Gonyaulax spinifera from the Adriatic sea: toxin production and phylogenetic analysis

The north-western coasts of Adriatic sea have long been subjected to recurring cases of harmful algae blooms and concomitant mussel contamination. In 1995, yessotoxins (YTXs) were detected for the first time in Adriatic mussels and their presence was subsequently linked to the presence of Protoceratium reticulatum and occasionally Lingulodinium polyedrum in seawater. Since then, farmed molluscs are yearly found to contain YTXs for long periods with a severe impact on aquaculture activities. In 2004, unusually high amounts of homoYTXs were detected in mussels. In this period, a careful look at the phytoplankton composition highlighted that P. reticulatum and L. polyedrum were nearly absent in seawater whereas the dinoflagellate Gonyaulax spinifera was present at high densities. G. spinifera has been recently associated with YTX production in New Zealand. The present work was performed in order to ascertain whether Adriatic G. spinifera was a toxic species. Determination of the toxin content of natural samples (mussels and net haul plankton samples) as well as monitoring of the dinoflagellate species present in seawaters allowed us to correlate the presence of G. spinifera to that of homoYTX in mussels. Cultures of G. spinifera were set up which gave us the opportunity to investigate toxin content and profile of the dinoflagellate, for the first time, by liquid chromatography-mass spectrometry (LC-MS) and to perform a molecular characterization of the species through rDNA sequencing. Two G. spinifera isolates, sampled in different years, were analyzed and resulted to be different in both toxin content and gene sequence: the isolate sampled in 2005 produced only low levels of YTX (5.4 pg cell-1) while that sampled in 2006 contained higher toxin amounts, namely 33.4 pg cell-1 of homo-YTX and 3.6 pg cell-1 of YTX; the two isolates also differed in 111 and 176 nucleotide positions, respectively, in the SSU and partial LSU rDNA genes. Comparing similar sequences of G. spinifera strains contained in GenBank, divergences among the Adriatic strains and strains of different geographical origin emerged; on the contrary, a good similarity was evidenced (1.3%) between the Adriatic isolate sampled in 2006 and two New Zealand strains; the similarity was confirmed also by the presence in all strains of YTXs, although the New Zealand strains were analyzed by ELISA which could not distinguish among the different YTX analogues. Due to an originally confusing description of the species and to the availability of very low number of deposited sequences the doubt arise whether the studied G. spinifera strains belong to the same species. However, although the taxonomy of the species complex G. spinifera remains largely unresolved, this study allowed to unambiguously link the presence of G. spinifera to recurring mussel toxicity episodes.