Molecular discrimination of toxic and non-toxicAlexandriumspecies (Dinophyta) in natural phytoplankton assemblages from the Scottish coast of the North Sea

Abstract

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were speciesspecific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermo¨hl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Growth and bioactive secondary metabolites of arctic Protoceratium reticulatum (Dinophyceae)

“…Genomic DNA was extracted from cell pellets using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions as described in detail by Toebe et al (2013). From the resultant DNA the D1/D2 hypervariable region of the large sub-unit (LSU) of the ribosomal operon was PCR amplified and Sanger sequenced with the primers D1R (forward) and D2C (reverse) (Scholin et al, 1994) and PCR chemistry, cycling and sequencing conditions as in Akselman et al (2015).…”

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

“…John et al, 2005) or recently developed ribotype-specific real time-quantitative PCR (RT-qPCR) techniques (e.g. Toebe et al, 2013).…”

“…The LSU rDNA sequences of all Alexandrium tamarense and Alexandrium tamutum isolates and their phylogenetic position were determined after extracting genomic DNA from cell pellets with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and as detailed in Toebe et al (2013). Genomic DNA was used to amplify the D1/D2 hypervariable region of the LSU of the ribosomal operon by PCR with primers D1R (forward) and D2C (reverse) under PCR chemistry and cycling conditions as in Toebe et al (2013) Table 2).…”

“…Genomic DNA was used to amplify the D1/D2 hypervariable region of the LSU of the ribosomal operon by PCR with primers D1R (forward) and D2C (reverse) under PCR chemistry and cycling conditions as in Toebe et al (2013) Table 2). Both alignments were generated in MAFFT v7.017 (Katoh and Kuma, 2002) using a plugin for Geneious Pro.…”

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

“…A sandwich hybridization assay, involving cell lysis with two hybridization reactions, has proved useful in obtaining near real-time mapping of the distribution of Alexandrium species when used onboard a ship . Quantitative real-time PCR (qRT-PCR) can provide accurate and reproducible quantification of gene copy formation during exponential phase of the reaction Toebe et al, 2013). Further advances have led to the development of DNA-biosensors for electrochemical detection of phytoplankton and their toxins .…”