Preprint volume - SIBM

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PLANKTON COMMITEE
using eukaryotic primers targeting the ITS-5.8S gene followed by a second PCR using
genus- or species-specific primers (i.e. nested PCR) as described in Battocchi et al.
(2010).
Results – All the environmental samples analyzed contained mixed microphytobenthic
assemblages including target taxa. The PCR method detected the presence of
Ostreopsis spp. cells even when target cells were not observed by microscopy
examination. The positive detection by PCR assay was higher than microscopy
determinations by 19% for the macrophye samples and 32% for the net and surface
seawater samples. Moreover, the PCR-based assay identified the two species O. ovata
and O. cf. siamensis, while species-specific identification of Ostreopsis cells was quite
difficult using LM microscopy. Using the PCR based assay, O. ovata cells were
detected at a rate of 67% and 66% in macrophyte and seawater samples, respectively,
and O. cf. siamensis cells were detected at the lower rates of 16% and 29%,
respectively (Fig. 1).
100%
80%
60%
40%
20%
0%
a) b) c)
Macrophyte
samples
Sea water
samples
%-PCR/-Micro
%-PCR/+Micro
%+PCR/-Micro
%+PCR/+Micro
100%
80%
60%
40%
20%
0%
O. ovata O. cf. siamensis
Fig. 1. a) Analyses of positive and negative PCR amplifications of Ostreopsis spp. compared with the
corresponding positive and negative microscopy analyses of the environmental samples
collected in 2007. PCR analysis of macrophyte (b), and net and surface seawater (c) samples
for detecting O. ovata and O. cf. siamensis. Data are expressed as percentages of the total PCR
positive and negative determinations.
a) Confronto dei dati di positività e negatività ottenuti con le due diverse tecniche applicate a
campioni ambientali raccolti nel 2007. Analisi di PCR dei campioni di macrofite (b) e retinate
e acqua di mare superficiale (c) per l’identificazione di O. ovata e O. cf. siamensis. I dati sono
espressi come percentuale sul totale dei campioni analizzati.
Conclusions –The PCR technique developed in this study efficiently identified both
species of Ostreopsis and it was more sensitive in detecting Ostreopsis spp. than
microscopy analyses. As for the statistical evidence, the proportions of false negatives
by microscopy relative to PCR-based data were found to be significantly larger than
the expectation under the hypothesis of equal power for microscopy and PCR-based
identification, thus showing that the latter method is significantly more reliable.
References
BATTOCCHI C., TOTTI C., VILA M., MASÓ M., CAPELLACCI S., ACCORONI S., REÑÉ A.,
SCARDI M., PENNA A. (2010) - Monitoring toxic microalgae Ostreopsis (dinoflagellate)
species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined
with light microscopy. Mar. Pollut. Bull. (in press).
TOTTI C., ACCORONI S., CERINO F., CUCCHIARI E., ROMAGNOLI T (2010) – Ostreopsis
ovata bloom along the Conero Riviera (northern Adriatic Sea): relationship with environmental
conditions and substrata. Harmful Algae, 9: 233-239.
41 st S.I.B.M. CONGRESS Rapallo (GE), 7-11 June 2010
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100%
80%
60%
40%
20%
0%
O. ovata O. cf. siamensis
-PCR/-Micro
+PCR/-Micro