Identification and quantification of toxic and nontoxic strains of the harmful dinoflagellate Alexandrium tamarense using fluorescence in situ hybridization and flow cytometry

Abstract

The co-occurrence of morphologically identical toxic and nontoxic ribotypes of the biotoxin producing marine dinoflagellate Alexandrium tamarense presents a significant problem for its identification and enumeration, particularly in a regulatory monitoring context. To address this, we have developed a fluorescence in situ hybridization-flow cytometry (FISH-FC)-based method of cell identification and enumeration. This employed the taxa specific oligonucleotide probes TamToxC and TamA to fluorescently label (with the fluorochromes CY.3 and FITC) Group I (toxic) and Group III (nontoxic) A. tamarense ribotypes, respectively. Detection was by fluorescence activated flow cytometric analysis. The FISH-FC method allowed effective discrimination between laboratory cultures of Group I and Group III ribotypes, with toxic and nontoxic cells creating distinct, easily identifiable, clusters in a flow cytometer bi-plot of side scatter (SSC) versus the green (FL1) fluorescence detection channel. Comparison of estimates of cell abundance obtained by the FISH-FC technique with those obtained by microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. Subsequently, the methodology was successfully applied on natural seawater samples, spiked with known concentrations of toxic and nontoxic A. tamarense cells at environmentally relevant concentrations. AcknowledgmentsThis work was funded by a NERC (CASE) studentship to LES at SAMS/UHI. KD was part funded by the Food Standards Agency (project FS241058), the EU FP7 project Asimuth, and the Culture Collection for Algae and Protozoa (CCAP). We thank the crew of the RV Seol Mara for their help with water collection. David Green, Sharon McNeill, Elaine Mitchell, and Mark Hart (SAMS) for their experience and expertise and Jean-Pierre Lacaze (Marine Scotland Science) for useful discussions.

Individual-based modelling of the development and transport of a Karenia mikimotoi bloom on the North-west European continental shelf

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Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species

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A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

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