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JPR Advance Access published online on October 4, 2006
Journal of Plankton Research, doi:10.1093/plankt/fbl053
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received February 15, 2006
Accepted September 27, 2006

Article

Monitoring of HAB species in the Mediterranean Sea through molecular methods

Antonella Penna 1 *, Elena Bertozzini 1, Cecilia Battocchi 1, Luca Galluzzi 2, Maria Grazia Giacobbe 3, Magda Vila 4, Esther Garces 5, Antonella Lugliè 6, and Mauro Magnani 7

1 Centro Biologia Ambientale, University of Urbino, 61100 Pesaro, Italy
2 Center of Biotechnology, University of Urbino, 61032 Fano, Italy
3 CNR-IAMC, Istituto per l’Ambiente Marino Costiero, 98122 Messina, Italy
4 Institut de Ciències del Mar-CSIC, 08003 Barcelona, Spain
5 IRTA, Institut de Recerca i Tecnologia Agroalimentaries-Centre d'Aqüicultura, 43540 Sant Carles de la Ràpita, Spain
6 DBEV, Dip. Botanica ed Ecologia Vegetale, University of Sassari, 07100 Sassari, Italy
7 Istituto Chimica Biologica, University of Urbino, 61029 Urbino, Italy

* To whom correspondence should be addressed.
Antonella Penna, E-mail: a.penna{at}uniurb.it


  Abstract

A qualitative and semi-quantitative PCR based assay was developed for the detection of several potentially HAB species and genera belonging to Dinophyceae, Bacillariophyceae and Raphydophyceae. Oligonucleotide primers were designed based on ITS-5.8S rDNA sequences available on public database or identified in this study. The specificity and sensitivity of the PCR assay were validated using clonal cultures and then natural seawater samples, as well as the known copy number of plasmids containing the target ITS-5.8S rDNA regions. A filter system for collecting mixed phytoplankton cells coupled to a target species-specific PCR assay was performed on spatial and temporal series of net and surface seawater samples during coastal water monitoring carried out in several localities of the Mediterranean Sea. The application of PCR allowed a rapid detection of various genera and species-specific potential HAB taxa in all examined natural samples. Qualitative and semi-quantitative PCR results obtained from field samples were compared with microscopic (LM) examinations. The two methods gave comparable results and the molecular assay was able to detect HAB target cells at concentrations not detectable by microscopy or those of uncertain identity. The highest values of positive detection of potential HAB taxa presence obtained by PCR assay compared to the microscopic examination ranged from 67 to 8.0%.

Communicating Editor: KJ Flynn


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