Seasonal production and molecular characterization of microcystins in Oneida Lake, New York, USA

Abstract

Oneida Lake, northeast of Syracuse, New York, in the United States, is a shallow eutrophic lake with a well-established toxic cyanobacterial population. Samples for DNA, toxin, and phycological analyses were collected from six stations throughout the summers of 2002 (78 samples) and 2003 (95 samples). DNA was amplified by PCR using primer sets specific to the nonribosomal microcystin synthetase complex (mcyB and mcyD). PCR analysis in 2002 indicated that the microcystin genes were present in the water column from mid-June through October, as 88% of the samples tested positive for mcyB and 79% of the samples tested positive for mcyD. In both years the onset of microcystin production was detected as early as mid-July by the protein phosphatase inhibition assay, reaching a maximum in 2002 of 2.9 microg L(-1) and in 2003 of 3.4 microg L(-1). Beginning in mid- to late August of both years the microcystin level at all six stations was in excess of the World Health Organization (WHO) advisory level of 1.0 microg L(-1). In the present study we compared microcystin occurrence and potential production at the six stations using protein phosphatase inhibition assay, high-performance liquid chromatography, and polymerase chain reaction analyses.

The occurrence of cyanobacterial toxins in New York lakes: Lessons from the MERHAB-Lower Great Lakes program

“…For DNA analysis, a 1-cm diameter subsample was taken from the original filter using a cork borer. DNA was extracted using a modified phenol:chloroform:isoamyl alcohol (25:24:1) protocol as described in Hotto et al (2005). DNA was amplified by PCR using 4 different primer sets, separated by 1.5% agarose gel electrophoresis and visualized using ethidium bromide.…”

“…Primer sequences and conditions used for PCR were specific to the cyanobacterial 16S rRNA (CYA), Microcystis spp. 16S rRNA (MIC), and two toxin biosynthetic genes (mcyB and mcyD) and are given in Hotto et al (2005).…”

Molecular Characterization of Potential Microcystin-Producing Cyanobacteria in Lake Ontario Embayments and Nearshore Waters

“…Multiple studies have focused on detection of the mcy genes using a PCR approach with species-specific primers based on differences within the mcy gene clusters (18,20,26,32,36,47) as well as universal primers targeting conserved sequences of the MC operon (14,17). The common mcy genes targeted are mcyA, -B, -C, -D, and -E, with many studies using a combination of the genes (15,26,27,33,44,48). Several studies have indicated that MC-producing Microcystis spp.…”

“…Extracts were clarified by centrifugation at 27,000 ϫ g followed by filtration through a 0.45-m nylon syringe filter and stored at Ϫ20°C. MC concentrations were determined using a PPIA, modified from Carmichael and An (7), run in 96-well plates (15). MC variants were identified from concentrated samples (10 to 20ϫ) by highperformance LC (Ace 5 C 18 column, 4.6 by 250 mm) with PDA and MS detectors using a gradient of 30 to 70% acetonitrile in water, both acidified to 0.1% (vol/vol) with trifluoroacetic acid.…”

“…DNA analysis. DNA was extracted from a 1.1-cm-diameter subsample of the original 47-mm filter using a protocol modified from Rudi et al (39) described previously (15). Briefly, filters were placed in Tris-EDTA buffer (pH 8.0) and digested with lysozyme and RNase A, followed by a proteinase K digestion.…”