ECOHAB 2002: 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). 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). 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). 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). 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 LC₅₀for 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). 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). 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 anophagefferensoccur, 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). 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). 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.) 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). 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). 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.) 9/1/02-8/31/05. NOAA/COP. Email: Lewitusa@mrd.dnr.state.sc.us

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 Kryptoperidiniumclade, 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). 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. shumwayaein 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). 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). 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). 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). 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.) 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.