1999 Progress Report: 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 Period Covered by this Report: October 1, 1998 through September 30, 1999
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
Objective:The overall objective of this project is to develop complex-and species-specific PCR primers and DNA probes to facilitate identification of Pfiesteria-complex organisms in cultures, experimental aquaria and environmental samples. DNA sequence data is obtained from clonal cultures of Pfiesteria-complex organisms found in Chesapeake Bay and its tributaries for phylogenetic analyses and development of DNA-based molecular diagnostics. DNA sequence comparisons among the organisms allows PCR primers and DNA probes to be developed for use in screening cultures and environmental samples.
Progress Summary:The only reliable method to accurately identify and distinguish Pfiesteria piscicida and other Pfiesteria-like organisms (PLOs) has been through 3-D reconstructions of the thecal plate structures by scanning electron microscopy (SEM). We are doing a comprehensive survey of DNA sequences for the internal transcribed spacer (ITS) region and portions of both the small (SSU) and large (LSU) subunit genes of the ribosomal DNA complex for available PLO clonal cultures. Sequence information is being used for phylogenetic studies and to develop DNA-based diagnostics. Sequence comparisons among the PLO cultures and to other dinoflagellates and protozoans allowed design of species-specific and genus-specific PCR primers and DNA probes. Clonal cultures of Pfiesteria piscicida, Pfiesteria sp. "B", Cryptoperidiniopsis spp., "Shepherd's crook" and "Lucy-like" PLOs were first analyzed and identified by SEM and then obtained for molecular analysis. DNA was isolated from a total of 18 clonal PLO cultures and 3 food source cultures. DNA sequences were obtained from the food sources to assure that PLO, rather than food source DNA clones, were selected for analysis. SSU, ITS and LSU DNA sequences from the PLOs were aligned and subjected to phylogenetic analyses. The SSU gene was highly conserved while the ITS region (excluding the 5.8S portion) and portions of the LSU gene fragment demonstrated considerable sequence variation. Comparison of DNA clone sequences obtained from clonal cultures demonstrated degenerate sites in the ITS region and the LSU gene and even a few in the SSU gene. Overall the molecular data strongly supported identifications made by SEM. Phylogenetic analyses grouped together all the cultures identified as P. piscicida and placed Pfiesteria sp. "B" between the Pfiesteria piscicida clade and Cryptoperidiniopsis spp. In addition, cultures identified by SEM as "Lucy" or "Lucy-like", grouped together in the DNA-based phylogenies.
Sequence alignments and comparisons were used to design four sets of PCR primers to specifically amplify P. piscicida, Pfiesteria sp. "B", Cryptoperidiniopsis spp. or the "Lucy" group clonal culture DNAs. PCR primers and reaction conditions were optimized for specificity with clonal culture DNAs and then tested for their ability to amplify these DNAs from water and sediment samples. In addition, specific DNA probes were designed for in situ hybridization. Samples from fish bioassay tanks and the environment have been tested with the DNA probes and PCR primers. Results of molecular diagnostic assays agreed with SEM identifications and PCR assays confirmed the presence of PLO species detected by in situ hybridization. For example, the Pfiesteria sp. "B" probe hybridized to dinoflagellate cells found in the lateral line canal and alimentary tract of fish from an experimental tank with high cell counts of a PLO identified by PCR assay as Pfiesteria sp. "B". DNA probe analyses indicated that dinoflagellates found in the alimentary tracts of menhaden collected from two sites in North Carolina were "Lucy-like" cells. PCR amplification of DNA from water samples collected at the same sites as these menhaden indicated the presence of "Lucy-like" DNA. Amplicons obtained with P. piscicida, Pfiesteria sp. "B" or "Lucy" group PCR primers from environmental samples were analyzed by DNA sequencing and digestion with several restriction endonucleases to confirm that the primers were specifically amplifying the targeted DNAs. The PCR assays, which have been shown to be specific for the target organisms, will enable rapid identification of PLOs from the environment. When these assays are coupled with the appropriate equipment, they will also permit quantification of those PLOs. In addition, DNA probes will permit identification of PLOs in fish filtering water that harbors dinoflagellates and may provide a biological indicator of PLO activity and abundance.
Future Activities:We are optimizing the molecular diagnostics and using them to screen new cultures for molecular identification for cross-validation with SEM identifications. We will sequence the SSU, ITS and LSU regions of additional selected cultures particularly targeting Pfiesteria sp. "B", Cryptoperidiniopsis spp. or the "Lucy" group clonal cultures. In addition, PCR assay optimization with aquaria and environmental samples including water and sediment will continue.
We recently have purchased a CytospinTM centrifuge to facilitate the in situ hybridization assays with cultured cells and aquaria and environmental water samples. We will continue to analyze dinoflagellates in the alimentary canals, gills and lateral lines of fish from toxic bioassay aquaria using in situ hybridization assays. In addition, the alimentary canals of menhaden both with and without lesions collected from several estuaries in North Carolina and Virginia waters are being examined for the presence of PLOs.
Two manuscripts from this work are presently in preparation. We have worked in collaboration with Wayne Litaker of the University of North Carolina to examine polymorphisms in the ITS region of clonal culture DNAs and are writing a manuscript describing the implications of this variation for designing species or genus-specific PCR primers. A second manuscript is being prepared on molecular characterization of the "Lucy"-group of PLOs. In addition, we plan to prepare a manuscript in the next year describing the molecular diagnostics that we have developed for PCR and in situ hybridization assays.
Journal Articles:No journal articles submitted with this report: View all 20 publications for this project
Supplemental Keywords:genetics, Virginia, VA, North Carolina, NC., RFA, Scientific Discipline, Geographic Area, Waste, Water, Ecosystem Protection/Environmental Exposure & Risk, Contaminated Sediments, State, Oceanography, Environmental Microbiology, algal blooms, Ecology and Ecosystems, phylogenetic analyses, dinoflagellates, DNA based molecular diagnostics, fish kills, Virginia (VA), contaminated sediment, phytoplankton, polymerase chain reaction, pfiesteria, Maryland (MD), ECOHAB, hybridization assays, Chesapeake Bay