Alexandrium catenella is a paralytic shellfish poisoning-causing dinoflagellate that overwinters as a resting cyst and germinates into the water column in the spring-summer. Using winter cyst distribution, a Gulf of Maine forecast and an early warning system in Puget Sound, Washington were developed to mitigate human health risks and economic effects of shellfish closures. The current protocol for A. catenella cyst enumeration by fluorescent microscopy is time consuming and requires highly specific training for cyst identification. This MERHAB project, funded by NOAA, includes development of a new quantitative polymerase chain reaction (qPCR) methodology for A. catenella cysts that is being evaluated against the standard microscopy protocol with the goal of producing more rapid and accurate cyst abundance data. Comparison of microscopy- and qPCR-based cyst abundances shows a strong correlation, but quantitative estimates of cysts using the qPCR method tends to overestimate cyst numbers. Assay development showed the importance of using a qPCR standard curve based on cysts instead of a diluted amplicon, necessitating concentration of a large number of A. catenella cysts from the environment at the time of the survey. Furthermore, interannual and regional variability in rDNA gene copy number requires development of a new standard curve for each cyst survey. qPCR standard curves were constructed and compared across three regions using cysts from the Gulf of Maine, Puget Sound and Kodiak, Alaska. To assess temporal variability in rDNA gene copy number, seasonal comparison of qPCR standard curves was completed for Quartermaster Harbor, Puget Sound over one year.
Greengrove et al. (Fri,) studied this question.