Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.