ECOHAB: DNA-Based Molecular Diagnostics for Pfiesteria-Complex Organisms in Chesapeake BayEPA Grant Number: R826791
Title: ECOHAB: DNA-Based Molecular Diagnostics for Pfiesteria-Complex Organisms in Chesapeake Bay
Investigators: Reece, Kimberly S. , Burreson, Eugene M. , Stokes, Nancy A.
Institution: Virginia Institute of Marine Science , College of William and Mary-VA
EPA Project Officer: Packard, Benjamin H
Project Period: October 1, 1998 through September 30, 2001
Project Amount: $279,202
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text | Recipients Lists
Research Category: Water Quality , Harmful Algal Blooms , Water , Ecosystems
Description: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). 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 objectives of this project are to obtain DNA sequence data from clonal cultures of Pfiesteria-complex organisms found in Chesapeake Bay and its tributaries for phylogenetic analyses and development of DNA-based molecular diagnostics. Comparisons of sequences among the organisms will allow complex- and species-specific PCR primers and DNA probes to be developed for use in screening cultures and environmental samples.
Approach: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 (ITS) region and a portion of the large subunit (LSU) gene of the ribosomal DNA complex of each Pfiesteria-like species will be PCR amplified and sequenced. In addition, cultures used as food sources for PCOs will be obtained for DNA isolation, PCR amplification and sequence analysis. Comparison of food source sequences to PCO sequences will allow design of PCO-specific primers, facilitating amplification of PCO DNA from the clonal cultures which contain both PCO and food source cells. Species-specific PCO primers and probes will be designed based on analysis of sequence alignments and tested for specificity and sensitivity against each of the PCO species and other dinoflagellate species. Environmental sediment and water samples will be used to verify utility of the PCR and in situ hybridization assays.
Expected Results:Specific and sensitive molecular diagnostics will be developed to identify Pfiesteria-complex organisms with particular emphasis on those species found in Chesapeake Bay and its estuaries. Complex-and species-specific PCR primers and DNA probes will facilitate identification of Pfiesteria-complex organisms in cultures and in aquaria and environmental samples.
Publications and Presentations:Publications have been submitted on this project: View all 20 publications for this project
Journal Articles:Journal Articles have been submitted on this project: View all 5 journal articles for this project
Supplemental Keywords:Virginia, Maryland, recombinant DNA, clones, protist, Rhodomonas, Cryptoperidiniopsis, Shepherd?s Crook., RFA, Scientific Discipline, Geographic Area, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Contaminated Sediments, Oceanography, State, Environmental Microbiology, algal blooms, Ecology and Ecosystems, phylogenetic analyses, dinoflagellates, DNA based molecular diagnostics, fish kills, Virginia (VA), contaminated sediment, polymerase chain reaction, phytoplankton, Maryland (MD), pfiesteria, ECOHAB, hybridization assays, Chesapeake Bay