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NOAA ECOHAB Project Abstracts

For past and other agency sponsored ECOHAB projects, visit the WHOI website.

Fiscal Year 2011 ECOHAB Projects

CIGUAHAB: Ciguatera Investigations in the Greater Caribbean Region: Ecophysiology, Population Connectivity, Forecasting, and Toxigenesis

Institutions: Florida Gulf Coast University (lead), Woods Hole Oceanographic Institution, University of Texas at Austin, Dauphin Island Sea Lab, Universidad Veracruzana (Veracruz, México), U. S. Federal Drug Administration, University of the Virgin Islands

Investigators: Michael L. Parsons (lead), Donald M. Anderson, Deana L. Erdner, Ronald Kiene, Yuri B. Okolodkov, Mindy L. Richlen, Alison Robertson, Tyler Smith

Ciguatera fish poisoning (CFP) is the most common form of phycotoxin-borne seafood illness across the globe, including the Greater Caribbean Region (i.e., the Caribbean, Yucatan, Gulf of Mexico, Florida Keys, and Bahamas), hereafter Greater Caribbean Region (GCR). At a global level, Fleming et al. (1998) estimate that there are 50,000 - 500,000 poisonings per year. The average annual economic impact of ciguatera in the United States has been estimated to be $21 million, far surpassing public health impacts from other illnesses associated with toxic algae. CFP is caused by the consumption of seafood (primarily reef fish) contaminated with ciguatoxins. Gambiertoxins, the precursors of ciguatoxins produced by the (sub)tropical benthic dinoflagellate genus, Gambierdiscus, enter coral reef food webs when herbivores and detritivores consume Gambierdiscus during grazing on substrate macroalgae. These precursors are transferred to higher trophic levels by bioaccumulation, bioconversion and biomagnification until they reach predatory finfish species that are targeted in many fisheries. People are exposed to the toxins when they consume the fish, thereby experiencing CFP.

Objectives:

  1. Characterize Gambierdiscus population diversity and connectivity on regional and local scales;
  2. Determine effects of environmental factors on the growth and toxicity of representative strains of Gambierdiscus;
  3. Investigate Gambierdiscus population dynamics and the environmental conditions that contribute to blooms in several representative locations for the study region;
  4. Investigate the fate of ciguatera precursors, toxins and metabolites in the reef food web;
  5. Model the population dynamics and toxin production of Gambierdiscus under different environmental forcings, including those associated with natural and human-induced perturbations such as pollution, reef destruction, and climate change;
  6. Communicate project results and discuss applications to resource management with stakeholders in the GCR, including medical personnel, natural resources officials, fishermen, and others, and develop a website to serve as an information clearinghouse for information on CFP.

Approach:

The GCR is a region with a large and expanding problem of CFP. Consistent with the underlying philosophy of the ECOHAB program, this project will increase understanding of the ecology and oceanography of Gambierdiscus populations, leading to a predictive capability for CFP in the region. The GCR study area covers a range of environments and habitats, some pristine and others with clear anthropogenic impacts. A suite of new genetic tools and ecological methods are now available to examine Gambierdiscus populations and their environments in detail, and this information can be used to parameterize a population dynamics model of Gambierdiscus in the GCR that will have predictive value.

Expected results:

CIGUAHAB will result in a comprehensive understanding of the diversity, physiology and ecology of Gambierdiscus in the GCR, will increase our knowledge and educate managers and the public about the risks of CFP, and will develop a model of bloom dynamics and toxin production which will provide information of clear management value and lead towards a predictive capability and an opportunity to estimate effects of global warming and other climactic or environmental perturbations on this important public health issue.

Brevetoxin Metabolism and Physiology - A Freshwater Model of Morbidity in Endangered Sea Turtles

Institutions: Florida Atlantic University (lead), Georgia Aquarium and Harbor Branch Oceanographic Institute, Mote Marine Laboratory, Florida Fish and Wildlife Conservation Commission/Fish and Wildlife Research Institute

Investigators: Sarah L. Milton (lead), Gregory Bossart, Deborah Fauquier, Catherine J. Walsh, Leanne Flewelling

Karenia brevis, the “Florida red tide” organism that frequently blooms in some areas of the Gulf of Mexcio, produces a suite of brevetoxins that cause human respiratory illness along beaches, accumulate in shellfish, which, when consumed, cause Neurotoxic Shellfish Poisoning, and cause mass mortality of fish and a number of protected and endangered species. Among the species impacted are a variety of threatened and endangered marine turtles.  For example, in the severe Florida red tides of 2005 and 2006 at least 179 loggerhead turtles died, but other species may be impacted as well, including leatherback, green, hawksbill, and Kemp’s ridley. Exposure to red tide outbreaks is thus a major threat to sea turtles off the coast of Florida, with reported effects on the pulmonary, neuromuscular, and immune systems, though neither acute nor sublethal effects of brevetoxins on sea turtle health have been well-characterized. During red tide outbreaks, however, it cannot be determined how large or longstanding toxin exposure is prior to rescue and thus exposure levels cannot be correlated with morbidity and mortality outcomes.

Understanding the risk of brevetoxin exposure to sea turtle health is critical as such impacts may affect survival of these endangered population. Due to the nature of study on endangered sea turtles, however, these questions cannot be addressed directly, as they require experimental investigation with controlled toxin doses. This makes it difficult to establish appropriate treatment methods, and none can be devised in advance.

Objectives:

The purpose of these studies is to use a non-endangered turtle model to delineate the pathways of toxin metabolism, determine the impacts of toxins on individual organisms, and develop new methods of treating brevetoxicosis in turtles.

Approach:

In this project non-endangered turtle models will be used to determine toxin effects on critical organ systems.   As exposure in sea turtles may occur through ingestion and/or inhalation, with potentially different distributions, effects, and rates of metabolism, brevetoxins will be administered by both mechanisms to anesthetized turtles. After exposure, tissue toxin levels will be measured over time in order to investigate the uptake and excretion.  Tissues will also be sampled over time for histology to determine the impacts on specific organ systems.  Additional studies will characterize the impact of brevetoxin on immune function.  Turtle neuronal cultures will be utilized to determine the concentrations and mechanisms of neurotoxicity in turtles.  Finally, the information will be used to develop methods of treating and rehabilitating endangered turtles suffering from brevitoxicosis.

Expected results:

Developing an animal model leading to an understanding of how brevetoxins cause illness and death in turtles will provide a sound scientific basis for treatment at the numerous rescue facilities that rehabilitate sea turtles in the Gulf Coast states.

A Regional Comparison of Upwelling and Coastal Land Use Patterns on the Development of HAB Hotspots Along the California Coast

Institutions: University of California at Santa Cruz (lead), University of Southern California, Monterey Bay Aquarium Research Institute, California State University/Moss Landing Marine Laboratories, University of California at Los Angeles, Southern California Coastal Water Research Project, NOAA National Ocean Service/National Centers for Coastal Ocean Science

Investigators: Raphael Kudela (lead), Clarissa Anderson, Dave Caron, Burt Jones, Gaurav Sukhatme, Chris Scholin, John Ryan, Jim Birch, Kanna Rajan, Heather Kerkering, G. Jason Smith, Yi Chao, Meredith Howard, Greg Doucette

Blooms of harmful and toxic algae have increased in frequency and severity along the 1000 mile long California coast during the past few decades, causing diverse impacts on the economy through commercial fisheries and tourism, as well as via direct impacts on marine birds and mammals.  Many of these occur at hot spots scattered along the west coast.  Pseudo-nitzschia produces a potent neurotoxin, domoic acid, which can accumulate in shellfish, other invertebrates, and, sometime fish, leading to illness and death in a variety of birds and marine mammals and necessitating shellfish harvesting closures to protect human health.  Regional ECOHAB or MERHAB projects are examining hot spots in Washington and Oregon; these regions are thought to be primarily influenced by natural processes.  While MERHAB projects in southern and central California have, among other activities, provided information from shore-based sampling about HAB hot spots in California, there have been no oceanographic studies on the west coast aimed at predictive understanding of coastal processes where human activities as well as upwelling may be stimulating HABs.  This project will compare HAB initiation and development at two California hot spots where the relative importance of upwelling and human activities (land use and runoff) differ.  Although the project will focus on Pseudo-nitzschia, the occurrence of other HABs, especially Alexandrium, which produces toxins that can cause Paralytic Shell Fish Poisoning, will also be investigated if they occur during the study.

Objective:
  1. To develop a better understanding of HAB initiation and bloom dynamics in California in response to physical and environmental forcing factors by simultaneously comparing two “hot spots”, Monterey Bay and San Pedro, California, leading to predictive models; and
  2. To establish a cutting-edge HAB alert detection system and demonstrate the utility of an integrated observing system for HABs as part of the Regional Coastal Ocean Observing System.

Approach:

The project will rely on a cutting-edge, high tech, tiered sampling methodology in collaboration with the Central and Northern California Ocean Observing System (CeNCOOS) and the Southern California Coastal Ocean Observing System (SCCOOS): existing shore-based monitoring efforts, deployment of 2 Slocum gliders with chlorophyll sensors and 2 Environmental Sample Processors (ESPs) with sensors for HAB cell/toxin detection (1 in each region), satellite imagery, statistical models, and data assimilation into ROMS model to identify when and where bloom events begin.  When a possible bloom is identified by these methods, or if a bloom is identified by another means (e.g., sudden increase in reported marine bird or mammal strandings; increase in toxicity or cell density from the shore-based network), intensive adaptive sampling will be conducted.  The sampling will include ship-based field experiments to assess species assemblage, growth conditions, response to fundamental parameters such as temperature, light, nutrients, and will be adjusted as the bloom evolves.  The enhanced understanding of conditions before and during blooms will be used to improve models for predicting future blooms.

Expected results:

By identifying blooms while they are still offshore before they impact near shore areas, this study will immediately improve the management response to HABs.  It will also lead to development of a HAB alert detection system and bloom forecasts that will provide early warning that will further improve shellfish, wildlife, and public health management.  If human activities are found to cause HABs it will lead to the development of new strategies for HAB prevention.

Species and Strain Differences in the Toxicity of Caribbean Gambierdiscus Species: Implications for Ciguatera Fish Poisoning in the Caribbean

Institutions: NOAA National Ocean Service/National Centers for Coastal Ocean Science (lead), North Carolina State University at Raleigh

Investigators: R. Wayne Litaker, Patricia Tester, Damian Shea

Ciguatera fish poisoning (CFP) is caused by the bioaccumulation of toxins, produced by tropical dinoflagellates in the genus Gambierdiscus, into herbivorous and carnivorous fish. Globally, CFP causes more human illness than all other harmful algal bloom species combined. Despite this fact, relatively little is known about the variety of ciguatoxin congeners produced by different Gambierdiscus species and the overall differences in toxicity among and between species. Possessing this knowledge is crucial for understanding CFP and how it may differ between the Atlantic and Pacific basins. For example, ciguatoxins (CTXs) isolated from Atlantic and Pacific fishes are structurally different. It has been proposed that these differences are due to the production of different toxin precursors by the resident species. Supporting evidence for this hypothesis comes from a recent study that confirmed distributional differences in Gambierdiscus species found in each basin. These differences may also account for why CFP symptoms tend to vary between the two regions. The project hypothesis is that relatively few Gambierdiscus species are highly toxic and that they disproportionately contribute to the flux of toxins that enter the food chain. This appears to be the case with Pacific Gambierdiscus species where G. polynesiensis is generally far more toxic on a per cell basis than other co-occurring species. If this hypothesis is correct for the Caribbean, then it may be possible to assess CFP potential by monitoring these key species. Interestingly, total toxicity per cell also appears to be inversely proportional to the latitude from which a Gambierdiscus clone was isolated. If true, this could also influence the severity and frequency of CFP occurrences in an area.

Objectives:

  1. Establish the relative per cell toxicity of the five known Caribbean Gambierdiscus species and one Gambierdiscus ribotype using genetically characterized clones currently in culture;
  2. Provide detailed analysis of the CTX congeners produced by the most toxic species;
  3. Characterize and compare the toxins produced by Caribbean isolates of G. caribaeus, a globally distributed species, to toxins isolated from other Pacific species;
  4. Assess the extent to which CTX toxicity varies within a species and whether per cell toxicity varies systematically with latitude or region.

Approach:

This project will draw on the large Gambierdiscus culture collection established by members of the Tester/Litaker laboratory over the past 8 years as well as many years experience isolating and culturing Gambierdiscus cells. The approach involves the following steps: 1) Screen Caribbean species using receptor binding bioassay to determine if Gambierdiscus ribotype 2 is the most toxic; 2) Screen Gambierdiscus caribaeus cultures from the Caribbean and Pacific to characterize the toxin suite being produced; 3) Grow large scale cultures of Gambierdiscus ribotype 2 and other toxic Caribbean Gambierdiscus species for detailed toxin analysis; and 4) Simultaneously characterize within species differences in toxicity and test the hypothesis that per cell toxicity increases with latitudinal gradients in the Caribbean.

Expected results:

The results of these studies will provide valuable information about ciguatoxins entering the food chain, and for the first time, will systematically address whether the amounts and types of ciguatoxins being produced in the Caribbean are different from those in the Pacific, and whether isolates from lower latitudes are more toxic. These data will also help determine the likelihood of success for a cell-based monitoring system for predicting CFP risk.

Fiscal Year 2010 ECOHAB Projects

The Ecophysiology and Toxicity of Heterosigma akashiwo in Puget Sound: A Living Laboratory Ecosystem Approach

Institutions: NOAA Northwest Fisheries Science Center, San Francisco State University, The University of Western Ontario, University of Maine, Norwegian National Veterinary Institute, Rensel Associates Aquatic Sciences, America Gold Seafood

Investigators: Vera L. Trainer, William P. Cochlan, Charles G. Trick, Mark L. Wells, Chris O. Miles, Jack Rensel, Kevin Bright

Over one half of the world’s fish production for human consumption currently comes from aquaculture while wild fisheries’ yields are either stable or declining. Recurring threats from the raphidophyte, Heterosigma akashiwo Hada (Sournia) have caused extensive damage ($2-6 million per episode) to wild and net-penned fish of Puget Sound, Washington, and are believed to be increasing in scope and magnitude in this region, and elsewhere in the world over the past two decades. The mechanism of H. akashiwo toxicity is not well understood. The toxic activity of H. akashiwo has been attributed to the production of reactive oxygen species, brevetoxin-like compound(s), excessive mucus, or hemolytic activity; however these mechanisms are not confirmed consistently in all fish-killing events or cultured strains. The difficulty of conducting research with active, toxin-producing field populations of H. akashiwo have resulted in conflicting findings from those obtained in lab culture studies, thereby limiting the ability of managers and fish farmers to respond to these episodic blooms. The overall goal of this project is to identify the primary toxic element and the specific environmental factors that stimulate fishkilling H. akashiwo blooms, and thereby provide managers with the fundamental tools needed to help reduce the frequency and toxic magnitude of these harmful algal events. Studies to date have provided incomplete and conflicting observations on the mode of toxicity and the environmental stimulation of toxification. A three-pronged approach is proposed to study the environmental controls of H. akashiwo growth and toxin production; laboratory culture experiments, field observations, and bottle and mesocosm manipulation experiments.

Objectives:

  1. The element(s) of toxic activity (inorganic, organic, or synergistic) associated with blooms of H. akashiwo and its various cellular morphologies.
  2. Determine the environmental parameters that stimulate the growth success and expression of cell toxicity in the H. akashiwo populations of Puget Sound.

Approach:

Because previous studies have used H.akashiwo cultures with little or no toxic activity, this approach uses a "living laboratory" to study H. akashiwo bloom ecology and toxicity using natural assemblages. A mobile lab at field sites where H. akashiwo cells are regularly found will enable full characterization of the toxic element(s) responsible for fish mortality, and the environmental factors influencing toxicity. Findings from annual field studies in late June and two rapid response deployments during major bloom events will be confirmed using laboratory studies with fresh (< 6 mo. old) isolates.

Expected results:

  1. Determination of the key elements of toxicity of H. akashiwo.
  2. Characterization of the environmental variables that influence either the induction or depression of elements of toxic activity in H. akashiwo.
  3. Strategy for realistic mitigation of H. akashiwo activities in Puget Sound, Washington.

Modeling favorable habitat areas for Alexandrium catenella in Puget Sound and evaluating the effects of climate change

Institutions: NOAA Northwest Fisheries Science Center, Seattle WA, Woods Hole Oceanographic Institution, University of Washington

Investigators: J.E. Stein, S.K. Moore, D.M. Anderson, E.P. Salathé, Jr., N.J. Mantua, N.S. Banas, C.L. Greengrove, B.D. Bill, V.L. Trainer

The dinoflagellate Alexandrium catenella produces a suite of potent neurotoxins that accumulate in shellfish and cause severe illness or death if contaminated shellfish are consumed by humans. Alexandrium catenella form dormant cysts that overwinter on the seafloor and provide the inoculum for toxic blooms the following summer when conditions become favorable again for growth of the motile cell. A 2005 survey of A. catenella cyst distribution in Puget Sound, Washington State, identified “seedbeds” with high cyst abundances that correspond to areas where shellfish frequently attain high levels of toxin. However, even at these sites, interannual variability in the magnitude of toxic events is high. In order to provide advanced warning of A. catenella blooms, managers need to know how much “seed” is available to initiate blooms, where this seed is located, and when/where this seed could germinate and grow. Evaluating how favorable habitat areas for cyst germination and vegetative growth will be altered by climate change would allow for risk assessments of A. catenella blooms through until the late 21st century.

Objectives:

  1. Determine interannual variations in A. catenellacyst distribution in Puget Sound.
  2. Quantify rates of cyst germination and vegetative growth for a range of temperature, salinity, and light conditions.
  3. Determine the presence/absence of an endogenous clock that regulates cyst germination.
  4. Model favorable habitat areas for cyst germination and vegetative growth.
  5. Evaluate climate change impacts on favorable habitat areas.
  6. Establish a time series with sufficient depth to provide seasonal forecasts of toxic blooms.

Approach:

To achieve these goals, they will conduct annual cyst surveys at ~80 stations throughout Puget Sound and in the Strait of Juan de Fuca. Laboratory experiments will be performed to determine the optimal ranges of environmental parameters that maximize rates A. catenella cyst germination and vegetative growth. This information will be combined with output from an existing numerical ROMS-based model of Puget Sound circulation to model favorable habitat areas for A. catenella at ~200 m resolution.  Downscaled regional climate change projections for Washington State will be used to construct new forecast scenarios the ROMS model and evaluate changes to favorable habitat areas for A. catenella through the late 21st century. Finally, once a time series with sufficient depth is established, the ability to provide seasonal bloom forecasts will be determined by relating cyst abundances with the magnitude of toxic events the following summer/fall.

Expected results:

The expected outcomes of this project include the production of seamless maps indicating favorable habitat areas for A. catenella in Puget Sound now and in a future warmer climate. These maps will be used by shellfish farmers and managers to guide harvesting and monitoring practices in space and time


Establishing the sources of phosphorus promoting toxic cyanobacteria blooms in the US Great Lakes using gene expression assays

Institutions: Stony Brook University, New York Sea Grant Extension, Cornell Univ. Cooperative Extension

Investigators: Christopher J. Gobler, James W. Ammerman, Charles O'Neil

Toxic cyanobacteria blooms represent a serious threat to human health, natural resources, and economies dependent on the US Great Lakes. Cyanobacteria blooms are common within eutrophic systems with elevated phosphorus levels and recent studies have found that P enrichment can specifically promote toxic strains of the harmful cyanobacteria, Microcystis.  Management plans focused on improving the quality of freshwater systems typically strive to reduce concentrations of total phosphorus (TP), despite the variable distribution and lability of different components of TP, including particulate, dissolved, organic, and inorganic. While dissolved inorganic P is the most bioavailable, some information suggests that dissolved organic P (DOP) may be important for promoting blooms of Microcystis. However, the extent to which Microcystis blooms utilize DOP during blooms has not been established as the methods for measuring alkaline phosphatase activity are not species-specific and/or include the activity of heterotrophic bacteria. Recently, the genomes of two clones of Microcystis have been sequenced. Our exploration of these genomes and our own clones of Microcystis have revealed that Microcystis possesses an alkaline phosphatase gene, although it is not the gene present in model bacteria (phoA) but rather it possesses phoX. Furthermore, these Microcystis clones also contain a high affinity phosphate transporter, pstS, sphX, and other P transporters. Prior research has shown that alkaline phosphatase genes and phosphate transporters are tightly regulated in response to changes in ambient P conditions.

Objectives:

Therefore, the objectives of this proposal are to develop quantitative gene expression assays for Microcystis alkaline phosphatase and phosphate transporters and to quantify how the expression of these genes is regulated by changes in exogenous P concentration and sources. Within regions of the Great Lakes prone to Microcystis blooms, we will establish gene expression patterns in parallel with the dynamics of nutrients, phophatase enzyme activity, microcystin, and densities of toxic and non-toxic Microcystis cells. Finally, we will examine expression patterns of P transport and metabolism genes during fieldbased, nutrient amendment experiments within regions of the Great Lakes prone to Microcystis blooms using differing sources of organic and inorganic P. These combined approaches will provide an indication of the role of organic and inorganic P in the occurrence Microcystis in the Great Lakes. Our final objective is an outreach campaign based on our findings. Co-PI O’Neill  ill work with Gobler and Ammerman to develop a factsheet and to host workshops focused on the role of P in the dynamics of Microcystis blooms. End users of the fact sheet and participants in the workshops will include state, local, and federal water treatment facilities, health departments, resource management agencies, stakeholders who constitute sources of phosphorus to the Great Lakes, educators promoting public outreach and education and the news media.


Fiscal Year 2009 ECOHAB Projects

Title: Deposition and resuspension of Alexandrium fundyense resting cysts in the Gulf of Maine: Phase II

Institutions: Woods Hole Oceanographic Institution, University of Massachusetts, United States Geological Survey

Investigators:  D. Anderson, D McGillicuddy, C. Pilskaln, R. Signell, B.Butman, A. Solow

The Gulf of Maine (GOM) supports productive shellfisheries frequently impacted by paralytic shellfish poisoning (PSP) - a serious threat to human health caused by the toxic dinoflagellate Alexandrium fundyense. PSP is the most widespread of the poisoning syndromes associated with harmful algal blooms (HABs). Blooms of A. fundyense in the GOM are highly seasonal, consistent with the view that life history transformations between cysts and vegetative cells are major regulatory factors. The ecology and oceanography of A. fundyense have been relatively well studied, but encystment and excystment dynamics remain poorly understood. This project is the second phase of a continuing study focusing on several aspects of that dynamic – the processes controlling the delivery, deposition, resuspension, and accumulation of resting cysts. In the parent project, researchers mapped the distribution of A. fundyense cysts in GOM bottom sediments and the benthic nepheloid layer (BNL) and obtained trap data on the sedimentation and resuspension fluxes of cysts through time.

Objectives:

  1. Determine the sinking characteristics and depositional behavior of resuspended cysts in bottom sediments;
  2. Incorporate the USGS Community Sediment Transport Model into the existing physical-biological model for Alexandrium dynamics in the GOM;
  3. Use this model formulation to explore the relationship between existing maps of cyst distribution and sediment type, and the thickness and cyst content of the BNL at different locations in the GOM;
  4. Characterize cyst content and residence time in the BNL at different locations in the GOM with varying BNL thicknesses, taking into account both resuspension fluxes and lateral advection;
  5. Conduct numerical experiments to explore the processes involved in the sedimentation of newly formed A. fundyense cysts, their deposition, reworking, and eventual redeposition in major cyst seedbeds in the GOM; and
  6. Optimize cyst mapping strategies to minimize cost without unacceptable loss of accuracy in model forecasts.

Approach:

Researchers will use observations from the parent project as well as new laboratory experiments and numerical model simulations to characterize cyst dynamics in surface waters, the BNL, and bottom sediments of the GOM.

Expected results:

The expected results of this project will support NOAA’s planned operational forecasting system for PSP in the GOM, and fit perfectly with NOAA and ECOHAB priorities to provide “Quantitative understanding of HABs…… in relation to the surrounding environment ….. leading to development of operational ecological forecasting capabilities in areas with severe, recurrent blooms along the US coast”. Continued expansion and refinement of the coupled numerical models will greatly enhance the capability for HAB forecasting in the GOM.


Title: Mechanism of harmful algal bloom initiation in the western Gulf of Mexico

Institutions: Texas A&M University, NOAA, Woods Hole Oceanographic Institution

Investigators: L.Campbell, R. Hetland, R. Stumpf; R. Olson; H. Sosik

The toxic dinoflagellate Karenia brevis is the primary harmful algal bloom (HAB) species in the Gulf of Mexico. One curious feature of K. brevis blooms is that although they occur regularly in the eastern Gulf, they occur only sporadically in the western Gulf along the Texas coast. This difference is unexpected since temperature, salinity, and nutrient conditions are similar in the two regions.

Objectives:

The central hypothesis of this project is that the primary mechanism of bloom initiation in the western Gulf of Mexico is convergence and consequent downwelling at the coast, which physically concentrates Karenia cells because they swim upwards toward light. This mechanism differs from the blooms in the eastern Gulf off Florida, where blooms result from upwelling favorable winds and concentration at nearshore frontal boundaries. Specific objectives are to (1) correlate wind and bloom events using an existing hydrodynamic model to test the hypothesis that K. brevis bloom events are linked to seasonal downwelling along the Texas coast and that upwelling events may play an important role in dispersing the bloom; (2) test downwelling index predictions with field data on bloom development, including sampling targeted by the model predictions; and (3) develop a “Downwelling Index” for improved predictive capability based on local wind conditions.

Approach:

A multi-investigator interdisciplinary program is proposed to develop better tools for prediction and to apply a novel imaging technology for detection and quantification of K. brevis. A numerical simulation of surface currents in the Gulf of Mexico suggested that increases in algal concentrations due to downwelling circulation may be comparable to (or, exceed) population increases due to growth alone. To investigate the relationship between K. brevis blooms and wind events, field samples will be collected by an extensive volunteer network combined with a targeted sampling program guided by model results and satellite data. Cell abundances will be analyzed in near-real time with the Imaging FlowCytobot (IFCB) and automated classification. Finally, results will enable creation of a ‘downwelling index’ based on local wind conditions that will provide a new tool to predict the likelihood of K. brevis bloom formation. This index will take into account the net accumulation of plankton near the coast due to downwelling circulation. Measured environmental conditions and observed bloom events, as well as simulated bloom events using the hydrodynamic model, will be used to develop and refine the downwelling index.

Expected results:

Important outcomes of this project include new fundamental knowledge on the mechanism of bloom formation in the western Gulf and demonstration of the IFCB as a powerful technique for identification and quantification of HABs in near real time. Once validated, the downwelling Index will be made available to managers via the NOAA HAB Forecast System for prediction/ early warning of HAB events. A better understanding of how K. brevis blooms form will lead to improved prediction of harmful algal blooms throughout the Gulf.


Linking biogeochemistry to harmful algal bloom nutritional physiology with gene expression analyses: a case study with Aureococcus anophagefferens

Institutions: Woods Hole Oceanographic Institution (lead); SUNY Stony Brook (subcontractor)

Investigators: S. Dyhrman, C. Gobler

Project summary:

Harmful algal blooms (HABs) represent a significant threat to fisheries, public health, and economies around the world. Despite many years of study, fundamental questions remain regarding how nutrients drive HABs, such as the brown tides caused by Aureococcus anophagefferens. What species of dissolved inorganic nitrogen and phosphorus, or dissolved organic nitrogen and phosphorus are preferentially transported and metabolized by cells during blooms? How does nutrient transport and metabolism change as a function of ambient conditions as blooms initiate, are sustained, and decline? This project capitalizes on the recent completion of the first HAB genome sequence (A. anophagefferens) and our preliminary gene expression work with this species to develop and apply assays of gene expression to track the nutritional physiology of A. anophagefferens in its natural environment. By concurrently examining nutrient dynamics (e.g. supply, composition etc.), bloom dynamics, and gene expression we will create a clearer understanding of how nutrients influence HABs.

Objectives:

  1. Optimize qRT-PCR methods for the quantification of the expression of genes involved in nutrient transport and metabolism.
  2. In laboratory experiments, validate how the expression of the target genes are regulated by nutritional physiology and changes in exogenous nutrients.
  3. Track gene expression patterns in parallel with nutrient dynamics through the course of a bloom.
  4. In field incubations, examine gene expression patterns during nutrient addition bioassays.

Approach:

In this targeted research study we propose the validation and application of quantitative gene expression assays to examine how nutrients influence bloom initiation, sustenance, and decline. We will link nutrient dynamics to the expression of genes involved in nutrient transport and metabolism in culture controls and through the course of a brown tide.

Expected results:

Parameterization of nutrient influences on HABs provided by the development and application of the approach described herein would 1) provide information that will enhance HAB forecasting efforts (e.g. better modeling of how eutrophication and nutrient inputs influence bloom dynamics), 2) provide decision makers with the information needed to control and mitigate blooms (e.g. assays of the effects of nutrient loading), and 3) help facilitate bloom prevention through an advanced understanding of how nutrients promote bloom formation, sustenance, and decline in different systems. Although this study uses brown tide as a model, we underscore that the approach developed and validated herein has never been conducted for any algal species and may be used as a blue-print for application to other HABs.


PNWTOX–The Columbia River plume and HABs in the Pacific Northwest: bioreactor, barrier, or conduit?

Institutions: University of Washington; Department of Fisheries and Oceans, Pacific Region, Institute of Ocean Sciences, Sidney, B.C., Canada; University of California, Santa Cruz.

Investigators: B. Hickey, E. Lessard, P. MacCready, N. Banas, M. Foreman, R. Thomson,  D.Masson; R. Kudela

Funding Partner: National Science Foundation

Objective:

The overall program objective is to improve predictability of Harmful Algal Bloom (HAB) events on Pacific Northwest (PNW) coastal beaches by advancing our understanding of HAB development/dissipation and transport and mixing processes using existing data in parallel with state of the art physical and bio-physical models that include, for the first time, both the Columbia River (CR) plume and potential HAB source regions off both Oregon and Washington.

Approach:

This project makes use of results and data from two recently completed, temporally overlapping 5-year, multi-institutional, interdisciplinary studies–ECOHAB PNW (The Ecology and Oceanography of Harmful Algal Blooms in the PNW) and RISE (River Influences on Shelf Ecosystems), to improve predictability of arrival of HABs, in particular, toxigenic Pseudo-nitzschia (PN), on PNW beaches. The overriding conclusion from both studies is that lack of understanding of the effect of the CR plume on cross-shelf and alongshelf transport and mixing is the greatest impediment to understanding how phytoplankton, in particular, HABs, arrive on coastal beaches. In this project, we will build on the wealth of complementary information and enhanced knowledge generated in these two programs to study the transport and mitigation of HABs to the Washington coast from both northern and southern sources and extend our analyses to species other than PN. Hypotheses include: 1) The CR plume is a bioreactor for growth but not for toxin production, 2) During downwelling winds, the CR plume inhibits shoreward transport of toxic blooms, 3) During upwelling winds the CR plume enhances cross-shelf transport of toxic blooms below the surface layer, and 4) The CR plume enhances northward transport of toxic blooms along the coast.

Studies will include idealized process studies addressing these hypotheses, hindcasts of selected HAB events, and development of a forecasting ability. The new Northwest Association of Networked Ocean Observing Systems (NANOOS) will be used for model verification and also model improvement via data assimilation.

Expected Benefits:

A large group of Government and Tribal bodies with interests in coastal shellfish resources will benefit from both the improved HABs understanding as well as a forecasting ability. New information on Alexandrium may allow managers to shorten annual PSP beach closures. Models in ECOHAB PNW did not include the CR plume; those in RISE did not include two known source regions for toxic PN, the Juan de Fuca eddy region (north) and Heceta bank (south). The proposed forecast/hindcast models will include both source regions and the plume as well as the other freshwater sources that impact nutrient and freshwater supply in the region. The imbedded biological model will use a new approach based on measured biological rates, providing a ten fold improvement in skill over most existing models.


Title: Causes and consequences of cell death in the toxic dinoflagellate Alexandrium tamarense

Institutions: University of Texas Marine Science Institute

Investigators: D. Erdner

Harmful Algal Blooms (HABs) in estuarine and coastal waters can endanger both public and ecosystem health, and their incidence and extent are increasing worldwide. The toxic dinoflagellate Alexandrium tamarense is responsible for outbreaks of paralytic shellfish poisoning, one of the most widespread HAB syndromes. While numerous laboratory and field studies have greatly increased our understanding of the biological and physical processes that lead to the initiation of blooms and their subsequent growth and transport, very little is known about the causes of bloom decline and termination. Preliminary results suggest that Alexandrium may initiate programmed cell death in response to nutrient stress, leading to the hypothesis that an active cell death pathway in Alexandrium may contribute to the decline of blooms in situ.

Objectives:

The overall goal of this project is to evaluate the relationship between nutrient stress, programmed cell death (PCD), and encystment in Alexandrium by: (1) documenting the PCD process in Alexandrium, including the genetic, biochemical, and morphological changes that occur; (2) identifying the triggers of PCD in Alexandrium; (3) investigating the link between PCD and the encystment process; and (4) assessing the presence and magnitude of PCD in a natural Alexandrium bloom.

Approach:

A suite of genetic and biochemical assays will be used to determine the existence of the PCD process in Alexandrium under conditions that result in nutrient stress or encystment. These include measures of caspase activity, DNA fragmentation, maintenance of membrane integrity, inversion of phosphatidylserine in the cell membrane, and metacaspase gene expression. These analyses will also be used with natural bloom populations, to assess the role of PCD in bloom decline in situ.

Expected Results:

The end results of the work will be: a description of the PCD process in a toxic dinoflagellate; an understanding of the environmental triggers of PCD; elucidation of the link between PCD and encystment pathways in Alexandrium; and assessment of the role of PCD in bloom decline in situ. The results of this project will provide valuable information on the links between nutrient conditions and bloom termination, contributing directly to all three of EPA’s desired outcomes: 1) assessment of the role of PCD in bloom decline will contribute to better modeling of harmful blooms, thereby providing information that will enhance HAB forecasting efforts; 2) data on the links between nutrient conditions and bloom decline (or persistence) will help to provide decision makers with the information needed to control and mitigate blooms; and 3) knowledge of cell death processes in toxic dinoflagellate will help facilitate bloom prevention through an advanced understanding of the conditions and processes that promote their formation, maintenance, and decline.


Title: Bloom dynamics of Alexandrium: the roles of resource competition and allelopathy

Institutions: University of Maine

Investigators: L. Karp-Boss, D. Townsend

Toxic species of the dinoflagellate genus Alexandrium are responsible for outbreaks of Paralytic Shellfish Poisoning (PSP), a recurrent and serious problem in the Gulf of Maine (GOM). Hence, understanding bloom dynamics of Alexandrium spp. is a major research focus in the GOM and other coastal areas. Previous ECOHAB-funded studies have documented that the highest Alexandrium cell concentrations are located in offshore waters, well away from most coastal shellfish beds, and are delivered to inshore waters by physical mechanisms. An intriguing question is: What restricts Alexandrium from blooming in inshore waters? One hypothesis suggested by Townsend et al. (2005), is that Alexandrium bloom dynamics may be controlled not only by physical and chemical factors but also by biological interactions with other phytoplankton taxa – in particular, diatoms.

Objectives:

This research will test the hypothesis that bloom dynamics of Alexandrium are influenced by competitive interactions with diatoms, and that the interactions are reciprocal. That is, while field observations and preliminary lab studies indicate that high densities or growth rates of diatoms impede the growth of Alexandrium in early spring and in near-shore waters, either by virtue of their rapid growth rate and exploitation of essential resources or via alleopathic interactions, Alexandrium blooms that have been established after the decline of the diatom bloom can prevent a second diatom bloom via allelopathy.

Approach:

This project will conduct detailed laboratory studies that will 1) examine allelopathic interactions between the toxic dinoflagellate A. fundyense and diatoms; 2) obtain ecophysiological parameters on nutrient-dependent growth kinetics of A.fundyense and diatoms common to the GOM, and apply them to a resource-based competition model to predict outcome of competition between A. fundyense and diatoms; and, 3) conduct competition experiments between A. fundyense and diatoms over a range of nitrate and silicate concentrations and compare the experimental results to model predictions. Laboratory efforts will be supplemented with field data for the evaluation of distributions of Alexandrium with respect to distributions of other phytoplankton taxonomic groups, in particular diatoms, nutrients and hydrographic condition.

Expected results:

Results from this study will provide new information on the interactions of HAB species with other members of the phytoplankton community, which could lead to new insights into potentially novel mechanisms by which HAB blooms may be controlled. On a more basic level, this study will provide physiological data on uptake kinetics of A. fundyense (which are currently lacking) that is necessary for the development and improvement of forecast models for Alexandrium blooms. The study will also provide information on inter- and intra- variations in physiological parameters between Alexandrium strains, new reference material, and will support the training of graduate and undergraduate students.


Title: Effects of Chronic Domoic Acid Exposure on Gene Expression in the Vertebrate CNS

Institutions: NOAA Northwest Fisheries Science Center, University of Washington

Investigators: K. Lefebvre, M. Myers, F. Farin, T. Bammler, R. Beyer

The potential impacts of chronic algal toxin exposure have long been a concern. One HAB toxin, domoic acid (DA), is a potent neurotoxin that interacts with the vertebrate central nervous system (CNS). Although the clinical signs of acute DA toxicity have been well defined, virtually nothing is known about the impacts of chronic, low-level toxin exposure, primarily due to the difficulties associated with long-term exposure studies. It is known that vertebrates such as fish, seabirds, marine mammals, and humans are repeatedly exposed to DA at levels below those that cause outward signs of toxicity, yet it remains unknown how these chronic sub-acute exposures may impact these organisms. This project plans to quantify gene expression patterns in whole brain and characterize histopathological aberrations in major organs as endpoints to examine the effects of chronic exposure in zebrafish. The overall goal of this project is to develop a general model for the characterization of gene expression effects in the vertebrate CNS and morphological damage in major organs associated with long-term, low-level toxin exposure.

Objectives:

The objectives of this project are to 1) quantify gene expression changes in the vertebrate CNS and characterize differentially expressed genes based on function to identify potential pathways of chronic disease associated with long-term, low-level algal toxin exposure, 2) quantify circulating blood toxin levels associated with changes in gene expression, and 3) perform histologic examinations of all major organ systems to characterize pathological impacts of chronic toxicity.

Approach:

The toxicogenetic approach will be to use microchip gene array technology to quantify differential gene expression in whole brain during a one-year DA exposure study using a vertebrate model system (zebrafish, Danio rerio). Through pilot studies, the investigators have quantified appropriate sub-acute doses, developed effective repetitive dosing procedures, and developed a statistically rigorous experimental design. RNA isolation methods, microchip array procedures, qRT-PCR confirmation procedures, and bioinformatics processes for grouping and identifying gene clusters of interest have also been perfected. In addition to gene expression analyses, this research will employ standard histology procedures to visualize potential pathological aberrations caused by chronic DA exposure.

Expected results:

This research is expected to yield several results that will directly aid assessments of HAB impacts on marine biota. First, this research will provide the only available data on the impacts of chronic, low-level algal toxin exposure using a realistic long-term exposure time scale. It is also likely that new pathways of DA toxicity will be identified since a single dose exposure pilot study has already revealed gene expression patterns unique to sub-acute exposure. The gene lists generated will be widely disseminated and publicly available for researchers to use as a starting point for species-specific studies on chronic HAB toxin exposure effects. Finally, the study will quantify circulating blood toxin levels that are associated with the observed gene expression effects. These blood toxin levels can be used for characterizing the potential risk to other vertebrates exposed to DA in the field.

Fiscal Year 2006 ECOHAB Projects

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

Investigators: L. Connell, V.M. Bricelj, P. Rawson

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.


Engineering Upgrades and Field Trials of the Autonomous Microbial Genosensor

Institution: University of South Florida, http://www.marine.usf.edu/microbiology/genosensor.shtml

Investigators: J.H. Paul, D.P. Fries, M. Smith.

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".


Identifying Regulatory Mechanisms for Heterosigma akashiwo Bloom Formation: Predation Interactions with Algal Behavior and Resource Use

Institution: Western Washington University

Investigators: S. L. Strom and S. Menden-Deuer

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.

Intraspecific Variation in a Toxin-producing Dinoflagellate

Institution: Texas A&M University

Investigators: L. Campbell, J.R., Gold

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:

  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; and
  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.


Relation Between Grazer Toxin Dynamics and Resistance to Toxic Dinoflagellates

Institution: University of Connecticut

Investigator: Hans G. Dam

Abstract:

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.

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.

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.

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.


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

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,

Abstract:

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.


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

Investigators: C. A. Heil, D Bronk, L.K. Dixon, G. Hitchcock, G. Kirkpatrick, M. Mulholland, J. O'Neil, J.J. Walsh, R. Weisberg

Abstract:

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.

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.

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.


Understanding Shellfish Resistance Strategies as a Means to Predict and Manage PSP Toxicity

Institutions: University of Washington, NOAA Northwest Fisheries Science Center, National Research Council Canada

Investigators: T. Scheuer, W.A.Catterall, V. Trainer, V.M. Bricelj

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.


Investigating Chronic Toxicity and Bioaccumulation of Microcystins in Freshwater Fish Using Toxicogenomics and Histopathology

Institution: The University of Tennessee

Investigators: T.B. Henry, G.S. Sayler, S.W. Wilhelm, R. J. Strange

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.

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.

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.