Monitoring of HAB species in the Mediterranean Sea through molecular methods

Abstract

A qualitative and semi-quantitative polymerase chain reaction (PCR)-based assay was developed for the detection of several potentially Harmful Algal Bloom (HAB) species and genera belonging to Dinophyceae, Bacillariophyceae and Raphydophyceae. Oligonucleotide primers were designed based on Internal Transcribed Spacer (ITS)-5.8S ribosomal DNA (rDNA) sequences available in 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 [light microscope (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 taxon presence obtained by PCR assay compared with the microscopic examination ranged from 67 to 8.0%.