A. Andersen, Bigelow Laboratory for Ocean Sciences, P.O. Box 475, McKown Point Rd., West Boothbay Harbor, ME, 04575; Randersen@bigelow.org; 207-633-9600.

  • Anderson, D.M. (WHOI), R. Pierce, (Mote Marine Laboratory), R.M. Greene (EPA Gulf Ecology Division), M. Lewis (EPA Gulf Ecology Division), P. Chapman (EPA Gulf Ecology Division), M. Bricelj (Institute for Marine Biosciences, NRC Canada). "ECOHAB: Control of harmful algal blooms using clay." 11/23/98-11/22/01 first year of a 3 year study. EPA Award CR827090.

    This project will investigate the feasibility of a promising control strategy - the removal of cells from the water column using clay flocculation. The approach relies on the ability of certain clays to scavenge particles, including algal cells, from seawater, carrying them to bottom sediments where they are buried and decomposed. The project will begin with small-scale laboratory experiments in which algal cultures in test tubes are treated with clays and cell removal, cell viability, toxin release, and other parameters are measured. Laboratory experiments will also examine nutrient uptake and release by clays applied to seawater containing no algae. These and other laboratory experiments will be conducted on each of the targeted HAB species, (the Florida red tide dinoflagellate Gymnodinium breve, the New York brown tide chrysophyte Aureococcus anophagefferens, and fish-killing Pfiesteria-like dinoflagellates). Work will then shift to aquaria or mesocosm tanks where the clay treatments will be applied to natural communities of planktonic and benthic organisms. No studies have yet been conducted on the loadings needed to remove U.S. HAB organisms, on the suitability of readily available U.S. clays, or of the possible environmental impacts of flocculation and sedimentation of bloom organisms, especially those containing toxins. The near-term results of this study will be used to evaluate the engineering requirements, economic costs and environmental clearances needed for a pilot program for field application of this mitigation strategy. The eventual conclusion from the investigations proposed here will be of great value in evaluating the feasibility and potential environmental impacts of this promising bloom mitigation strategy. Knowledge will have been gained that can steer us towards related strategies that may someday help us minimize the impacts of some HABs.

    Lead PI: D. Anderson, Biology Department, WHOI, Woods Hole, MA, 02543; danderson@whoi.edu; 508-289-2351.

  • Bissett, W.P. (Florida Environmental Research Institute). "Hyperspectral modeling of harmful algal blooms on the West Florida Shelf." 08/01/98-07/31/01. ONR Award N00014-98-1-0844.

    Each red tide occurrence of Gymnodinium breve costs an estimated $20 million dollars in economic damage, plus untold damage to the marine ecological systems through massive fish, bird and mammal kills. The management of marine resources, e.g., closure of shellfish beds, in response to these outbreaks requires the ability to predict their occurrence. As the natural life cycle of G. breve occurs within an autotrophic community, predictions must incorporate the interactions of G. breve within a complete phytoplankton assemblage, not just the growth and mortality of G. breve. In addition, proposed mitigation of these blooms will require realistic numerical simulations that incorporate the dynamics of the entire marine ecosystem, in order to investigate possible deleterious feedbacks. It is hypothesized that the prediction of G. breve must include the competitive interactions amongst multiple phytoplankton populations for spectral light energy, as well as the competitive interactions for multiple nutrient resources. The project will develop a three-dimensional Ecological Simulation of the West Florida Shelf (Eco-Sim-WFS) that includes a hyperspectral model of coastal ocean optics. Eco-Sim-WFS will incorporate the competitive feedback mechanisms of phytoplankton spectral light absorption and scattering onto a realistic autotrophic ecosystem. This work builds on the currently funded NOAA/COP intensive study of harmful algal blooms on the West Florida Shelf (ECOHAB-Florida), and a NRL 6.1 Accelerated Research Initiative on hyperspectral coastal optics (Spectral Signature of Optical Properties in the Littoral Zones). The model will be an enhancement of an existing Ecological Simulation (EcoSim 1.0) developed for oligotrophic waters. The model’s 60 m spatial resolution and 10 nm spectral resolution will enable it to directly utilize the water-leaving radiance data stream from a new ONR/NRL satellite instrument, the Coastal Ocean Imaging Spectrometer, to be launched in May, 2000. Deliverables from this work include - 1) A numerical simulation of G. breve within a complete phytoplankton ecosystem, which can be used to predict G. Breve blooms under realistic physical forcing. It will also provide a platform to test the effects of potential G. breve mitigation schemes on the ecosystem of the West Florida Shelf; 2) A prognostic simulation of the dept-dependent inherent and apparent optical properties of the West Florida Shelf, which includes predicting upwelling radiance, L_u(l), for a given downwelling irradiance and solar zenith angle.

    Lead PI: W.P. Bissett, Florida Environmental Research Institute, The Florida Aquarium, 701 Channelside Drive, Tampa, FL 33602; pbissett@marine.usf.edu; 813-273-4161.

  • Garman, G. (VCU), Webb, S. (VCU), B. Browne (VCU), and S. McIninch (VCU). "Free-living, pathogenic amoebae in Chesapeake Bay tributaries." 07/01/98-12/31/99. EPA Award R82-7191.

    During the Summer and Fall of 1997, occurrences of moribund and dead fish in Maryland and Virginia coastal rivers were reported widely by the popular press. Because many of the afflicted fish exhibited lesions and related characteristics, these outbreaks were attributed initially to toxins produced by the dinoflagellate Pfiesteria piscicida or by related Pfiesteria-complex organisms (PCO’s). Although toxins from PCO’s have been confirmed as the likely cause of some recent fish kills (e.g., Pocomoke River), examinations of fish lesions and environmental samples collected during the same period in other coastal rivers of the Chesapeake Bay drainage (e.g., Rappahannock and James rivers in Virginia) have produced no evidence of PCO’s. This proposal presents preliminary evidence that free-living, pathogenic amoebae of the genera Acanthamoeba, and possibly Naegleria, may represent currently unrecognized agents of ulcerative disease in freshwater and estuarine fishes of the Chesapeake region, and may be comparable in effect to Harmful Algal Blooms (HABs) in coastal estuaries. We hypothesize that at least some of the reported outbreaks of dead and moribund fish with lesions in Virginia rivers during the Summer and Fall of 1997 may be attributable to free-living pathogenic amoebae and not to Pfiesteria or other HAB-related organisms. If true, pathogenic amoebae may represent a previously unrecognized agent of fish and human diseases in coastal rivers. The proposed study would expand significantly the scope of a present VCU project, funded by the Virginia Department of Environmental Quality (DEQ).

    Lead PI: G. Garman, Center for Environmental Studies, Virginia Commonwealth University, P.O. Box 843050, Richmond, VA, 23284; ggarman@saturn.vcu.edu; 804-828-7202.

  • Glibert, P.M. (HPL, U.MD), W.C. Boicourt (HPL, U.MD), R.M. Harrell (HPL, U.MD), H.R. Harvey (CBL, U.MD), R.R. Hood (HPL, U.MD), M.R. Roman (HPL, U.MD), L.P. Sanford (HPL, U.MD), D.K. Stoecker (HPL, U.MD), J.M. Burkholder (NCSU), S.C. Cary (U. DE), D.A. Hutchins (U. DE), and A. Lewitus (USC). "ECOHAB: Toxic dinoflagellates and nutrients. Toward a mechanistic understanding of outbreaks of Pfiesteria and related dinoflagellates. A regional, comparative study." 09/01/98-08/31/99 first year of 5 year study. NOAA Awards NA86OP0493, NA86OP0510, and NA86OP0495.

    This research project is a regional, comparative study of the physical, nutritional, and trophodynamic mechanisms that contribute to blooms of Pfiesteria. The study will test the hypothesis that certain mechanisms are interdependent, and together contribute to the development and persistence of these blooms. Through comparative field and mesocosm experiments, spanning a range of conditions under which Pfiesteria and other Pfiesteria-like species are found to occur or to bloom, the environmental conditions and factors that contribute to the populations' success will be described. Parameters to be measured include nutritional requirements, fish detection, turbulence impacts, predator role, and metabolite production that reduces grazing pressure or induces stress in fish; molecular techniques will also be developed for life-stage detection. Field sites in MD, NC, DE, and SC will be studied to identify common features that might explain environmental requirements of Pfiesteria-like organisms. The laboratory and field results will be incorporated into physiological and ecosystem models that can be used to predict the abundance of Pfiesteria in the mid-Atlantic region.

    Lead PI: P. Glibert, Horn Point Laboratory, University of MD, P.O. Box 775, Cambridge, MD, 21613; glibert@hpl.umces.edu; 410-221-8422.

  • Lin, S. and E.J. Carpenter (SUNY, Stony Brook). "ECOHAB: Factors affecting growth of Pfiesteria piscicida." 09/01/98-08/31/99, first year of 3.5 yr study. NOAA Award NA86OP0491.

    The project will focus on development of molecular techniques to measure in situ growth rates of Pfiesteria piscicida from Eastern Shore tributaries of the Chesapeake Bay. One technique will focus on a cell cycle protein method, (including identification of the cell cycle related genes, proteins, and development of antibodies), another surface antibodies, and a third the development and application of specific gene probes for identification of life stages and growth status in the water column and sediments. These results will then be used with environmental variables from the field in the construction of a box model that relates environmental conditions to field growth rates. It is anticipated that the research will not only provide new detection tools but also determine the mechanisms through which environmental factors control life cycle transformations, growth rate, and abundance of planktonic and benthic stages of P. piscicida.

    Lead PI: S. Lin, Marine Sciences Research Ctr., SUNY, Stony Brook, NY, 11794; Slin@ccmail.sunysb.edu; 516-632-8697.

  • Maske, H. (CHORS, SDSU), J. Mueller (CHORS, SDSU), C. Trees (CHORS, SDSU), R. Iglesias (CHORS, SDSU). "ECOHAB: Hyperspectral optical properties of a red tide bloom." 07/01/98-06/30/01, first year of a 3 year study. ONR Award N00014-98-1-0778.

    Red tide blooms derive their name from the red color observed on the sea surface. Red tides are in general composed of species that contain phycobiliproteins or by dinoflagellates. Phycobiliproteins are pigments with absorption spectra that filter out wavelength other than the red light thus allowing the residual reflected light appear on the sea surface. The objective of our research under this project is to characterize the bio-optical properties of dinoflagellate red tide blooms and explain what is the source of the red color in naturally occurring blooms, and why does the color not appear in cultures of these species? Our main hypothesis is that the red color originates from chlorophyll fluorescence and that the fluorescence can be observed from above the sea surface because of the high concentration of dinoflagellates near the surface. Our main tools for investigating the optical properties are in situ measurements of inherent and apparent optical properties at high vertical resolution near the surface with a free falling/rising profiler that includes a CTD, radiometers and photometer. Also we will take water samples at about 0.2 vertical resolution near the surface with water bottles arranged in a rigid vertical array. These samples will be used for pigment analysis and physiological measurements. The data will be compared with radiative transfer calculations trying to reach closure by adjustment of the quantum yield of fluorescence emission.

    Lead PI: H. Maske, Centre for Hydro-Optics and Remote Sensing, San Diego State University, 6505 Alvarado Rd. Suite 206 San Diego, CA 92120; hmaske@chors.sdsu.edu, hmaske@cicese.mx; (from USA): 011 52 617 45050 ext. 24260.

  • Oldach, D. (U. MD School of Medicine) and P. Rublee (NCSU at Greensboro). "ECOHAB dinoflagellate molecular ecology." 10/08/98-8/31/01, 3 years. EPA Assistance ID No. R 827084-01-0.

    Pfiesteria piscicida 18s rRNA gene sequence pools will be amplified from cultures and environmental samples, followed by heteroduplex mobility assay (HMA) for gauging sequence diversity within the amplified pool of cDNA. HTA (heteroduplex tracking assay) will then be undertaken to identify characterized and novel dinoflagellate gene sequences. PCR primers will be developed for assays of Pfiesteria piscicida, and assays will also be undertaken with universal dinoflagellate primes followed by HTA mapping. It is anticipated that quantitative PCR analyses will be follow, with initial work in the laboratory using kinetic thermal cycling assays and fluorogenic probes in the 5'exonuclease assay. Subsequent studies will include field assays in MD and NC with battery powered miniaturized analytical thermal cycling instruments (MATCI, Cepheid, Inc.). Detection of species in the field will be correlated with environmental variables as well as observed health effects in ‘exposed’ individuals in cohort studies from impacted waterways.

    Lead PI: D. Oldach, University of Maryland School of Medicine & Institute of Human Virology,

    Room 556, U of MD Medical Biotechnology Center, 725 W. Lombard Street, Baltimore, MD 21201; oldach@umbi.umd.edu; 410 706-4609.

  • Paul, J.H. (USF). "ECOHAB-Exploring lytic and temperate viruses of Gymnodinium breve as a mechanism of controlling red tide blooms." 09/01/98-08/31/99. NOAA Award NA86OP0494.

    Red tides caused by the toxin-producing dinoflagellate Gymnodinium breve are common along Florida’s Gulf Coast. The observation that some blooms undergo sudden dissipation suggests a catastrophic event has occurred with the bloom. Viruses are capable of producing such rapid mass mortality. The goal of our research is to investigate both lytic and temperate viruses as a potential mechanism of red tide termination. During December, we sampled water from a red tide bloom near Ft. Myers, FL. Water samples were either prefiltered through a 1.2 or a 0.2 um filter, and the viral fraction concentrated by ultrafiltration. Aliquots (1 to 1.5 ml) of the concentrated fraction were added to 25 ml G. breve cultures and the culture growth monitored by fluorescence for 10 days. Nearly every culture that received the concentrated fraction showed a dramatic decrease in fluorescence after 5 to 7 days, whereas controls continued to grow normally. The "lytic agent" was transferred to fresh cultures, and the period of time required to cause a decrease in fluorescence ("crash") decreased to 3 to 4 days. The lytic agent remained active after filtration through a 0.2 um filter. The lytic agent has been serially passaged at least five times and remains active/infective. We have observed virus-like particles by epifluorescence microscopy but these may be from bacteria present in the cultures. Transmission electron microcopy of thin sectioned samples has not yet detected viral particles in G. breve cells. We are now analyzing some biological and physical properties of the lytic agent.

    Lead PI: J.H. Paul, Department of Marine Sciences, University of South Florida, St. Petersburg, FL, 33701; jpaul@seas.marine.usf.edu; 813-553-1168.<

  • Reece, K.S. (VIMS), E.M. Burreson (VIMS), and N.A. Stokes (VIMS). "ECOHAB: DNA-based molecular diagnostics for Pfiesteria-complex organisms in Chesapeake Bay." 07/01/98-06/30/01, EPA Award R826791.

    The newly discovered heterotroph Pfiesteria piscicida and other closely related toxic dinoflagellates have been blamed for many of the harmful algal blooms resulting in fish kills in the US Atlantic Coast estuaries during the past seven years. Pfiesteria piscicida and other Pfiesteria-like dinoflagellates are commonly referred to as the Pfiesteria-complex organisms (PCOs). Pfiesteria piscicida has been found associated with fish kills in the Neuse and Pamlico estuaries of North Carolina and has been implicated in human health effects including neurocognitive impairment and skin lesions. Fish kills in Chesapeake Bay tributaries and health complaints from individuals having frequent exposure to waters were attributed to a Pfiesteria- like dinoflagellate and prompted closure of the Pocomoke River for 2 months during the summer of 1997. The discovery of several Pfiesteria-like species with complex life cycles has highlighted the necessity for accurate identification and characterization of these organisms. Currently the only method available to accurately identify Pfiesteria-like species is analysis of the thecal plate structure of cysts by scanning electron microscopy (SEM). This method cannot be used for identification of all life stages and is time-consuming and tedious precluding its use for large-scale monitoring programs. The objective of this project is to develop DNA-based diagnostics for use in screening cultures and environmental samples. Primers for use in the polymerase chain reaction (PCR) and DNA probes for use in in situ hybridizations that are group-specific to the Pfiesteria-complex organisms (PCOs) and specific to species within the complex will be designed and tested. Following species identification by SEM, DNA will be isolated and the internal transcribed spacer region and a portion of the large subunit gene of the ribosomal DNA complex of each Pfiesteria-like species will be PCR amplified and sequenced. Primers and probes will be designed based on analysis of sequence alignments and tested for specificity and sensitivity against each of the PCOs and other dinoflagellate species. Environmental sediment and water samples will be used to verify utility of the PCR and in situ hybridization assays.

    Lead PI: K.S. Reece, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA, 23062; kreece@vims.edu; 804-684-7407

  • Repeta, D.J. (WHOI). "ECOHAB: Dissolved organic nitrogen and brown tide blooms in Long Island coastal waters." 01/01/98 - 03/31/01, NSF Award OCE 9730015.

    Blooms of the `Brown Tide` unicellular algae (Aureococcus anophagefferens) have occurred sporadically since 1985 in coastal waters of Eastern Long Island and have devastated the local commercial scallop fishery. Analysis of a 10 year time series data set from the Peconic Estuary indicates that bloom intensity is inversely correlated with the discharge of high nitrate groundwater and associated with higher salinities. Laboratory and field data suggest that salinity is unlikely to represent a direct physiological control on Brown Tide blooms. However budget calculations indicate that nitrogen supply from groundwater is 1 to 2 orders of magnitude higher than other external sources for this ecosystem. Data collected in 1995 demonstrated that Brown Tide blooms utilize dissolved organic nitrogen (DON) for growth as evidenced from the large decrease in DON parallel with cell increase. We hypothesize that bloom initiation is regulated by the relative supply of inorganic and organic nitrogen, determined to a large extent by groundwater flow variability. An appropriate first step to test this hypothesis is the demonstration that there are compounds in the DON that can be efficiently utilized by A. anophagefferens giving it a competitive advantage over other endemic species. We will identify the source of DON that is available to A. anophagefferens via field and laboratory studies. The laboratory work will involve the identification of the DON components from the Peconic Estuary that can support growth of the alga and characterization of the DON uptake systems and utilization mechanisms that make this alga competitive at utilizing nitrogen. Immunological probes to major proteins involved in the utilization of DON will be prepared. In the field we will characterize the DON fraction utilized by A. anophagefferens during a bloom as well as follow the nitrogen nutrition of this algae using immunological probes. Weekly nutrient bioassays and analysis of various dissolved and particulate nitrogen pools will complement the more detailed field sampling.

    Lead PI: D.J. Repeta, Woods Hole Ocean Institution, Woods Hole, MA 02543; drepeta@whoi.edu; 508-548-1400

  • Roesler, C. (UConn, Bigelow), "ECOHAB: Ecophysiology of subpopulations of Alexandrium tamarense."09/01/98 -08/31/01, 01/01/99-12/31/01. NASA Awards NAG5-7654 and NAG5- 7872.

    Toxic algal blooms, often called red or brown tides for the color they impart to the water, pose a serious threat to ecosystem and public health, as well as to the economy, as they are responsible for fish kills, shellfish poisoning, and human illness. While the reports of toxic outbreaks is escalating worldwide, early detection remains elusive, impeding progress towards understanding the forces responsible for bloom initiation, development and advection. The long term goals of this project are to identify those factors which lead to the initiation and development of toxic blooms and the variability in bloom toxicity using controlled laboratory experiments in concert with in situ detection of toxic blooms in the natural environment. This project specifically focuses on Alexandrium tamarense, a toxic dinoflagellate responsible for recurrent episodes of paralytic shellfish poisoning (PSP) from northeastern Canada to the coast of New Jersey. We will investigate the ecophysiology of the northern and southern subpopulations, which demonstrate significant differences in toxicity and bloom initiation, posing the questions of why Long Island Sound is bereft of extreme A. tamarense blooms and why the isolate in Long Island appears to have reduced toxicity. To answer these questions we will (1) measure quantitative toxin profiles during all growth stages of the two isolates under a range of environmental conditions and (2) investigate natural outbreaks of A. tamarense in New England waters using an optical models to assess the spatial and temporal distributions in concert with environmental parameters. The basis of the optical models is the extraction of the algal inherent optical properties (absorption, scattering, backscattering coefficients) from ocean color in the presence of a mixed algal community. These properties provide sufficient information for detection, species identification and bloom monitoring. It is anticipated that the approach will supplement our understanding of toxic bloom dynamics as development can be tracked backwards in time via imagery and the actual initiating environmental conditions can be determined. This approach can also be used to forecast the progress of existing blooms providing early detection for "down stream" waters.

    Lead PI: C. Roesler, Bigelow Laboratory for Ocean Sciences, McKown Point Road, P.O. Box 475, West Boothbay Harbor, ME 04575; CRoesler@bigelow.org; 207-633-9654.

  • Trainer, V.L. (NOAA) and M. Bricelj (NRC, Canada). "ECOHAB: Mechanisms and control of toxin accumulation in shellfish." 09/01/98-08/31/99, first year of 3. Internal transfer of funds to NW Fisheries Science Center, NOAA.

    The project will characterize mechanisms regulating toxin sensitivity and accumulation in bivalve molluscs, specifically butter and softshell clams Mya arenaria and the razor clam Siliqua patula. Mya, isolated from toxic and non-toxic regions, will be exposed to toxic and non-toxic Alexandrium in the laboratory. Behavioral and feeding responses will be determined, as well as nerve sensitivity to all paralytic shellfish toxins present; grazing inhibition will be examined as a ‘sensitivity’ measure to toxins and toxin uptake and elimination will be estimated. Using a similar approach, razor clams will be exposed to toxic Pseudo-nitzschia sp.; domoic acid uptake and elimination kinetics will be measured. A unique feature of the study will be the isolation of inducible binding proteins, necessary structural elements for the development of quick, sensitive, receptor-based analytical toxin detection methods. Overall, project findings will provide insights on the potential for acclimation and/or genetic adaptation of bivalve populations to toxins, and possibly provide sentinels of ecosystem health at the level of the primary consumers.

    Lead PI: V.L. Trainer, Northwest Fisheries Science Center, F/NWC2, NOAA, 2725 Montlake Blvd. E., Seattle, WA, 98122-2097; Vera.L.Trainer@noaa.gov; 206-860-3387.

  • Zohar, Y., G. Vasta, R. Belas, and A. Place (COMB, U. MD). "ECOHAB: Molecular approaches to Pfiesteria-complex dinoflagellates in Chesapeake Bay." 09/01/98-08/31/00, first 2 years of 5. NOAA Award NA86OP0492.

    This research project is designed to develop a Dinoflagellate Culture Core Facility to maintain Pfiesteria-complex dinoflagellate (PCD) stock cultures, optimize culture conditions, and provide toxic and non-toxic PCD cells, supernatants, and extracts to the project PIs and the HAB community. Research projects in the program will also develop molecular probes to detect PCDs, determine the role of dinoflagellate associated bacteria in PCD physiology and toxigenesis, and determine the molecular nature and physiology of the elicitors of toxigenicity. The project will deliver a set of molecular probes and biosensors specific to PCDs and provide basic scientific data on the genetics and physiology of PCDs, dinoflagellate associated bacteria, and toxin elicitors.

    Lead PI: Y. Zohar, Center of Marine Biotechnology, University of Maryland, 701 East Pratt St., Baltimore, MD, 21202; zohar@umbi.umd.edu; 410-234-8800.

  ECOHAB1997: PROJECT SUMMARIES

  • Anderson, D.M. (WHOI), D.T. Townsend (U. ME), J.H. Churchill (WHOI), J.J. Cullen (Dalhousie Univ.), G.J. Doucette (NOAA), W.R. Geyer (WHOI), M.D. Keller (Bigelow), T.C. Loder (UNH), D.R. Lynch (Dartmouth), J.L. Martin (DFO Canada), D.J. McGuillicuddy (WHOI), J.E. O’Reilly (NOAA), N.R. Pettigrew (U. ME), R.P. Signell (USGS), A.C. Thomas (U. ME), and J.T. Turner (U. MA, Dartmouth). "ECOHAB: Gulf of Maine - The ecology and oceanography of toxic Alexandrium blooms in the Gulf of Maine." 8/1/97-3/31/98, NOAA Sea Grant Award NA46RG0470 (first 18 months of 4.5 year award); 6/1/98-5/31/99, NSF Award OCE-9808173.

    The overall objective of the ECOHAB-Gulf of Maine project is to understand and model the dynamics of the toxic dinoflagellate Alexandrium in the Gulf of Maine by investigating the physical, biological, chemical, and behavioral mechanisms underlying population abundance and distribution in several key habitats or regimes and by characterizing the transport pathways that link them. Field studies include both moored and shipboard hydrographic observations, nutrient conditions, and population distributions (including benthic resting cysts) within the Casco Bay region, the Eastern Maine Coastal Current, and the southern Bay of Fundy. The Casco Bay region including the Kennebec River plume is an area where initiation of blooms is likely to occur. Inoculation of Alexandrium cells into the plume waters that form the Western Maine Coastal Current explain the southerly transport of vegetative populations along the coasts of southern Maine, New Hampshire, and Massachusetts, but the mechanisms of bloom initiation are not understood. Larger scale offshore surveys extending along the Maine coast from Casco Bay to the Bay of Fundy will determine the coupling of the physics with the Alexandrium biology of the northern Gulf of Maine-Bay of Fundy region and linkages, if any, to southern Gulf of Maine populations initiated near Casco Bay. Field studies will also determine offshore cyst distributions along the Maine coast as well as distributions within several important inshore embayments (e.g., Casco Bay). Field and laboratory excystment experiments will determine the requirements leading to bloom initiation and coupled with the hydrographic data will identify the physical features governing delivery of the inoculum to areas favorable for growth. Laboratory mesocosm studies will also be conducted to determine diel population responses to light and nutrients, critical to resolving nutrition and substrate supply for advected populations. All data will be merged into a physical-biological coupled model for the western Gulf of Maine toward generating a forecasting tool for better understanding of the seasonal population dynamics and the mechanisms for delivery of toxic cells to shellfish within the region.

    Lead PI: D.M. Anderson, Biology Department, WHOI, Woods Hole, MA, 02543; danderson@whoi.edu; 508-289-2351

  • Doucette,G.J. (Medical University of. SC). "ECOHAB: Algicidal bacteria and the regulation of Gymnodinium breve blooms in the Gulf of Mexico" 9/1/97-8/31/00, Second year of 3 year award. NSF Award OCE-9726260.

    The focus of this project is to investigate the possible role of algicidal bacteria in promoting the decline and dissipation of Gymnodinium breve red tides along the Florida Gulf coast. Blooms of G. breve are especially problematic along the Florida coast, where costs associated with these annual events are estimated at about $20 million per episode. As a result, there is a growing interest in exploring strategies for the management, mitigation, and/or control of G. breve blooms, which will rely, in part, on acquiring knowledge of natural mechanisms involved in regulating population growth of this dinoflagellate. Several reports of algicidal bacteria occurring in high concentrations at the onset of bloom termination suggest that such microbes may directly or indirectly lead to the decline of these events, yet the issue of bacterial involvement in terminating harmful algal blooms has not been addressed in U.S. coastal waters. Our approach is to test the hypothesis that algicidal bacteria specific for G. breve are present in microbial assemblages indigenous to the Florida Gulf coast. This effort will involve attempts to isolate bacterial strains from both bloom and non-bloom waters, and screen for their ability to inhibit or preclude the growth of G. breve laboratory cultures. Bacterial isolates showing algicidal activity against G. breve will then be classified using both classical and molecular techniques, the latter yielding 16S rRNA sequence data needed to design strain-specific oligonucleotide probes. Molecular probes will provide a means to track the population dynamics of these bacteria in controlled (e.g., mesocosms) as well as natural systems, thereby helping to assess their involvement in promoting bloom decline. Concurrent laboratory studies will define the algicidal target specificity among phytoplankton taxa closely- and more distantly-related to G. breve, evaluate the conditions promoting algicidal activity of the bacteria, and begin to characterize the algicidal agent and its mechanism of action. The knowledge gained from this study represents an essential first step in evaluating the potential use of algicidal bacteria as a means of controlling or regulating blooms of G. breve.

    Lead PI: G.J. Doucette, Marine Biomedical & Environmental Sciences, Medical University of South Carolina, Charleston, SC 29412; greg.doucette@noaa.gov; 843-762-8528.

  • Durbin, E.G. (GSO-URI), R.G. Campbell (GSO-URI), and A.D. Cembella (NRC-Canada). "ECOHAB: Zooplankton grazing of toxic Alexandrium spp. as a mechanism in the control of bloom formation and toxin transfer." 09/15/97 — 08/31/00, NSF Award OCE 9726261.

    Toxic Alexandrium spp. dinoflagellates cause paralytic shellfish poison (PSP) outbreaks in the Gulf of Maine each year, posing a public health threat and resulting in economic loss. The population dynamics of Alexandrium spp. are at present poorly understood; in order to understand and model these dynamics basic information on the mechanisms that allow blooms to form must be obtained. This study will determine the role of grazing by the dominant zooplankton (copepods) in the population dynamics of toxic Alexandrium spp. The study will conduct laboratory experiments to elucidate the mechanisms responsible for specific grazing behavior of dominant zooplankton species. Coordinated in situ field studies focusing on the spring period of bloom initiation will also be conducted, at sites where high PSP levels occur early and regularly.

    Lead PI: E.G. Durbin, Graduate School Oceanography, University of Rhode Island, Narragansett, RI, 02882; edurbin@gso.uri.edu; 401-874-6580.

  • Kvitek, R.G. (CSU-Monterey Bay). "ECOHAB: Influence of harmful algal blooms on the distribution and ecology of high level marine predators." 02/01/98-01/31/00. NSF Award OCE-9726263.

    The purpose of this project has been to test the general hypothesis that the foraging behavior and distribution of high level marine predators (sea otters and shorebirds) under natural conditions are mediated by benthic prey toxicity due to harmful algal blooms (HAB's). The research has had two taxonomic foci (sea otters and shorebirds) in two geographic regions (California and Alaska). In southeast Alaska, the recent and rapid expansion of the sea otter population into the more HAB-prone inside passage provided an ideal opportunity to test the influence of HAB toxins on the distribution and feeding ecology of this important marine predator. The preliminary results from the first Alaska field season show that sea otters generally shun or leave areas where their primary prey (butter clams) are toxic. However, when otters do forage at toxic prey sites, they either shift their diet to less profitable prey (e.g., smaller clams found in deeper water) that are not toxic and ignore the larger more abundant and accessible butter clams, or they capture, open and discard the toxic butter clams. During our second Alaskan field season we will seek to confirm these findings and to determine whether otters discriminate between more and less toxic prey at the level of the individual. That is, are otters that do capture butter clams at toxic sites testing each clam that they capture, and discarding all or part of it depending on the toxin level. The second component of the project focuses on the response of common northern California shore birds (Dowitchers, Godwits, Oystercatchers, Sanderlings, Whimbrels) to the predictable seasonal increase in the paralytic shellfish poisoning toxin (PSPT) content of two major intertidal invertebrate prey (mole crabs and mussels). To test the general hypothesis that the foraging behavior of these avian predators is mediated by PSPT, we are monitoring and correlating seasonal changes in invertebrate prey toxicity with changes in the shorebird behavior and diet over a two year period at several widely separated sites along the California coast. Each paired study site includes rocky habitat where Black Oystercatchers forage on sea mussels, and exposed sandy beach where the other shorebirds forage on mole crabs. During 1998, prey toxicity resulting from PSPT HAB activity was much lower than normal and no change in bird foraging behavior was observed. This condition was likely due to the 1997/98 El Niño event that resulted in very low summer primary productivity. While this situation did not provide an opportunity to compare shore bird foraging under toxic and non-toxic conditions, it did yield an excellent set of low toxicity baseline observations for comparison with the upcoming non-El Niño HAB season in 1999.

    Lead PI: R.G. Kvitek, California State University - Monterey Bay, 100 Campus Center, Seaside, CA, 93955-8001; rikk_kvitek@monterey.edu; 408-582-3529.

  • Paul, V.J. (UGuam), "ECOHAB: Chemical ecology of cyanobacterial blooms on the tropical reefs of Guam." 12/15/97-12/14/00, EPA Award R82-6220.

    Harmful algal blooms (HABs) of many types have increased in abundance and severity in the United States and worldwide in recent years. Of particular concern in coral reef habitats are the frequent and persistent seaweed blooms. These blooms can have many negative impacts including; overgrowing corals, negatively affecting seagrass communities, and washing up on beaches in areas where tourism is economically essential. An additional concern for cyanobacterial blooms are the toxins they produce and their impacts on other reef organisms and humans. The production of deterrent and toxic secondary metabolites by benthic cyanobacteria probably facilitates bloom formation on coral reefs because most generalist grazers avoid this potential food source. Almost nothing is known about the temporal and spatial patterns of bloom formation in reef habitats or about environmental factors affecting bloom formation and persistence. Additionally, secondary metabolite types and concentrations can vary considerably among different collections of cyanobacteria, but environmental factors influencing this chemical variation are not understood. This project will investigate the temporal and spatial patterns of cyanobacterial blooms on Guam, eight reef sites will be monitored biweekly and cyanobacterial populations will be measured. Secondary metabolites associated with these blooms will be isolated by chromatographic methods and characterized by spectroscopic methods including 2D NMR techniques. Effects of these compounds on feeding by herbivores such as fishes and invertebrates will be examined in laboratory and field bioassays. Compounds released by the cyanobacteria into seawater will be characterized and examined for their effects on competitors and other microorganisms in laboratory and field bioassays. Effects of grazing, light, and nutrients (nitrogen, phosphorus, iron) on secondary metabolite production will be examined in a combination of field and laboratory experiments. The results of this research will yield information on the dynamics of cyanobacterial blooms in tropical reef environments; the production and toxicity of their secondary metabolites and effects on herbivores, competitors, and microorganisms.

    Lead PI: V.J. Paul, University of Guam Marine Laboratory, Mangilao, GU 96923, USA; vpaul@uog9.uog.edu; 671-735-2186.

  • Stabile, J.E. (NYU Med. Center), and I.I. Wirgin (NYU Med. Center). "ECOHAB: Population genetics of brown tide blooms." 10/01/97-09/30/99, NSF Award OCE 9726262.

    During the past decade blooms of the brown tide microalgae, Aureococcus anophagefferens, have occurred sporadically in Peconic Bay and Great South Bays of Long Island, N.Y. Blooms of the brown tide vary annually in the timing of their onset, duration and intensity. We hypothesize that temporal and spatial variability in bloom characteristics is due to underlying genetic variation among populations of A. anophagefferens. This hypothesis was tested by DNA sequence analysis of the two internal transcribed spacer regions (ITS1 and ITS2), the 5.8S subunit and the first hypervariable region of the large subunit of rDNA. Brown tide PCR primers were developed and used to amplify A. anophagefferens DNA directly from pre-bloom and bloom water samples and from cultured isolates. PCR products were cloned into the pCR2.1 >plasmid vector and 20 recombinants per water sample or cultured isolate were sequenced. Sequence data were obtained from 1995 summer bloom water samples from West Neck Bay and Flanders Bays in the Peconic Bay system and cultured isolates CCMP 1784 (L.I., N.Y.), CCMP 1785 (L.I., N.Y.), CCMP 1790 (L.I., N.Y.) and CCMP 1794 (Barnegatt Bay, N.J.). Extremely high levels of ITS1 sequence variability were observed among and within water samples and cultured isolates of the brown tide microorganism. A total of 25 polymorphic nucleotide sites were observed among 191 bp of ITS1 DNA sequence. Cloned PCR fragments were assigned ITS1 composite types on the basis of unique combinations of polymorphic nucleotides. A total of 37 different ITS1 types were observed among the 120 cloned fragments. Cultured isolates of A. anophagefferens were observed to have between 6-16 different ITS1 types. Water samples were observed to have ITS1 types not found in the current cultured isolates. CCMP 1794 from Barnegatt Bay had unique ITS1 types suggesting the presence of geographic differentiation between the New Jersey and Long Island sites. High levels of sequence variability were also observed in ITS2, 5.8S and the large subunit of rDNA. However, variability in these regions was less than that of ITS1. We are currently working on alternative methods to more clearly evaluate the spatial and temporal variability of the brown tide microorganism.

    Lead PI: J. Stabile, New York University Medical Center, Nelson Laboratory for Environmental Medicine, Long Meadow Road, Tuxedo, NY 10987; jstabile@iona.edu; 914-633-2253.

  • Steidinger, K.A. (FMRI), J.J. Walsh (USF), C.R. Tomas (USF), J.H. Landsberg (USF), G.A. Vargo (USF), R.H. Pierce (Mote), G.J. Kirkpatrick (Mote), J.D. Buck (Mote), R.H. Weisburg (USF), J. Fournie (EPA), F. VanDolah (NOAA), P.A. Tester (NOAA), T. Whitledge (U. AK), R. Wanninkhof (NOAA), G. Janowitz (NCSU), D. Kamykowski (NCSU), T. Hopkins (NCSU), T. Wolcott (NCSU), D. Wolcott (NCSU), G. Fahnenstiel (NOAA), D.F. Millie (USDA), O.M. Schofield (Rutgers), K.A. Fanning (USF), S.E. Lohrenz (U. S. Miss.), and D.G. Redalje (U. S. Miss.). "ECOHAB: Florida." 09/01/97-03/31/99, first 1.5 years of 4.5 year project, NOAA Grant NA960P0084. EPA Awards R826792 to G. Vargo, 06/01/98-05/31/01 and R827085 to K. Steidinger, 10/15/98-10/14/01.

    ECOHAB: Florida is a multidisciplinary project to determine biological and physical factors that lead to recurrent blooms of NSP-producing Gymnodinium breve on Florida’s western shelf. Using moored instrumentation for collection of in situ physical data, routine hydrographic surveys, intensive field and laboratory studies on the ecology, physiology, toxicity, and behavior of the organism, and robust mathematical modeling, the research will determine those factors like responsible for transport and accumulation of Gymnodinium in the region, as well as trophodynamics and toxin transfer in the coastal food web. Biological measurements include life cycle measurements with flow cytometry, diel vertical migration studies in controlled and field environments, optical characteristics of coastal waters and populations, assemblage and species-specific productivity, nutrient uptake, toxicity and toxin distributions, and grazing rates of planktonic herbivores. The study is now being used as a base for many other regional and targeted studies on Gymnodinium (e.g., roles of bacteria and viruses in bloom dynamics) and circulation in the Gulf, including complementary remote sensing projects with multi- and hyperspectral sensors.

    Lead PI: K.A. Steidinger, Florida Department of Environmental Protection, Florida Marine Research Institute, 100 8^th Ave., SE, St. Petersburg, FL, 33701-5095; steidinger_k@epic7.dep.state. Fl.us; 813-896-8626.

  • Wells, M.L. (UCSC), D.L. Garrison (NSF), and R. Tjeerdema (UCSC). "ECOHAB: Ecophysiology studies of Pseudo-nitzschia species." 01/01/98-12/31/01, EPA Award R82-6306.

    Domoic acid-producing diatoms belonging to the genus Pseudo-nitzschia cause serious toxic algal blooms on the west coast of the United States that have resulted in deaths of seabirds in California water and human poisonings in Oregon and Washington. Presently, little is known about the growth requirements of the individual species and the environmental conditions that promote toxicity. This proposal addresses the need for detailed information on how Pseudo- nitzschia species respond to various levels of macro- and micronutrients. These data are fundamental to developing the capability of predicting where and when these toxic blooms are likely to develop. We are specifically testing the hypotheses that 1) domoic acid production changes as a function of silicate (Si)/nitrogen(N) limitation of the organisms, varying for different species in conjunction with their N and SI metabolic requirements; 2) cellular production and release rates of domoic acid vary systematically with the iron (Fe) nutrient status of organisms, with rates being high under Fe deficient conditions and low in iron replete conditions; and 3) Pseudo-nitzschia species have a high Fe requirement, so that toxic blooms in coastal waters are probably restricted to regions and times when Fe concentrations in surface waters are high. In addition, we will examine the novel idea that domoic acid has a roles in trace metal uptake. This information could provide a predictive capability for assessing the conditions that allow toxic species to bloom and out compete those species which lack the ability to produce domoic acid. To determine nutrient requirements of Pseudo-nitzschia species, nutrient uptake kinetics, and to assess the effect of nutrients on domoic acid production, we will use a variety of continuous and batch culture approaches, utilizing locally-isolated cultures. The proposed research will make s significant contribution to management strategies for future domoic acid blooms in coastal waters of the Pacific and elsewhere. Being able to predict where and when blooms of toxigenic Pseudo-nitzschia species are likely to develop, would lead to considerably more efficient monitoring strategies and an enhanced ability to plan appropriate responses. If our hypotheses are correct, serious toxic bloom problems may be restricted to specific regions of the coast system where micro- or macronutrient concentrations are sufficiently high to support the development of high-density blooms. These same conditions may also affect the levels of toxin produced by bloom species.

    Lead PI: M.L. Wells, Institute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, CA 95064; mwells@cats.ucsc.edu; 408-459-3877.

  • Wikfors, G.H. (NEFSC, NOAA), C. Martin (NEFSC, NOAA), S.E. Shumway (Bigelow), D.G. Dam (UConn), G. McManus (UConn), and R.M. Smolowitz (UPenn). "ECOHAB: Trophic effects of two dinoflagellates." 01/15/98-07/14/99, NOAA FOP and EPA Award R826219-01-0.

    Most HAB dinoflagellates grow relatively slowly; therefore, accumulation of their biomass (a bloom) is likely attributable in large part to reduced grazing. Among the grazing organisms that normally limit phytoplankton biomass accumulation are pelagic consumers, such as protozoans, copepods, and larvae of benthic invertebrates, as well as benthic filter-feeders, chiefly the bivalve mollusks. Knowledge of direct, harmful effects of an algal species upon consumers would explain the mechanism by which a bloom of that alga can occur, and provide predictive capability of the types of ecosystems, dominated by benthic or pelagic consumers, that are most susceptible to blooms of that alga. We propose to investigate systematically, under controlled laboratory conditions, effects of two cultured HAB dinoflagellates, Prorocentrum minimum and Gyrodinium aureolum, upon a suite of representative consumer organisms, including three protozoans, two copepods, and a larval and post-set bivalve. Effects of these dinoflagellates, both alone and in various combinations with " good food" algae, upon feeding, behavior, population dynamics, and histological condition of individual organisms will be documented. This work will benefit from a team approach utilizing, in all experiments, identical algal cultures produced in the unique Milford Microalgal Mass Culture Facility. Results will provide information critical to interpretation of field studies of HAB dynamics and food-web effects.

    Lead PI: G.H. Wikfors, North East Fishery Science Center, NMFS, NOAA, Milford, CT., 06460; Gary.Wikfors@NOAA.gov; 203-783-4217.