Certain aspects of research on Pfiesteria and Pfiesteria-like algae may be enhanced by the availability of culture isolates. In particular, laboratory experimentation would benefit from the availability of cultures to investigate the autecology and toxicology of these dinoflagellates as well to investigate interactions of Pfiesteria and Pfiesteria-like with other organisms. Presumably, laboratory results will have implications for understanding blooms, predicting occurrences, and minimizing deleterious effects. The proposed research is to first establish and maintain cultures of Pfiesteria and Pfiesteria-like organisms. Second, basic culturing experiments are proposed to determine optimal growth conditions and to establish the range of tolerance for temperature and salinities. Pfiesteria and Pfiesteria-like organisms appear to be obligate phagotrophs, but another common dinoflagellate phagotroph, Oxyrrhis marina, has been found capable of growing on a defined medium. Thus, the third aspect of the proposed research is to develop a defined organic medium that will support the growth of Pfiesteria and Pfiesteria- like organisms. Finally, most microscopic organisms in culture gradually "evolve" over time, sometimes changing in their growth characteristics, toxicity, and ability to exhibit alternate life stages. Cryopreservation offers relief from these undesirable attributes of "evolving" culture strains. The fourth aspect of this research is to establish cryopreservation protocols and to maintain cryopreserved cultures for culture strains of Pfiesteria and Pfiesteria-like organisms.
Lead PI: R.A. Andersen, Bigelow Laboratory for Ocean Sciences, P.O. Box 475, McKown Point Rd., West Boothbay Harbor, ME, 04575; Randersen@bigelow.org; 207-633-9600.
Anderson, D.M. (WHOI), R. Pierce, (Mote Marine Laboratory), R.M. Greene (EPA Gulf Ecology Division), M. Lewis (EPA Gulf Ecology Division), P. Chapman (EPA Gulf Ecology Division), M. Bricelj (Institute for Marine Biosciences, NRC Canada). "ECOHAB: Control of harmful algal blooms using clay." 11/23/98-11/22/01 first year of a 3 year study. EPA Award CR827090.
This project will investigate the feasibility of a promising control strategy - the removal of cells from the water column using clay flocculation. The approach relies on the ability of certain clays to scavenge particles, including algal cells, from seawater, carrying them to bottom sediments where they are buried and decomposed. The project will begin with small-scale laboratory experiments in which algal cultures in test tubes are treated with clays and cell removal, cell viability, toxin release, and other parameters are measured. Laboratory experiments will also examine nutrient uptake and release by clays applied to seawater containing no algae. These and other laboratory experiments will be conducted on each of the targeted HAB species, (the Florida red tide dinoflagellate Gymnodinium breve, the New York brown tide chrysophyte Aureococcus anophagefferens, and fish-killing Pfiesteria-like dinoflagellates). Work will then shift to aquaria or mesocosm tanks where the clay treatments will be applied to natural communities of planktonic and benthic organisms. No studies have yet been conducted on the loadings needed to remove U.S. HAB organisms, on the suitability of readily available U.S. clays, or of the possible environmental impacts of flocculation and sedimentation of bloom organisms, especially those containing toxins. The near-term results of this study will be used to evaluate the engineering requirements, economic costs and environmental clearances needed for a pilot program for field application of this mitigation strategy. The eventual conclusion from the investigations proposed here will be of great value in evaluating the feasibility and potential environmental impacts of this promising bloom mitigation strategy. Knowledge will have been gained that can steer us towards related strategies that may someday help us minimize the impacts of some HABs.
Lead PI: D. Anderson, Biology Department, WHOI, Woods Hole, MA, 02543; firstname.lastname@example.org; 508-289-2351.
Bissett, W.P. (Florida Environmental Research Institute). "Hyperspectral modeling of harmful algal blooms on the West Florida Shelf." 08/01/98-07/31/01. ONR Award N00014-98-1-0844.
Each red tide occurrence of Gymnodinium breve costs an estimated $20 million dollars in economic damage, plus untold damage to the marine ecological systems through massive fish, bird and mammal kills. The management of marine resources, e.g., closure of shellfish beds, in response to these outbreaks requires the ability to predict their occurrence. As the natural life cycle of G. breve occurs within an autotrophic community, predictions must incorporate the interactions of G. breve within a complete phytoplankton assemblage, not just the growth and mortality of G. breve. In addition, proposed mitigation of these blooms will require realistic numerical simulations that incorporate the dynamics of the entire marine ecosystem, in order to investigate possible deleterious feedbacks. It is hypothesized that the prediction of G. breve must include the competitive interactions amongst multiple phytoplankton populations for spectral light energy, as well as the competitive interactions for multiple nutrient resources. The project will develop a three-dimensional Ecological Simulation of the West Florida Shelf (Eco-Sim-WFS) that includes a hyperspectral model of coastal ocean optics. Eco-Sim-WFS will incorporate the competitive feedback mechanisms of phytoplankton spectral light absorption and scattering onto a realistic autotrophic ecosystem. This work builds on the currently funded NOAA/COP intensive study of harmful algal blooms on the West Florida Shelf (ECOHAB-Florida), and a NRL 6.1 Accelerated Research Initiative on hyperspectral coastal optics (Spectral Signature of Optical Properties in the Littoral Zones). The model will be an enhancement of an existing Ecological Simulation (EcoSim 1.0) developed for oligotrophic waters. The model’s 60 m spatial resolution and 10 nm spectral resolution will enable it to directly utilize the water-leaving radiance data stream from a new ONR/NRL satellite instrument, the Coastal Ocean Imaging Spectrometer, to be launched in May, 2000. Deliverables from this work include - 1) A numerical simulation of G. breve within a complete phytoplankton ecosystem, which can be used to predict G. Breve blooms under realistic physical forcing. It will also provide a platform to test the effects of potential G. breve mitigation schemes on the ecosystem of the West Florida Shelf; 2) A prognostic simulation of the dept-dependent inherent and apparent optical properties of the West Florida Shelf, which includes predicting upwelling radiance, Lu(l), for a given downwelling irradiance and solar zenith angle.
Lead PI: W.P. Bissett, Florida Environmental Research Institute, The Florida Aquarium, 701 Channelside Drive, Tampa, FL 33602; email@example.com; 813-273-4161.Garman, G. (VCU), Webb, S. (VCU), B. Browne (VCU), and S. McIninch (VCU). "Free-living, pathogenic amoebae in Chesapeake Bay tributaries." 07/01/98-12/31/99. EPA Award R82-7191.
During the Summer and Fall of 1997, occurrences of moribund and dead fish in Maryland and Virginia coastal rivers were reported widely by the popular press. Because many of the afflicted fish exhibited lesions and related characteristics, these outbreaks were attributed initially to toxins produced by the dinoflagellate Pfiesteria piscicida or by related Pfiesteria-complex organisms (PCO’s). Although toxins from PCO’s have been confirmed as the likely cause of some recent fish kills (e.g., Pocomoke River), examinations of fish lesions and environmental samples collected during the same period in other coastal rivers of the Chesapeake Bay drainage (e.g., Rappahannock and James rivers in Virginia) have produced no evidence of PCO’s. This proposal presents preliminary evidence that free-living, pathogenic amoebae of the genera Acanthamoeba, and possibly Naegleria, may represent currently unrecognized agents of ulcerative disease in freshwater and estuarine fishes of the Chesapeake region, and may be comparable in effect to Harmful Algal Blooms (HABs) in coastal estuaries. We hypothesize that at least some of the reported outbreaks of dead and moribund fish with lesions in Virginia rivers during the Summer and Fall of 1997 may be attributable to free-living pathogenic amoebae and not to Pfiesteria or other HAB-related organisms. If true, pathogenic amoebae may represent a previously unrecognized agent of fish and human diseases in coastal rivers. The proposed study would expand significantly the scope of a present VCU project, funded by the Virginia Department of Environmental Quality (DEQ).
Lead PI: G. Garman, Center for Environmental Studies, Virginia Commonwealth University, P.O. Box 843050, Richmond, VA, 23284; firstname.lastname@example.org; 804-828-7202.Glibert, P.M. (HPL, U.MD), W.C. Boicourt (HPL, U.MD), R.M. Harrell (HPL, U.MD), H.R. Harvey (CBL, U.MD), R.R. Hood (HPL, U.MD), M.R. Roman (HPL, U.MD), L.P. Sanford (HPL, U.MD), D.K. Stoecker (HPL, U.MD), J.M. Burkholder (NCSU), S.C. Cary (U. DE), D.A. Hutchins (U. DE), and A. Lewitus (USC). "ECOHAB: Toxic dinoflagellates and nutrients. Toward a mechanistic understanding of outbreaks of Pfiesteria and related dinoflagellates. A regional, comparative study." 09/01/98-08/31/99 first year of 5 year study. NOAA Awards NA86OP0493, NA86OP0510, and NA86OP0495.
This research project is a regional, comparative study of the physical, nutritional, and trophodynamic mechanisms that contribute to blooms of Pfiesteria. The study will test the hypothesis that certain mechanisms are interdependent, and together contribute to the development and persistence of these blooms. Through comparative field and mesocosm experiments, spanning a range of conditions under which Pfiesteria and other Pfiesteria-like species are found to occur or to bloom, the environmental conditions and factors that contribute to the populations' success will be described. Parameters to be measured include nutritional requirements, fish detection, turbulence impacts, predator role, and metabolite production that reduces grazing pressure or induces stress in fish; molecular techniques will also be developed for life-stage detection. Field sites in MD, NC, DE, and SC will be studied to identify common features that might explain environmental requirements of Pfiesteria-like organisms. The laboratory and field results will be incorporated into physiological and ecosystem models that can be used to predict the abundance of Pfiesteria in the mid-Atlantic region.
Lead PI: P. Glibert, Horn Point Laboratory, University of MD, P.O. Box 775, Cambridge, MD, 21613; email@example.com; 410-221-8422.
Lin, S. and E.J. Carpenter (SUNY, Stony Brook). "ECOHAB: Factors affecting growth of Pfiesteria piscicida." 09/01/98-08/31/99, first year of 3.5 yr study. NOAA Award NA86OP0491.
The project will focus on development of molecular techniques to measure in situ growth rates of Pfiesteria piscicida from Eastern Shore tributaries of the Chesapeake Bay. One technique will focus on a cell cycle protein method, (including identification of the cell cycle related genes, proteins, and development of antibodies), another surface antibodies, and a third the development and application of specific gene probes for identification of life stages and growth status in the water column and sediments. These results will then be used with environmental variables from the field in the construction of a box model that relates environmental conditions to field growth rates. It is anticipated that the research will not only provide new detection tools but also determine the mechanisms through which environmental factors control life cycle transformations, growth rate, and abundance of planktonic and benthic stages of P. piscicida.
Lead PI: S. Lin, Marine Sciences Research Ctr., SUNY, Stony Brook, NY, 11794; Slin@ccmail.sunysb.edu; 516-632-8697.
Maske, H. (CHORS, SDSU), J. Mueller (CHORS, SDSU), C. Trees (CHORS, SDSU), R. Iglesias (CHORS, SDSU). "ECOHAB: Hyperspectral optical properties of a red tide bloom." 07/01/98-06/30/01, first year of a 3 year study. ONR Award N00014-98-1-0778.
Red tide blooms derive their name from the red color observed on the sea surface. Red tides are in general composed of species that contain phycobiliproteins or by dinoflagellates. Phycobiliproteins are pigments with absorption spectra that filter out wavelength other than the red light thus allowing the residual reflected light appear on the sea surface. The objective of our research under this project is to characterize the bio-optical properties of dinoflagellate red tide blooms and explain what is the source of the red color in naturally occurring blooms, and why does the color not appear in cultures of these species? Our main hypothesis is that the red color originates from chlorophyll fluorescence and that the fluorescence can be observed from above the sea surface because of the high concentration of dinoflagellates near the surface. Our main tools for investigating the optical properties are in situ measurements of inherent and apparent optical properties at high vertical resolution near the surface with a free falling/rising profiler that includes a CTD, radiometers and photometer. Also we will take water samples at about 0.2 vertical resolution near the surface with water bottles arranged in a rigid vertical array. These samples will be used for pigment analysis and physiological measurements. The data will be compared with radiative transfer calculations trying to reach closure by adjustment of the quantum yield of fluorescence emission.
Lead PI: H. Maske, Centre for Hydro-Optics and Remote Sensing, San Diego State University, 6505 Alvarado Rd. Suite 206 San Diego, CA 92120; firstname.lastname@example.org, email@example.com; (from USA): 011 52 617 45050 ext. 24260.
Oldach, D. (U. MD School of Medicine) and P. Rublee (NCSU at Greensboro). "ECOHAB dinoflagellate molecular ecology." 10/08/98-8/31/01, 3 years. EPA Assistance ID No. R 827084-01-0.
Pfiesteria piscicida 18s rRNA gene sequence pools will be amplified from cultures and environmental samples, followed by heteroduplex mobility assay (HMA) for gauging sequence diversity within the amplified pool of cDNA. HTA (heteroduplex tracking assay) will then be undertaken to identify characterized and novel dinoflagellate gene sequences. PCR primers will be developed for assays of Pfiesteria piscicida, and assays will also be undertaken with universal dinoflagellate primes followed by HTA mapping. It is anticipated that quantitative PCR analyses will be follow, with initial work in the laboratory using kinetic thermal cycling assays and fluorogenic probes in the 5'exonuclease assay. Subsequent studies will include field assays in MD and NC with battery powered miniaturized analytical thermal cycling instruments (MATCI, Cepheid, Inc.). Detection of species in the field will be correlated with environmental variables as well as observed health effects in ‘exposed’ individuals in cohort studies from impacted waterways.
Lead PI: D. Oldach, University of Maryland School of Medicine & Institute of Human Virology,
Room 556, U of MD Medical Biotechnology Center, 725 W. Lombard Street, Baltimore, MD 21201; firstname.lastname@example.org; 410 706-4609.
Paul, J.H. (USF). "ECOHAB-Exploring lytic and temperate viruses of Gymnodinium breve as a mechanism of controlling red tide blooms." 09/01/98-08/31/99. NOAA Award NA86OP0494.
Red tides caused by the toxin-producing dinoflagellate Gymnodinium breve are common along Florida’s Gulf Coast. The observation that some blooms undergo sudden dissipation suggests a catastrophic event has occurred with the bloom. Viruses are capable of producing such rapid mass mortality. The goal of our research is to investigate both lytic and temperate viruses as a potential mechanism of red tide termination. During December, we sampled water from a red tide bloom near Ft. Myers, FL. Water samples were either prefiltered through a 1.2 or a 0.2 um filter, and the viral fraction concentrated by ultrafiltration. Aliquots (1 to 1.5 ml) of the concentrated fraction were added to 25 ml G. breve cultures and the culture growth monitored by fluorescence for 10 days. Nearly every culture that received the concentrated fraction showed a dramatic decrease in fluorescence after 5 to 7 days, whereas controls continued to grow normally. The "lytic agent" was transferred to fresh cultures, and the period of time required to cause a decrease in fluorescence ("crash") decreased to 3 to 4 days. The lytic agent remained active after filtration through a 0.2 um filter. The lytic agent has been serially passaged at least five times and remains active/infective. We have observed virus-like particles by epifluorescence microscopy but these may be from bacteria present in the cultures. Transmission electron microcopy of thin sectioned samples has not yet detected viral particles in G. breve cells. We are now analyzing some biological and physical properties of the lytic agent.
Lead PI: J.H. Paul, Department of Marine Sciences, University of South Florida, St. Petersburg, FL, 33701; email@example.com; 813-553-1168.<
Reece, K.S. (VIMS), E.M. Burreson (VIMS), and N.A. Stokes (VIMS). "ECOHAB: DNA-based molecular diagnostics for Pfiesteria-complex organisms in Chesapeake Bay." 07/01/98-06/30/01, EPA Award R826791.
The newly discovered heterotroph Pfiesteria piscicida and other closely related toxic dinoflagellates have been blamed for many of the harmful algal blooms resulting in fish kills in the US Atlantic Coast estuaries during the past seven years. Pfiesteria piscicida and other Pfiesteria-like dinoflagellates are commonly referred to as the Pfiesteria-complex organisms (PCOs). Pfiesteria piscicida has been found associated with fish kills in the Neuse and Pamlico estuaries of North Carolina and has been implicated in human health effects including neurocognitive impairment and skin lesions. Fish kills in Chesapeake Bay tributaries and health complaints from individuals having frequent exposure to waters were attributed to a Pfiesteria- like dinoflagellate and prompted closure of the Pocomoke River for 2 months during the summer of 1997. The discovery of several Pfiesteria-like species with complex life cycles has highlighted the necessity for accurate identification and characterization of these organisms. Currently the only method available to accurately identify Pfiesteria-like species is analysis of the thecal plate structure of cysts by scanning electron microscopy (SEM). This method cannot be used for identification of all life stages and is time-consuming and tedious precluding its use for large-scale monitoring programs. The objective of this project is to develop DNA-based diagnostics for use in screening cultures and environmental samples. Primers for use in the polymerase chain reaction (PCR) and DNA probes for use in in situ hybridizations that are group-specific to the Pfiesteria-complex organisms (PCOs) and specific to species within the complex will be designed and tested. Following species identification by SEM, DNA will be isolated and the internal transcribed spacer region and a portion of the large subunit gene of the ribosomal DNA complex of each Pfiesteria-like species will be PCR amplified and sequenced. Primers and probes will be designed based on analysis of sequence alignments and tested for specificity and sensitivity against each of the PCOs and other dinoflagellate species. Environmental sediment and water samples will be used to verify utility of the PCR and in situ hybridization assays.
Lead PI: K.S. Reece, Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, VA, 23062; firstname.lastname@example.org; 804-684-7407
Repeta, D.J. (WHOI). "ECOHAB: Dissolved organic nitrogen and brown tide blooms in Long Island coastal waters." 01/01/98 - 03/31/01, NSF Award OCE 9730015.
Blooms of the `Brown Tide` unicellular algae (Aureococcus anophagefferens) have occurred sporadically since 1985 in coastal waters of Eastern Long Island and have devastated the local commercial scallop fishery. Analysis of a 10 year time series data set from the Peconic Estuary indicates that bloom intensity is inversely correlated with the discharge of high nitrate groundwater and associated with higher salinities. Laboratory and field data suggest that salinity is unlikely to represent a direct physiological control on Brown Tide blooms. However budget calculations indicate that nitrogen supply from groundwater is 1 to 2 orders of magnitude higher than other external sources for this ecosystem. Data collected in 1995 demonstrated that Brown Tide blooms utilize dissolved organic nitrogen (DON) for growth as evidenced from the large decrease in DON parallel with cell increase. We hypothesize that bloom initiation is regulated by the relative supply of inorganic and organic nitrogen, determined to a large extent by groundwater flow variability. An appropriate first step to test this hypothesis is the demonstration that there are compounds in the DON that can be efficiently utilized by A. anophagefferens giving it a competitive advantage over other endemic species. We will identify the source of DON that is available to A. anophagefferens via field and laboratory studies. The laboratory work will involve the identification of the DON components from the Peconic Estuary that can support growth of the alga and characterization of the DON uptake systems and utilization mechanisms that make this alga competitive at utilizing nitrogen. Immunological probes to major proteins involved in the utilization of DON will be prepared. In the field we will characterize the DON fraction utilized by A. anophagefferens during a bloom as well as follow the nitrogen nutrition of this algae using immunological probes. Weekly nutrient bioassays and analysis of various dissolved and particulate nitrogen pools will complement the more detailed field sampling.
Lead PI: D.J. Repeta, Woods Hole Ocean Institution, Woods Hole, MA 02543; email@example.com; 508-548-1400
Roesler, C. (UConn, Bigelow), "ECOHAB: Ecophysiology of subpopulations of Alexandrium tamarense."09/01/98 -08/31/01, 01/01/99-12/31/01. NASA Awards NAG5-7654 and NAG5- 7872.
Toxic algal blooms, often called red or brown tides for the color they impart to the water, pose a serious threat to ecosystem and public health, as well as to the economy, as they are responsible for fish kills, shellfish poisoning, and human illness. While the reports of toxic outbreaks is escalating worldwide, early detection remains elusive, impeding progress towards understanding the forces responsible for bloom initiation, development and advection. The long term goals of this project are to identify those factors which lead to the initiation and development of toxic blooms and the variability in bloom toxicity using controlled laboratory experiments in concert with in situ detection of toxic blooms in the natural environment. This project specifically focuses on Alexandrium tamarense, a toxic dinoflagellate responsible for recurrent episodes of paralytic shellfish poisoning (PSP) from northeastern Canada to the coast of New Jersey. We will investigate the ecophysiology of the northern and southern subpopulations, which demonstrate significant differences in toxicity and bloom initiation, posing the questions of why Long Island Sound is bereft of extreme A. tamarense blooms and why the isolate in Long Island appears to have reduced toxicity. To answer these questions we will (1) measure quantitative toxin profiles during all growth stages of the two isolates under a range of environmental conditions and (2) investigate natural outbreaks of A. tamarense in New England waters using an optical models to assess the spatial and temporal distributions in concert with environmental parameters. The basis of the optical models is the extraction of the algal inherent optical properties (absorption, scattering, backscattering coefficients) from ocean color in the presence of a mixed algal community. These properties provide sufficient information for detection, species identification and bloom monitoring. It is anticipated that the approach will supplement our understanding of toxic bloom dynamics as development can be tracked backwards in time via imagery and the actual initiating environmental conditions can be determined. This approach can also be used to forecast the progress of existing blooms providing early detection for "down stream" waters.
Lead PI: C. Roesler, Bigelow Laboratory for Ocean Sciences, McKown Point Road, P.O. Box 475, West Boothbay Harbor, ME 04575; CRoesler@bigelow.org; 207-633-9654.
Trainer, V.L. (NOAA) and M. Bricelj (NRC, Canada). "ECOHAB: Mechanisms and control of toxin accumulation in shellfish." 09/01/98-08/31/99, first year of 3. Internal transfer of funds to NW Fisheries Science Center, NOAA.
The project will characterize mechanisms regulating toxin sensitivity and accumulation in bivalve molluscs, specifically butter and softshell clams Mya arenaria and the razor clam Siliqua patula. Mya, isolated from toxic and non-toxic regions, will be exposed to toxic and non-toxic Alexandrium in the laboratory. Behavioral and feeding responses will be determined, as well as nerve sensitivity to all paralytic shellfish toxins present; grazing inhibition will be examined as a ‘sensitivity’ measure to toxins and toxin uptake and elimination will be estimated. Using a similar approach, razor clams will be exposed to toxic Pseudo-nitzschia sp.; domoic acid uptake and elimination kinetics will be measured. A unique feature of the study will be the isolation of inducible binding proteins, necessary structural elements for the development of quick, sensitive, receptor-based analytical toxin detection methods. Overall, project findings will provide insights on the potential for acclimation and/or genetic adaptation of bivalve populations to toxins, and possibly provide sentinels of ecosystem health at the level of the primary consumers.
Lead PI: V.L. Trainer, Northwest Fisheries Science Center, F/NWC2, NOAA, 2725 Montlake Blvd. E., Seattle, WA, 98122-2097; Vera.L.Trainer@noaa.gov; 206-860-3387.
Zohar, Y., G. Vasta, R. Belas, and A. Place (COMB, U. MD). "ECOHAB: Molecular approaches to Pfiesteria-complex dinoflagellates in Chesapeake Bay." 09/01/98-08/31/00, first 2 years of 5. NOAA Award NA86OP0492.
This research project is designed to develop a Dinoflagellate Culture Core Facility to maintain Pfiesteria-complex dinoflagellate (PCD) stock cultures, optimize culture conditions, and provide toxic and non-toxic PCD cells, supernatants, and extracts to the project PIs and the HAB community. Research projects in the program will also develop molecular probes to detect PCDs, determine the role of dinoflagellate associated bacteria in PCD physiology and toxigenesis, and determine the molecular nature and physiology of the elicitors of toxigenicity. The project will deliver a set of molecular probes and biosensors specific to PCDs and provide basic scientific data on the genetics and physiology of PCDs, dinoflagellate associated bacteria, and toxin elicitors.
Lead PI: Y. Zohar, Center of Marine Biotechnology, University of Maryland, 701 East Pratt St., Baltimore, MD, 21202; firstname.lastname@example.org; 410-234-8800.