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1 State University of New York at Stony Brook, Stony Brook, NY
2 Woods Hole Oceanographic Institution, Woods Hole, MA
* Corresponding author: sdyhrman{at}whoi.edu
The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense produces toxin that can accumulate in shellfish and cause paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern (1). Accurate measurements of A. fundyense distributions, at a low cell density, are critical to continued PSP monitoring and mitigation efforts.
Traditional assessments of A. fundyense cell number rely on microscopic counts of species abundance. However, it is difficult to visually distinguish A. fundyense from other dinoflagellates and other species of Alexandrium, as there are often only subtle morphological differences between taxa. Oligonucleotide probe-based methods (2) help to distinguish between Alexandrium and other genera and are commonly employed in the Gulf of Maine to map cell distribution. Although these approaches are useful, we developed and applied an assay that does not require microscopy. Various studies have used quantitative PCR (qPCR) to assay cell numbers of dinoflagellates such as Pfiesteria piscicida (3). In this study we mapped A. fundyense distribution using a qPCR assay that appears to be specific and sensitive.
Primers were designed to amplify a 174 base pair region of the large ribosomal subunit (LSU) gene (4). This gene was chosen because it has been sequenced from many species of Alexandrium, is commonly used for phylogenic analyses (5), and is present in high copy number.
Building on previous work (4), the specificity of the LSU primers was tested against DNA extracted from phytoplankton cultures with Qiagen’s DNEasy Kit, according to the manufacturer’s instructions, using standard hot start amplification conditions and a 60 °C annealing temperature. Under these conditions amplification occurred only with A. fundyense isolated from the Gulf of Maine (Fig. 1A).
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Using qPCR, the number of cells in a field sample was determined. In this study, qPCR was performed using Stratagene Brilliant SYBR Green QPCR Master Mix, and a fluorescence threshold was set by the analytical software for the BioRad iCycler. The PCR cycle during which this threshold was crossed for each sample designated the CT. Sample CT can be compared to the CT of standards with a known cell count to specify the number of cells present in the sample (4).
To ensure that different strains of A. fundyense have the same copy number of the LSU gene, standard curves were built from two Gulf of Maine strains in culture (GTCA28 and CB301). Both strains resulted in similar standard curves (y = -2.5281x + 30.664, R2 = 0.9514 for the first strain and y = -2.0291x + 27.61, R2 = 1 for the second strain). A dilution series of a known number of cells was also analyzed with a field sample matrix. No significant PCR inhibition of the target was detected from extraneous DNA in the field samples.
The number of cells detected in the Gulf of Maine ranged from 1 to 35 cells per liter (Fig. 1B). This range is similar to the range of cell numbers calculated from other methods (live counts done during the cruise and oligonucleotide probe counts [Anderson, unpubl. data]), which we did not expect to be exactly the same since the samples were taken from different Niskein bottles and processed differently. Of the stations tested, station 86 and offshore stations 104 and 105 had the highest concentrations of cells. To confirm amplification of A. fundyense from the field samples, PCR products generated from samples 89 and 128 were sequenced at the Marine Biological Laboratory, Woods Hole, Massachusetts, using the facility’s protocols and were shown to be A. fundyense.
In summary, qPCR appears to be a specific and sensitive approach to monitoring the abundance of A. fundyense. This method shows great promise for mapping the A. fundyense populations in the Gulf of Maine.
This research was supported by an EPA Star Award through the ECOHAB Program (R-83041501-0) and a NSF-REU site grant (OCE-0097498) to the Boston University Marine Program.
Literature Cited
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