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Abstract

This work focuses on a group of diatoms, belonging to the genus Pseudo-nitzschia, which is a globally important phytoplankton genus. Some species of the genus produce the toxin domoic acid, which is responsible for shellfish intoxication known as the amnesic shellfish poisoning (ASP). These diatoms have few morphological features that allow us to tell them apart, although more than 50 species are known. This work is the first attempt to discern the diversity of these diatoms in the Gulf of Trieste to which Slovenian waters also belong. Here, species of the genus Pseudo-nitzschia are common members of the local phytoplankton community, but we were mostly clueless as to which species occur here and when. This knowledge is important for the study of the ecology of the phytoplankton community but also to understand the potential threats from ASP in the area. Therefore, we first determined the species diversity of the genus using morphological methods, including electron microscopy, and genetic barcoding with three different markers (28S, ITS1 and ITS2, rbcL) on environmental isolates that were cultured over a four-year period. Then, we examined the toxicity potential of these cultures using both analytical methods (HPLC, LC-MS/MS) and ELISA assays. We also partnered with a French biotechnological company Microbia Environnement, with whom we developed a colorimetric sandwich-hybridization assay to study and monitor the genus in a more efficient and less costly way. Finally, we introduced the metabarcoding methodology to the study of phytoplankton to analyse the phytoplankton community including Pseudo-nitzschia with a state-of-the-art and high-throughput methodology that avoids the human factor in taxonomy and abundance assessment. In total, 9 species along with their seasonal occurrence patterns were identified from October 2016 to October 2020. Ecological associations with these species were also revealed through the study. Three of these species (P. multistriata, P. delicatissima, P. galaxiae) were found to produce domoic acid in culture, with P. galaxie the first time in the Adriatic. The development of the colorimetric assay yielded a set of nucleotide probes used with the assay that work best in easily and efficiently evaluating the status of the Pseudo-nitzschia population in the environment. A probe specific for P. galaxiae was also found and the assay setup with this probe accurately predicted the population dynamic of this species. Finally, the metabarcoding approach revealed a previously unknown diversity of phytoplankton groups, including a species of Pseudo-nitzschia that was not identified from the culturing effort. This work is the first to reveal the diversity and ecology of Pseudo-nitzschia in the Gulf of Trieste and one of the most robust to date in the wider Adriatic area, since it combines several morphological and molecular methods, which is essential in a group of organisms, where the concept of species is not well defined. Our work on the colorimetric assay resulted in a new tool that is cost-efficient and can be used in monitoring as well as aquaculture operations to help farmers assess the threat level from potential ASP poisoning of their product. It is also one of the first efforts to employ metabarcoding for phytoplankton in the Adriatic, and to use rbcL as a metabarcoding primer for diatoms in the marine environment. The results of this work have greatly improved our understanding of the local phytoplankton community with globally important implications and findings and will help guide further research into this topic.