Preprint volume - SIBM

Pre-print Volume – Posters
PLANKTON COMMITEE
C. BATTOCCHI, C. TOTTI 1 , M. VILA 2 , M. MASÒ 2 , S. CAPELLACCI, S. ACCORONI 1 ,
A. RENÉ 2 , M. SCARDI 3 , A. PENNA
Dep. of Biomolecular Science, University of Urbino, Viale Trieste, 296 - 61121 Pesaro, Italia.
c.battocchi@campus.uniurb.it
1 DISMAR, Università Politecnica delle Marche, Ancona, Italia.
2 ICM-CSIC, Barcelona, Spain.
3 Dep. of Biology, University of Roma “Tor Vergata”, Italia.
MONITORING OF TOXIC MICROALGA
OSTREOPSIS (DINOFLAGELLATE) SPECIES
IN MEDITERRANEAN COASTAL WATERS USING
THE PCR BASED-ASSAY COMBINED WITH LIGHT MICROSCOPY
MONITORAGGIO DELLA MICROALGA POTENZIALMENTE TOSSICA
OSTREOPSIS (DINOFLAGELLATA) IN AREE COSTIERE MEDITERRANEE
CON IL METODO MOLECOLARE DI PCR E MICROSCOPIA OTTICA
Abstract – A molecular PCR based-assay was developed and applied to macrophyte and seawater samples
containing mixed microphytobenthic and phytoplanktonic assemblages in order to detect toxic Ostreopsis
species in the Mediterranean Sea. The PCR allowed rapid detection of Ostreopsis cells, even if their
abundances are below the light microscopy’s detection limit. Species-specific identification was possible
only by PCR-based assay, due to the inherent difficulty of morphological identification in field samples.
During the monitoring of the toxic Ostreopsis blooms PCR based methods proved to be effective tools
complementary to microscopy for rapid and specific detection of Ostreopsis in marine coastal waters.
Key-words: coastal waters, Mediterranean Sea, monitoring, Ostreopsis, PCR.
Introduction – Harmful Algal Blooms (HABs) occur frequently in coastal waters
throughout the world causing negative impact on environmental quality, human health
and economical activities. HAB species include Ostreopsis, a benthic/epiphytic genus
known to produce palytoxin-like compounds. Two Ostreopsis species, as O. ovata and
O. cf. siamensis, are being found with increasing frequency in the Mediterranean Sea
(Battocchi et al., 2010; Totti et al., 2010). Correct identification of these two species is
quite difficult by microscopy analyses but it is particular important given that the two
species can produce different toxins, which are a potential risk to humans and other
organisms. In this study, an efficient PCR based assay was applied to environmental
samples in order to monitor the presence of Ostreopsis species in Mediterranean
coastal areas, and to compare molecular data with microscopy determinations.
Materials and methods – A total of 125 samples of macrophytes, net and surface
water samples were collected from April to November 2007 at 20 sites distributed in
northern Adriatic Sea and Catalan Sea where blooms of Ostreopsis have never been
detected before or commonly occur, respectively. Subsamples were settled for 24h in
Sedgwick-Rafter or Utermöhl chambers and Ostreopsis spp. were counted under
inverted microscopes (Axiovert 40 CFL and Axiovert 135H, Zeiss, Germany or a Leitz
DM-II, Germany) at 200× or 400× magnification on the half or entire sedimentation
chamber. Pellets obtained from macrophyte and seawater samples were used for total
DNA extraction and purification according to Battocchi et al. (2010). Genus and
species-specific primers were designed in the 5.8S rDNA-ITS regions. PCR reactions
were carried out directly using these primers or by following two steps: an initial PCR
41 st S.I.B.M. CONGRESS Rapallo (GE), 7-11 June 2010
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