Toxin-producing phytoplankton monitoring in the Adriatic Sea: microscopy vs molecular methods

Abstract Background Climate change is likely shifting the dynamics of phytoplankton, critical for mussel farming. Monitoring toxic microalgae is key for seafood safety, as their biotoxins can accumulate in shellfish and harm human health. While microscopy traditional method is used, molecular techniques such as Real-Time PCR and eDNA metabarcoding could offer better detection. This study combines these methods to monitor toxin-producing phytoplankton in the Adriatic Sea. Methods A monitoring program (2023-2024) analyzed 271 water samples from Ferrara, Forlì-Cesena, Ravenna, and Rimini Province (Emilia-Romagna Region) using microscopy (Utermöhl technique) and Real-Time PCR. DNA extraction was done with the DNeasy®PowerWater® kit (Qiagen), followed by PCR on Bio-Rad CFX96 instruments. The phytoplankton community of the LTER-SG01 station (located along the Senigallia-Susak transect, Marche Region) was analysed combining microscopy (Utermöhl technique) and eDNA metabarcoding (targeting V4 and V9 regions of the 18S rRNA gene, and using PR2 and SILVA as reference databases). Results In 2023, Alexandrium spp. peaked in May and June (10^2-10^3cells/L), Dinophysis spp. was sporadic in June (10^2cells/L), and Pseudo-nitzschia spp. was year-round (10^5-10^6cells/L). In 2024, blooms of Dinophysis spp. began earlier, from March (10^2cells/L). Real-Time PCR detected 12% more positives for Alexandrium spp. and Dinophysis spp. than microscopy, and it was less effective for Pseudo-nitzschia spp.. Metabarcoding method revealed greater phytoplankton diversity. Only a small overlap existed between microscopy and metabarcoding methods and between different genetic markers and databases. Conclusions Combining microscopy, Real-Time PCR, and eDNA metabarcoding methods is key to accurately monitor harmful species and ensure seafood safety. Integrated monitoring is crucial for safeguarding aquaculture and public health from toxin-producing phytoplankton. Key messages • Integrated monitoring using microscopy, PCR, and eDNA metabarcoding enhances detection of harmful microalgae. • Climate change is influencing the timing of harmful algal blooms, increasing risks to seafood safety.