STRIVE - Environmental Protection Agency

Molecular Diversity and Dynamics of Toxigenic Blue-green Algae in Irish Lakes
3.6 Cyanotoxins Detection in West and
Northwest Irish Lakes
3.6.1 Detection of Cyanotoxin Genes in Field
Samples
The nucleic acid extracts obtained from the lakes
sampled were subject to PCR analysis to determine
the presence of toxigenic cyanobacteria potentially
synthesising cyanotoxins. PCR products were
successfully amplified for microcystin genes only.
Bands of the expected size (~300 bp) were observed for
18 out of 50 samples, and varied in intensity (Fig. 3.23).
Double digestion was carried out on the amplicons using
the restriction enzymes Rsa.I and Hha.l, producing a
number of RFLP patterns.
The restriction profiles were converted into a binary
matrix and subsequent cluster analysis defined three
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groups A, B and C of water bodies containing 7, 9
and 2 lakes, respectively (Fig. 3.24). Contingency
tables showed there were no significant relationships
between the clustering of lakes based on their PCR
restriction profiles and the time period during which
they were sampled, or their distribution in the three
identified longitudinal ecoregions (X < 6.11, p = 0.05,
df = 4, n = 18). However, focusing on the samples
of clusters A and B only, the cumulated abundances
of Anabaena and Oscillatoria were significantly
lesser in cluster B than in cluster A (Mann-Whitney,
p = 0.04, n = 16). This was further corroborated using
the DGGE-derived data pertaining to the diversity
and abundance of filamentous cyanobacteria
(Mann-Whitney, p = 0.001, n = 16). A reverse pattern
was found for spherical cyanobacteria but with no
statistical support (Mann-Whitney, p = 0.462, n = 16).
Figure 3.23. Mcy-A PCR and RFLP analysis of field sample extracts. 1% agarose gel showing the
mcy-A amplicons with lanes 1-8 corresponding to samples 0210-0213-0218-0219-0220-0221-0229-0233,
respectively (a). 2% agarose gel showing the restriction profiles obtained for samples 0233 (1), 0221 (2), 0229
(3), 0220 (4) and 0210 (5), respectively (b).