Molecular Approaches to the Investigation of Viable Dinoflagellate Cysts in Natural Sediments from Estuarine Environments1

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Abstract
Molecular methods offer an efficient alternative to microscopic identification of dinoflagellate cysts in natural sediments. Unfortunately, amplification of DNA also detects the presence of dead cells and is not a reliable indication of cyst viability. Because mRNA transcripts are more labile than DNA, the presence of specific transcripts may be used as a proxy for cyst viability. Here, we evaluate mRNA detection capabilities for identification of viable cysts of the dinoflagellate, Pfiesteria piscicida, in natural sediment samples. We targeted transcripts for cytochrome c oxidase subunit 1, cytochrome b (COB), and Tags 343 and 277, recently identified by serial analysis of gene expression. Expression was confirmed in laboratory cultures and compared with natural sediment samples. Three of the transcripts were detected in sediments by RT-PCR. The fourth transcript, for COB, was not detected in sediments, perhaps because of down-regulation of the gene in anoxic conditions. Our results suggest that methods targeting specific mRNA transcripts may be useful for detection of viable cysts in natural sediment samples. In addition, dinoflagellate cysts, which sustain extended periods of anoxia, may provide an important source of data for studies of anoxia tolerance by microbial eukaryotes.