Molecular monitoring of Dinophysis species assemblage in mussel farms in the Northwestern Adriatic Sea

Several Dinophysis species can produce potent lipophilic toxins that pose a risk to human health when contaminated seafood is consumed, especially filter-feeding bivalve mussels. In the mussel farms of the Northwestern Adriatic Sea, seawater and seafood are regularly monitored for the presence of Dinophysis species and their associated toxins, but the current methodological approaches, such as light microscopy determinations, require a long time to make results available to local authorities. A molecular qPCR-based assay can be used to quantify various toxic Dinophysis species in a shorter timeframe. However, this approach is not currently employed in official testing activities. In this study, field samples were collected monthly or bi-weekly over one year from various mussel farms along the Northwestern Adriatic coast. The abundance of Dinophysis species in the seawater was determined using both traditional microscopy and qPCR assays. In addition, the concentration of lipophilic toxins for DSP in mussel flesh was quantified using LC-MS/MS focusing on the okadaic acid group. Dinophysis spp. site-specific single cells were isolated and analysed by qPCR yielding a mean rDNA copy number per cell of 1.21 × 10⁴ ± 1.81 × 10³. The qPCR assay gave an efficiency of 98 % and detected up to 10 copies of the rDNA target gene. The qPCR and light microscopy determinations in environmental samples showed a significant positive correlation (Spearman r_s = 0.57, p-value < 0.001) with a ratio of 2.24 between the two quantification methods, indicating that light microscopy estimates were generally 44.6 % lower than those obtained by the qPCR assay. The qPCR approach showed several advantages such as rapidity, sensitivity and efficiency over conventional microscopy analysis, showing its potential future role in phytoplankton monitoring under the Official Controls Regulations for shellfish.