ECOHAB 2003: Project Summaries

Edwards, K.A. (U.Washington). 9/1/03-8/31/06. NASA Award. Email: edwards@apl.washington.edu

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

Ferry, J.L. (U. South Carolina) and P. M. Moeller (NOAA). 9/1/03-8/31/06. EPA Award: RD83-1042. Email: ferry@mail.chem.sc.edu

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

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

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

Franks, P.J.S. (SIO) and Farooq Azam (SIO). 10/1/03-9/30/06. NOAA Award NA17RJ1231. Email: pfranks@ucsd.edu

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

Hutchins, D.A, S.C. Cary, and K.J. Coyne (U. Del.), and M. Doblin (ODU). 9/1/03-8/31/06. EPA Award RD83-1041. Email: dahutch@udel.edu

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

Landsberg, J., K. Steidinger, L. Flewelling, B.Richardson (FMRI), S. Hall (USFDA), G. Doucette (NOAA). 09/01/03- 08/31/06. NOAA Award NA03NOS4780196. Email: jan.landsberg@fwc.state.fl. us

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

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

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

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

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

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

Mitchell, B. G., M. Kahru, and C. Hewes (UCSD/SIO). 9/1/03 - 8/31/06. NASA Award. Email: gmitchell@ucsd.edu

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

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

Pierce, R.H. (Mote), R.W. Dickey (FDA), J.L.C. Wright (UNC), and K.A. Steidinger (FMRI). 9/1/03 - 8/31/06. NOAA Award NA03NOS4780197. Email: rich@mote.org

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

Van Dolah, F. M. (NOAA), G. R. DiTullio (U. Charleston, SC). 9/1/03 -8/31/06. NOAA Award NA03NOS4780198 (to U. Charleston). Email: Fran.Vandolah@noaa.gov

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

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

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

Villareal, T. A. (UT Austin), S. L. Morton and P. Moeller (NOAA). 9/1/03 - 8/31/06. NOAA Award NA03NOS4780238. Email: tracy@utmsi.utexas.edu

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

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