Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques

The toxic dinoflagellate Pfiesteria piscicida has been identified in coastal waters and estuaries along the Atlantic coast of the United States. Estuaries in the mid-Atlantic region, in particular, have been targeted as high-risk areas for toxic blooms since reports of Pfiesteria-related fish kills in the Pocomoke River, Maryland, in 1997. The development of monitoring strategies for these areas requires that the presence of Pfiesteria be rapidly and accurately assessed. Routine monitoring by light microscopy lacks both the sensitivity and accuracy required for species-specific detection and enumeration of Pfiesteria, especially at the low levels normally found in non-bloom conditions. In this study, we developed 2 molecular techniques to identify and enumerate P. piscicida in the Delaware Inland Bays and the Pocomoke River. The first technique, denaturing gradient gel electrophoresis (DGGE), was used to identify several similar but distinct strains of Pfiesteria in water and their benthic stages (cysts or amoebae) in sediment samples. A comparison of DGGE analyses of Pfiesteria community structure in the Pocomoke River and the Delaware Inland Bays revealed subtle differences in strain composition. A second technique, PCR-fluorescent fragment detection (PCR-FFD), was designed for quantitative enumeration of Pfiesteria in water samples. This technique offers a 1000-fold increase in sensitivity over microscopic techniques. To demonstrate the utility of PCR-FFD, we conducted a study of Pfiesteria at the Roosevelt Inlet, Lewes, Delaware. Pfiesteria concentrations over 2 tidal cycles were correlated to other physical, biological and chemical variables, Overall, our data establish the presence of Pfiesteria as a minor but prevalent member of the phytoplankton community in mid-Atlantic estuaries.