Molecular Phylogeny, Morphology, Growth and Toxicity of Three Benthic Dinoflagellates Ostreopsis sp. 9, Prorocentrum lima and Coolia monotis Developing in Strait of Gibraltar, Southwestern Mediterranean.

Few works have been carried out on benthic harmful algal blooms (BHAB) species in the southern Mediterranean and no data are available for the highly dynamic Strait of Gibraltar (western Mediterranean waters). For the first time, Ostreopsis sp. 9, Prorocentrum lima and Coolia monotis were isolated in this key region in terms of exchanges between the Atlantic Ocean and the Mediterranean and subject to intense maritime traffic. Ribotyping confirmed the morphological identification of these three dinoflagellates species. Monoclonal cultures were established and the maximum growth rate and cell yield were measured at a temperature of 24 °C and an irradiance of 90 µmol photons m-2 s-1, for each species: 0.26 ± 0.02 d-1 (8.75 × 103 cell mL-1 after 28 days) for Ostreopsis sp. 9, 0.21 ± 0.01 d-1 (49 × 103 cell mL-1 after 145 days) for P. lima and 0.21 ± 0.01 d-1 (10.02 × 103 cell mL-1 after 28 days) for C. monotis. Only P. lima was toxic with concentrations of okadaic acid and dinophysistoxin-1 measured in optimal growth conditions ranging from 6.4 pg cell-1 to 26.97 pg cell-1 and from 5.19 to 25.27 pg cell-1, respectively. The toxin content of this species varied in function of the growth phase. Temperature influenced the growth and toxin content of P. lima. Results suggest that future warming of Mediterranean coastal waters may lead to higher growth rates and to increases in cellular toxin levels in P. lima. Nitrate and ammonia affected the toxin content of P. lima but no clear trend was noted. In further studies, we have to isolate other BHAB species and strains from Strait of Gibraltar waters to obtain more insight into their diversity and toxicity.

New insights into the dynamics of causative dinoflagellates and the related contamination of molluscs by paralytic toxins in the southwestern Mediterranean coastal waters of Morocco.

The distribution of the two potentially toxic dinoflagellates Gymnodinium catenatum and Alexandrium spp. was investigated in the Mediterranean Moroccan Sea from March 2018 to March 2019. The cockle Acanthocardia tuberculata and the smooth clam Callista chione were collected at four stations, and their toxin levels were assessed using the mouse bioassay. The toxin profile was analysed by LC-MS/MS in G. catenatum and in the bivalves harvested in M'diq and Djawn. The species G. catenatum was present throughout the year, whereas Alexandrium spp. was less abundant. The paralytic shellfish toxin (PST) level in cockles was, on average, six times above the sanitary threshold; GTX5 was the major contributor to the total PST level, followed by dc-STX and STX. The toxin level of the smooth clam was considerably lower than that of the cockle; GTX5 and C-toxins were the dominating analogues. Our results suggest the responsibility of G. catenatum for the recurrent PST contamination in the Moroccan Mediterranean Sea, with a west-east gradient.

Development of harmful algal blooms species responsible for lipophilic and amnesic shellfish poisoning intoxications in southwestern Mediterranean coastal waters.

Mediterranean waters have undergone environmental changes during the last decades leading to various modifications of the structure of phytoplankton populations, especially Harmful Algal Blooms (HABs) species. Monitoring of the potentially toxic phytoplankton species was carried out biweekly in the western Mediterranean coast of Morocco from March 2018 to March 2019. Lipophilic Shellfish Toxins (LSTs) using LC-MS/MS and Domoic Acid (DA) using HPLC-UV were measured in the exploited mollusks, the cockle Acanthocardia tuberculata and the smooth clam Callista chione. We also determined the prevailing environmental factors in four surveyed sites (M'diq bay, Martil, Kaa Asras, and Djawn) selected to cover a variety of coastal ecosystems. Results showed that Pseudo-nitzschia spp. a DA producer species, was abundant with a pick of 50 × 103 cells l-1 on October 2018 in Djawn. Dinophysis caudata was the dominate Dinophysis species and showed a maximum density of 2200 cells l-1 on July in Djawn. Prorocentrum lima, an epibenthic dinoflagellate, appeared rarely in the water column with densities <80 cells l-1. Gonyaulax spinifera and Protoceratium reticulatum were found occasionally with a maximum density of 160 cells l-1. Karenia selliformis was detected only five times (<80 cells l-1) throughout the survey period. LC-MS/MS analyses revealed the presence of OA/DTX3, PTX-2, PTX-2 sa, and PTX-2 sa epi in the cockle at concentrations of up to 44.81 (OA/DTX-3+PTXs) ng g-1 meat. GYM-A was detected in the clam at concentrations of up to 4.22 ng g-1 meat. For the first time, AZAs and YTXs were detected in the southwestern Mediterranean with maximum values of 2.49 and 10.93 ng g-1 meat of cockle, respectively. DA was detected in moderate concentrations not exceeding 5.65 µg g-1 in both mollusks. Results showed that the observed toxic algae in the water column were responsible from the analysed toxins in the mollusks. It is likely that the southwestern Mediterranean waters could see the development of emergent species producing potent toxins (YTXs, AZAs, GYM-A). These dinoflagellates have to be isolated, ribotyped, and their toxin profiles determined.

Occurrence of three dominant epibenthic dinoflagellates (Ostreopsis spp., Coolia monotis and Prorocentrum lima) in relation to biotic substrates and environmental factors in a highly dynamic ecosystem, the Strait of Gibraltar (Southwestern Mediterranean).

No studies have been carried out on the benthic harmful algal blooms (BHABs) along the Strait of Gibraltar in the Mediterranean, and little is known about the diversity of blooming species. Here, epibenthic dinoflagellates were monitored at least biweekly over 18 months (May 2019-November 2020) in Oued Lihoud, Cap Malabata and Dalia on the thalli of five dominant macrophytes and in the water column. This is the first report on the seasonal distribution of BHAB species hosted by natural biotic substrates in the Strait of Gibraltar, which is known for high hydrodynamics, major entry of Atlantic waters and important maritime traffic. Three BHAB dinoflagellates were observed in the surveyed areas: Ostreopsis spp., Coolia monotis and Prorocentrum lima. The analysis of all data at the three sites showed that Dictyota dichotoma was the most favourable macroalgae host for these benthic dinoflagellates. The highest cell densities were observed in Cap Malabata for Ostreopsis spp. (2.7 × 105 cells/g fresh weight in September 2020), P. lima (4.57 × 104 cells/g FW in September 2020) and C. monotis (4.07 × 104 cells/g FW in June 2019). Phosphate and temperature were positively correlated to the abundances of the studied thermophilic BHAB species. In contrast, negative correlations were recorded with salinity, ammonium, nitrite, nitrate, DIN, nitrogen/phosphate ratio and suspended material, attesting of the complex relationships between environmental factors and BHAB species dynamic in each marine ecosystem. Toxin analyses of the natural phytoplankton assemblage during BHABs showed the presence of only lipophilic toxins, namely okadaic acid and dinophysistoxins produced by P. lima. These BHABs species have to be isolated to establish monoclonal cultures for ribotyping and ecophysiological investigations.