Grantee Research Project Results
Final Report: Detection and Identification of the Toxins from Pfiesteria and Related Harmful Algal Blooms
EPA Grant Number: R826655Title: Detection and Identification of the Toxins from Pfiesteria and Related Harmful Algal Blooms
Investigators: Gawley, Robert E. , Baden, Daniel G.
Institution: University of Miami , University of North Carolina at Chapel Hill
EPA Project Officer: Packard, Benjamin H
Project Period: August 1, 1998 through July 31, 2002
Project Amount: $353,975
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text | Recipients Lists
Research Category: Water Quality , Nanotechnology , Harmful Algal Blooms , Water , Aquatic Ecosystems
Objective:
The objectives of this research project were to:
- Grow cultures of Pfiesteria and related “Shephard’s Crook” dinoflagellates (a not-yet-assigned organism that is present in many Pfiesteria outbreaks), as well as Scripsiellas, Glenodiniums, and Gyrodiniums dinoflagellates. Concentrate the toxins on reversed-phase C-18 high-performance liquid chromatography columns by fractions by whole-fish bioassay using Tilapia fish fry. Purify the toxins to homogeneity, accumulate enough to allow chemical characterization, and elucidate the toxins’ structure using mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, and perhaps X-ray crystallography.
- Use the techniques of combinatorial chemistry to design ligand-selective host(s) that bind toxins from Pfiesteria and related dinoflagellates, while simultaneously effecting a visible signal of such binding in the form of enhanced fluorescence, seeking a host with optimum response to toxin and minimum response to other possible guests. Because the structures of the toxins are not known, this will be accomplished by seeking a correlation of toxicity in toxic fractions with fluorescence signaling of library members on polymer beads. The most highly fluorescent beads could then be produced in bulk for use as a toxin sensor.
- Once hosts that bind the toxin are known, build an affinity column to replace the C-18 column in the aquarium filter, and use this tool to collect the toxin for chemical characterization. This stationary phase could also be used for separating the toxins from other lipophilic components of the toxin extracts. The latter is a prerequisite for work on the biological aspects of these toxins. A large-scale application of this approach might be used in detoxification efforts.
Summary/Accomplishments (Outputs/Outcomes):
Objective 1
Samples of the Pfiesteria organisms and Pfiesteria-like organisms (PLO) were isolated from estuarine environments from North Carolina, Florida, Maryland, South Carolina, and Delaware. Water samples collected from areas where fish kills were active were also examined for all of the microalgae that were present. From each water sample, clonal cell cultures were obtained, including several non-Pfiesteria specimens.
Several of the above clones were assayed for cytotoxicity against a rat neuroblast line, and for toxicity (i.e., apparent mortality and morbidity) against mosquitofish (Gambusia holbrookii). These tests were negative for all samples tested.
Because our work over the last 4 years has failed to produce any positive evidence of ichthyotoxic compounds produced by Pfiesteria, we have been forced to ask a different question: Does Pfiesteria produce an ichthyotoxin? Scientists at the Virginia Institute of Marine Science (VIMS) at the College of William and Mary have shown that samples of live dinospores from P. shumwayae (Center for Culture of Marine Phytoplankton [CCMP] 2089) cultures, in organismal assays, lead to mortality of larval fish within as little as 24 hours. The apparent mechanism is predation on the fish by the dinoflagellate. In collaborative work with scientists from the VIMS, we conducted the following experiments.
We showed that cell-free water, obtained by centrifugation (with and without prior sonication) of these active fish-killing cultures of CCMP 2089, were consistently negative in ichthyotoxicity assays with no mortality of adult or larval fish observed over 7 days. This simple experiment demonstrates clearly that there is no ichthyotoxin in the bulk water, either before or after the cells are lysed by sonication.
It is conceivable that a toxin might have been adsorbed to the cell mass, but solvent extracts of lyophilized cell mass (methylene chloride, methanol), likewise, yielded no icthyotoxicity at concentrations equivalent to those present in cultures.
In addition to apparent lack of icthyotoxicity, the same cell-free extracts and fractions were not cytotoxic when tested against a rat neuroblast cell-line. These experiments suggest that there is also no cytotoxin being produced by CCMP 2089.
In collaboration with colleagues at Florida International University and at VIMS, we have used molecular biological techniques to search for biosynthetic genes, specifically polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes, associated with toxin production in dinoflagellates and other microalgae.
No amplification was observed with primers for PKS sequences in either PCR of total genomic DNA, or RT-PCR of total RNA, from P. shumwayae grown on algal prey, or the same culture exposed to larval fish (Cyprinodon variegatus) for 18 hours prior to DNA/RNA extraction. All ichthyotoxins known (to us) that are produced by dinoflagellates fall in the biosynthetic class of compounds known as polyketides (PK), which are biosynthesized via PKS enzymes. Lack of amplification with PKS primers suggests the lack of the typical PKS genes that have been identified in various, other polyketide-producing dinoflagellates.
Primers for NRPS genes amplified no sequences of the expected size for NRPS sequences (600 or 1,000 bp); however, they did amplify numerous other sequences. Several of these amplified products were cloned and sequenced. Analysis by BLAST revealed that one of the cloned sequences, a 401-bp product, shares homology to known and putative PKS or fatty acid synthase genes. Amplification of such sequences using the NRPS primers was very surprising. Control experiments confirmed that this amplification product originated in P. shumwayae and was not found in genomic or cDNAs of the cultured algal prey, Rhodomonas salina, or artificial seawater that had been incubated with fish in the absence of CCMP 2089. Sequencing of the full-length gene and further characterization will be needed before we can understand the function of the encoded enzyme.
Objective 1 Conclusions
Although these experiments do not eliminate polyketides as possible toxins produced by Pfiesteria, they strongly suggest that if Pfiesteria is capable of producing this class of secondary metabolites, the polyketide is either not excreted into the bulk medium and not released by cell lysis or is nontoxic. Taken together with the lack of ichthyotoxicity in centrifuged medium or solvent extracts, the conclusion is virtually inescapable that P. shumwayae does not produce an exogenous ichthyotoxin. This paradox of rapid mortality of fish exposed to live dinospores, but no apparent cell-free, ichthyotoxic compound can be resolved by invoking other mechanisms, such as micropredation as detailed by collaborators at VIMS.
These results were published in the Proceedings of the National Academy of Science in August. The impact of these findings is evidenced by a great deal of attention in the media including television coverage on CNN, and in articles in U.S. News & World Report, the Associated Press, Reuters, Chemical & Engineering News, Environmental Health Perspectives, Environmental News Network, Science, Science News, New Scientist, and the New York Times (see Relevant Web, Sites).
Objectives 2 and 3
Because we have concluded that there is no ichthyotoxin in Pfiesteria, developing a fluorescent sensor to detect it is moot. Nevertheless, detection of small molecule harmful algal bloom toxins is a more general goal of our work, and we have made significant progress on that front. Specifically, we have: (1) synthesized and tested 11 potential chemosensors for the dinoflagellate toxin saxitoxin, and (2) measured binding constants in the range of 104-105 M-1 for some of these crowns. These results were published recently in the Journal of the American Chemical Society. Though not directly related to the Pfiesteria, the chemosensors developed have received considerable attention, including articles in The Alchemist and Nature (see Relevant Web Sites).
Work on toxin sensors is continuing with funding from the U.S. Environmental Protection Agency and the U.S. Department of Agriculture.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 9 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Berry JP, Reece KS, Rein KS, Baden DG, Haas LW, Ribeiro WL, Shields JD, Snyder RV, Vogelbein WK, Gawley RE. Are Pfiesteria species toxicogenic? Evidence against production of ichthyotoxins by Pfiesteria shumwayae. Proceedings of the National Academy of Sciences of the United States of America 2002;99(17):10970-10975. |
R826655 (Final) R828225 (2000) R828225 (2001) R828225 (Final) |
Exit Exit Exit |
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Cardona CM, Gawley RE. An improved synthesis of a trifurcated Newkome-type monomer and orthogonally protected two-generation dendrons. Journal of Organic Chemistry 2002;67(4):1411-1413. |
R826655 (Final) R829599 (Final) |
not available |
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Gawley RE, Pinet S, Cardona CM, Datta PK, Ren T, Guida WC, Nydick J, Leblanc RM. Chemosensors for the marine toxin saxitoxin. Journal of the American Chemical Society 2002;124(45):13448-13453. |
R826655 (Final) R829599 (Final) |
not available |
Supplemental Keywords:
water, watersheds, sediments, marine, estuary, risk assessment, exposure, effects, bioavailability, vulnerability, dose-response, animal, organism, cellular, chemicals, organics, pathogens, environmental chemistry, biology, ecology, analytical, measurement methods, southeast, Atlantic coast, Chesapeake Bay, North Carolina, NC, Virginia, VA,, RFA, Scientific Discipline, Water, Geographic Area, Waste, Ecosystem Protection/Environmental Exposure & Risk, Limnology, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Contaminated Sediments, Chemistry, State, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, algal blooms, Southeast, Biology, Ecological Indicators, East Coast, ecological exposure, pfiesteria piscicida, North Carolina, agricultural runoff, fish kills, Shephard's Crook, whole-fish bioassay, ecology, suburban watersheds, bloom dynamics, harmful algal blooms, Gyrodiniums, economic assessments, fish lesions, ligand sensitive hosts, Everglades, pfiesteria, toxins, environmental chemistry, dinoflagellate, Glenodriniums, Tilapia, bioassay, fluorescence sensing, Virginia, Florida, ScripsiellasRelevant Websites:
http://www.rsmas.miami.edu/groups/niehs/ Exit
http://www.uncwil.edu/cmsr/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.