First Characterization of Protoceratium reticulatum (Dinophyceae) from Atlantic coast of Morocco: Growth experiment, molecular identification, and toxicity

Protoceratium reticulatum is a dinoflagellate recognized as a producer of yessotoxin and its analogues. It grows over a wide range of temperature, irradiance, nutrients, salinity, and pH. There is limited research on toxic dinoflagellates in Moroccan coastal areas. Therefore, for the first time, P. reticulatum was isolated from the Agadir coast in the Imiouaddar shellfish growing area (Morocco, Atlantic), and monoclonal cultures were established. For growth experiments, cultures were exposed to three temperatures (15, 20, and 25 °C), three levels of irradiance (20, 80, and 130μmol m−2 s−1), and five concentrations of nitrate/phosphate concentrations of L1 culture medium (L1, 1.5 × L1, 2 × L1, 2.5 × L1, 3 × L1). Molecular analysis of the 18S rDNA gene confirmed the identity of our P. reticulatum strain. The strain was grown only at 20 °C. Temperatures of 15 and 25 °C induced cell death. Different levels of irradiance and nutrients did not influence its maximum growth rate. However, the 3 × L1 culture medium significantly impacted maximum cell density. Despite the combination of all the more or less optimal parameters (20 °C, 80μmol m−2 s−1, L1 medium), the maximum growth rate remained low: 0.29 div. day−1. The cell lysate of P. reticulatum significantly affected the survival of Artemia salina nauplii. The analysis of toxins by liquid chromatography with tandem mass spectrometry (LC-MS/ MS) of P. reticulatum culture extract revealed that the species was able to produce yessotoxin at a concentration of 27.0 ± 2.60 pg YTX cell−1. Until now, no bloom of P. reticulatum has been reported on the Atlantic coast of Agadir. However, in Moroccan coastal waters, an “early warning” system has already been established to better control shellfish growing areas in the event of a bloom of this species.