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.

Modelling spatiotemporal distributions of Vulcanodinium rugosum and pinnatoxin G in French Mediterranean lagoons: Application to human health risk characterisation.

Consumption of seafood contaminated by phycotoxins produced by harmful algae is a major issue in human public health. Harmful algal blooms are driven by a multitude of environmental variables; therefore predicting human dietary exposure to phycotoxins based on these variables is a promising approach in health risk management. In this study, we attempted to predict the human health risks associated with Vulcanodinium rugosum and its neurotoxins, pinnatoxins (PnTXs), which have been regularly found in Mediterranean lagoons since their identification in 2011. Based on environmental variables collected over 1 year in four Mediterranean lagoons, we developed linear mixed models to predict the presence of V. rugosum and PnTX G contamination of mussels. We found that the occurrence of V. rugosum was significantly associated with seawater temperature. PnTX G contamination of mussels was highest in summer but persisted throughout the year. This contamination was significantly associated with seawater temperature and the presence of V. rugosum with a time lag, but not with dissolved PnTX G in seawater. By using the contamination model predictions and their potential variability/uncertainty, we calculated the human acute dietary exposures throughout the year and predicted that 25% of people who consume mussels could exceed the provisional acute benchmark value during the warmest periods. We suggest specific recommendations to monitor V. rugosum and PnTX G.

Artificial Substrates Coupled with qPCR (AS-qPCR) Assay for the Detection of the Toxic Benthopelagic Dinoflagellate Vulcanodinium rugosum.

Vulcanodinium rugosum is an emerging benthopelagic neuro-toxic dinoflagellate species responsible for seasonal Pinnatoxins and Portimines contaminations of shellfish and marine animals. This species is challenging to detect in the environment, as it is present in low abundance and difficult to be identified using light microscopy. In this work, we developed a method using artificial substrates coupled with qPCR (AS-qPCR) to detect V. rugosum in a marine environment. This sensitive, specific and easy-to-standardize alternative to current techniques does not require specialized expertise in taxonomy. After determining the limits and specificity of the qPCR, we searched for the presence of V. rugosum in four French Mediterranean lagoons using artificial substrates collected every two weeks for one year. The AS-qPCR method revealed its occurrences in summer 2021 in every studied lagoon and detected cells in more samples than light microscopy. As V. rugosum development induces shellfish contamination even at low microalga densities, the AS-qPCR method is accurate and relevant for monitoring V. rugosum in a marine environment.

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.

Liza ramada Juveniles after Exposure to the Toxic Dinoflagellate Vulcanodinium rugosum: Effects on Fish Viability, Tissue Contamination and Microalgae Survival after Gut Passage.

Pinnatoxins (PnTX) and Portimines (Prtn), two toxins produced by the benthic dinoflagellate Vulcanodinium rugosum, are known to be lethal to mice after intraperitoneal or oral administration. They are also known to accumulate in shellfish such as mussels and clams, but their effect on fish and the upper food chain remains unknown. In this work, juveniles of the fish Liza ramada (Mullet) were exposed to a strain of V. rugosum producing PnTX G and Prtn A. The fishes' viability and contamination were recorded at times interval. Results showed that L. ramada juveniles were able to feed on V. rugosum and that their tissues could be contaminated by PnTX G and Prtn A without impact on fish viability. Furthermore, the microalgae temporary cysts survived and germinated after fish gut passage. This study showed the potential of L. ramada to transfer PnTX and Prtn toxins to the upper food chain and to disseminate V. rugosum in environment.

Prediction of Alexandrium and Dinophysis algal blooms and shellfish contamination in French Mediterranean Lagoons using decision trees and linear regression: a result of 10 years of sanitary monitoring.

French Mediterranean lagoons are frequently subject to shellfish contamination by Diarrheic Shellfish Toxins (DSTs) and Paralytic Shellfish Toxins (PSTs). To predict the effect of various environmental factors (temperature, salinity and turbidity) on the abundance of the major toxins producing genera, Dinophysis and Alexandrium, and the link with shellfish contamination, we analysed a 10-year dataset collected from 2010 to 2019 in two major shellfish farming lagoons, Thau and Leucate, using two methods: decision trees and Zero Inflated Negative Binomial (ZINB) linear regression models. Analysis of these decision trees revealed that the highest risk of Dinophysis bloom events occurred at temperature <16.3°C and salinity <27.8, and of Alexandrium at temperature ranging from 10.4 to 21.5°C and salinity >39.2. The highest risk of shellfish contaminations by DSTs and PSTs occurred during the set of conditions associated with high risk of bloom events. Linear regression prediction enables us to understand whether temperature and salinity influence the presence of Alexandrium and affect its abundance. However, Dinophysis linear regression could not be validated due to overdispersion issues. This work demonstrates the tools which could help sanitary management of shellfish rearing areas.

Metal stresses modify soluble proteomes and toxin profiles in two Mediterranean strains of the distributed dinoflagellate Alexandrium pacificum.

HABs involving Alexandrium pacificum have been reported in metal-contaminated ecosystems, suggesting that this distributed species adapts to and/or can tolerate the effects of metals. Modifications in soluble proteomes and PST contents were characterized in two Mediterranean A. pacificum strains exposed to mono- or polymetallic stresses (zinc, lead, copper, cadmium). These strains were isolated from two anthropized locations: Santa Giusta Lagoon (Italy, SG C10-3) and the Tarragona seaport (Spain, TAR C5-4F). In both strains, metals primarily downregulated key photosynthesis proteins. Metals also upregulated other proteins involved in photosynthesis (PCP in both strains), the oxidative stress response (HSP 60, proteasome and SOD in SG C10-3; HSP 70 in TAR C5-4F), energy metabolism (AdK in TAR C5-4F), neoglucogenesis/glycolysis (GAPDH and PEP synthase in SG C10-3) and protein modification (PP in TAR C5-4F). These proteins, possibly involved in adaptive proteomic responses, may explain the development of these A. pacificum strains in metal-contaminated ecosystems. The two strains showed different proteomic responses to metals, with SG C10-3 upregulating more proteins, particularly PCP. Among the PSTs, regardless of the metal and the strain studied, C2 and GTX4 predominated, followed by GTX5. Under the polymetallic cocktail, (i) total PSTs, C2 and GTX4 reached the highest levels in SG C10-3 only, and (ii) total PSTs, C2, GTX5 and neoSTX were higher in SG C10-3 than in TAR C5-4F, whereas in SG C10-3 under copper stress, total PSTs, GTX5, GTX1 and C1 were higher than in the controls, revealing variability in PST biosynthesis between the two strains. Total PSTs, C2, GTX4 and GTX1 showed significant positive correlations with PCP, indicating that PST production may be positively related to photosynthesis. Our results showed that the A. pacificum strains adapt their proteomic and physiological responses to metals, which may contribute to their ecological success in highly anthropized areas.

Seasonal variations of phytoplankton assemblages in relation to environmental factors in Mediterranean coastal waters of Morocco, a focus on HABs species.

Studies on phytoplankton and in particular Harmful Algal Blooms (HABs) species in southern Mediterranean waters are scarce. We performed from April 2008 to June 2009 weekly investigations on microphytoplankton community structure and abundance in two contrasted marine ecosystems located in the western Moroccan Mediterranean coast, M'diq Bay and Oued Laou Estuary. Simultaneously, we measured the main physico-chemical parameters. Globally, the two studied areas showed comparable values of the assessed abiotic environmental factors. Temperature and salinity followed seasonal variation with values ranging from 13.5 °C to 21.4 °C and 31 to 36.8, respectively. Average nutrient values in surface water ranged from 0.7 to 45.76 µM for dissolved inorganic nitrogen, 0.02-2.10 µM for PO4 and 0.23-17.46 µM for SiO4 in the study areas. A total of 92 taxa belonging to 8 taxonomic classes were found. The highest number of microphytoplankton abundance reached 1.2 × 106 cells L-1 with diatoms being the most abundant taxa. Factorial Discriminant Analysis (FDA) and Spearman correlation test showed a significant seasonal discrimination of dominant microphytoplankton species. These micro-organisms were associated with different environmental variables, in particular temperature and salinity. Numerous HABs species were encountered regularly along the year. Although Dinophysis species and Prorocentrum lima were present in both sites, no Lipophilic Shellfish Poisoning was detected for the analyzed bivalve mollusks. Domoic acid (DA), produced by toxic species of Pseudo-nitzschia was found with concentrations up to 18 µg DA g-1 in the smooth clam Callista chione. Data showed that the observed persistent and dramatic Paralytic Shellfish Poisoning (PSP) intoxication of mollusks resulted probably of Gymnodinium catenatum proliferations in both studied areas. Contrary to C. chione, the cockle Achanthocardia tuberculatum showed a permanent and extremely high toxicity level during the 15 months survey with up to 7545 µg Equivalent Saxitoxin kg-1 flesh (ten times higher than the sanitary threshold of 800 µg eqSTX Kg-1flesh). The present work highlights for the first time the dynamic of microphytoplankton including HABs species and their associated toxin accumulation in the commercially exploited shellfish in the southern western Mediterranean waters of Morocco. Furthermore, the acquired data will help us to improve the monitoring of HABs species and related toxins in these coastal marine systems.

A novel index based on planktonic copepod reproductive traits as a tool for marine ecotoxicology studies.

Copepods are excellent bioindicators of climate change and ecosystem pollution in anthropized coastal waters. This work reviewed the results of previous studies examining changes in egg production rate (EPR), hatching success (HS), and nauplius survival rate (NSR) in natural conditions and in the presence of pollutants, including heavy metals and organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) and persistent organic pollutants (POPs). At high concentrations, cadmium and silver induce an increase in EPR in the copepods Acartia tonsa and Acartia hudsonica, while exposure to mercury decreases EPR in adults by 50%. All three metals affect HS, with mercury inducing a stronger effect than cadmium and silver. Cadmium affects reproductive traits in Centropages ponticus, decreasing EPR and particularly HS. Furthermore, copper and chromium at high concentrations induce significant decreases in eggs per female in Notodiaptomus conifer. In terms of organic contaminant and Polycyclic Aromatic Hydrocarbons (PAHs), Eurytemora affinis is reported to be affected by naphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, and 2,3,5-trimethylnaphthalene and can thus be used in ecotoxicity studies, but only if the exposure time is high. Acartia tonsa shows significant reductions in EPR and HS at high concentrations of fluoranthene, phenanthrene, and pyrene. However, the response to Persistent Organic Pollutants (POPs) such as pentachlorophenol (PCP) and 1,2-dichlorobenzene (DCB) differs. In E. affinis, EPR increases with DCB, but HS falls to <1%. EPR increases when the species is exposed overnight, but HS remains low in the presence of DCB. Based on these results, we developed a novel copepod reproductive trait index (CRT-Index) for use in marine ecotoxicology surveys and tested in some simple cases. We show that copepods are good candidates as models for ecotoxicology studies, in particular using reproductive traits (EPR, HS and NSR) because of their sensitivity to a wide range or pollutants.

Distribution of dinoflagellate cyst assemblages in recent sediments from a southern Mediterranean lagoon (Mellah, Algeria) with emphasis on toxic species.

This is the first study on the dinoflagellate cysts in Algerian waters and in Mellah Lagoon (South Western Mediterranean), located within a protected reserve. In total, 42 species of dinocysts belonging to 7 orders, 12 families and 23 genera, were identified in the 26 superficial sediment samples from Mellah Lagoon. The distribution of dinocysts in the sediment of this lagoon is heterogeneous. Indeed, their abundance oscillates between 1 and 315 cysts g-1 dry sediment (DS). Cyst morphotype assemblages were dominated by a few numbers of species: Alexandrium minutum (15.87%), Gonyaulax verior (9.81%), Protoperidinium spp. (7.74%), Alexandrium affine (7.05%), Scrippsiella trochoidea (6.67%), and Alexandrium pseudogonyaulax (6.19%). There is a positive correlation between the density of cysts and the depth (r = 0.61; p < 0.05), organic matter (r = 0.70; p < 0.05), water content (r = 0.71; p < 0.05), and the fine fraction of sediment (r = 0.74; p < 0.05). Surprisingly, although the Mellah Lagoon is almost semi-closed, it holds an important specific richness in dinocysts (42 species) higher than others observed in Mediterranean lagoons. However, cyst abundances are low compared to other lagoons in the Mediterranean Sea. Finally, the presence of dinocysts of Alexandrium catenella/tamarense, A. minutum, and Gymnodinium catenatum associated to paralytic shellfish toxins, A. pseudogonyaulax which produces goniodomin A, also Protoceratium reticulatum and Gonyaulax spinifera complex which produce yessotoxins, needs to implement a monitoring program to prevent a potential human intoxication due to the consumption of contaminated sea products by these potent neurotoxins.

Unsuspected intraspecific variability in the toxin production, growth and morphology of the dinoflagellate Alexandrium pacificum R.W. Litaker (Group IV) blooming in a South Western Mediterranean marine ecosystem, Annaba Bay (Algeria).

Physiological plasticity gives HABs species the ability to respond to variations in the surrounding environment. The aim of this study was to examine morphological and physiological variability in Alexandrium pacificum R.W. Litaker (Group IV) (former Alexandrium catenella) blooming in Annaba bay, Algeria. Monoclonal cultures of up to 30 strains of this neurotoxic dinoflagellate were established by the germination of single resting cysts from the surface sediment of this southern Mediterranean marine ecosystem. Ribotyping confirmed formally for the first time that A. pacificum is developing in Eastern Algerian waters. Toxin analyses of A. pacificum strains revealed substantial intraspecific variability in both the profile and toxin amount. However, the toxin profile of most strains is characterized by the dominance of GTX6 (up to 96 mol %) which is the less toxic paralytic molecule. The toxin concentrations in the isolated strains varied widely between 3.8 and 30.82 fmol cell-1. We observed an important variation in the growth rate of the studied A. pacificum strains with values ranging from 0.05 to 0.33 d-1. The lag time of the studied strains varied widely and ranged from 4 to 20 days. The intraspecific diversity could be a response to the selection pressure which may be exerted by different environmental conditions over time and which can be genetically and in turn physiologically expressed. This study highlights, for the first time, that the sediment of a limited area holds an important diversity of A. pacificum cysts which give when germinate populations with noticeable physiological plasticity. Consequently, this diversified natural populations allow an exceptional adaptation to specific environmental conditions to outcompete local microalgae and to establish HABs which could explain why this dinoflagellate is successful and expanding worldwide.

Intraspecific variability in membrane proteome, cell growth, and morphometry of the invasive marine neurotoxic dinoflagellate Alexandrium pacificum grown in metal-contaminated conditions.

Over the past decades, the occurrence, distribution and intensity of harmful algal blooms involving the dinoflagellate Alexandrium pacificum have increased in marine coastal areas disturbed by anthropogenic inputs. This invasive species produces saxitoxin, which causes the paralytic shellfish poisoning syndrome in humans upon consumption of contaminated seafood. Blooms of A. pacificum have been reported in metal-contaminated coastal ecosystems, suggesting some ability of these microorganisms to adapt to and/or resist in metal stress conditions. This study seeks to characterize the modifications in membrane proteomes (by 2-D electrophoresis coupled to LC-MS/MS), cell growth and morphometry (measured with an inverted microscope), in response to metal stress (addition of Zn2+, Pb2+, Cu2+ and Cd2+), in two Mediterranean A. pacificum strains: SG C10-3 and TAR C5-4F, respectively isolated from the Santa Giusta Lagoon (Sardinia, Italy) and from the Tarragona seaport (Spain), both metal-contaminated ecosystems. In the SG C10-3 cultures grown in a metal cocktail, cell growth was significantly delayed, and cell size increased (22% of 37.5 µm cells after 25 days of growth). Conversely, no substantial change was observed for cell growth or cell size in the TAR C5-4F cultures grown in a metal cocktail (P > 0.10), thus indicating intraspecific variability in the responses of A. pacificum strains to metal contamination. Regardless of the conditions tested, the total number of proteins constituting the membrane proteome was significantly higher for TAR C5-4F than for SG C10-3, which may help TAR C5-4F to thrive better in contaminated conditions. For both strains, the total number of proteins constituting the membrane proteomes was significantly lower in response to metal stress (29% decrease in the SG C10-3 proteome: 82 ± 12 proteins for controls, and 58 ± 12 in metal-contaminated cultures; 17% decrease in the TAR C5-4F proteome: 101 ± 8 proteins for controls, and 84 ± 5 in metal-contaminated cultures). Moreover, regardless of the strain, proteins with significantly modified expression in response to stress were mainly down-regulated (representing 45% of the proteome for SG C10-3 and 38% for TAR C5-4F), clearly showing the harmful effects of the metals. Protein down-regulation may affect cell transport (actin and phospholipid scramblase in SG C10-3), photosynthesis (RUBISCO in SG C10-3, light-harvesting protein in TAR C5-4F, and high-CO2-inducing periplasmic protein in both strains), and finally energy metabolism (ATP synthase in both strains). However, other modifications in protein expression may confer to these A. pacificum strains a capacity for adaptation and/or resistance to metal stress conditions, for example by (i) limiting the metal entry through the plasma membrane of the SG C10-3 cells (via the down-regulation of scramblase) and/or (ii) reducing the oxidative stress generated by metals in SG C10-3 and TAR C5-4F cells (due to down-regulation of ATP-synthase).

Occurrence of epibenthic dinoflagellates in relation to biotic substrates and to environmental factors in Southern Mediterranean (Bizerte Bay and Lagoon, Tunisia): An emphasis on the harmful Ostreopsis spp., Prorocentrum lima and Coolia monotis.

Harmful events associated with epibenthic dinoflagellates, have been reported more frequently over the last decades. Occurrence of potentially toxic benthic dinoflagellates, on the leaves of two magnoliophytes (Cymodocea nodosa and Zostera noltei) and thalli of the macroalgae (Ulva rigida), was monitored over one year (From May 2015 to April 2016) in the Bizerte Bay and Lagoon (North of Tunisia, Southern Mediterranean Sea). The investigated lagoon is known to be highly anthropized. This is the first report on the seasonal distribution of epibenthic dinoflagellates hosted by natural substrates, from two contrasted, adjacent coastal Mediterranean ecosystems. The environmental factors promoting the development of the harmful epibenthic dinoflagellates Ostreopsis spp., Prorocentrum lima and Coolia monotis were investigated. The highest cell densities were reached by Ostreopsis spp. (1.9 × 103 cells g-1 FW, in October 2015), P. lima (1.6 × 103 cells g-1 FW, in June 2015) and C. monotis (1.1 × 103 cells g-1 FW, in May 2015). C. nodosa and Z. noltei were the most favorable host macrophytes for C. monotis (in station L2) and Ostreopsis spp. (in station L3), respectively. Positive correlations were recorded between Ostreopsis spp. and temperature. Densities of the epibenthic dinoflagellates varied according to the collection site, and a great disparity was observed between the Bay and the Lagoon. Maximum concentrations were recorded on C. nodosa leaves from the Bizerte Bay, while low epiphytic cell abundances were associated with macrophytes sampled from the Bizerte Lagoon. The observed differences in dinoflagellate abundances between the two ecosystems (Bay-Lagoon) seemed not related to the nutrients, but rather to the poor environmental conditions in the lagoon.

Seasonal variations of phytoplankton community in relation to environmental factors in a protected meso-oligotrophic southern Mediterranean marine ecosystem (Mellah lagoon, Algeria) with an emphasis of HAB species.

The spatial and temporal variation of phytoplankton communities including HAB species in relation to the environmental characteristics was investigated in the protected meso-oligotrophic Mellah lagoon located in the South Western Mediterranean. During 2016, a biweekly monitoring of phytoplankton assemblages and the main abiotic factors were realized at three representative stations. Taxonomic composition, abundance, and diversity index were determined. In total, 227 phytoplankton species (160 diatoms and 53 dinoflagellates) were inventoried. There was a clear dominance of diatoms (62.9%) compared with dinoflagellates (36.8%). Diatoms dominated in spring and dinoflagellates developed in summer and early autumn in Mellah showing a marked seasonal trend. Data showed that the dynamic of the phytoplankton taxa evolving in the lagoon was mainly driven by temperature and salinity. For the first time, a number of potentially toxic species have been identified, including 2 diatoms (Pseudo-nitzschia group delicatissima, Pseudo-nitzschia group seriata) and 5 dinoflagellates (Alexandrium minutum, Alexandrium tamarense/catenella, Dinophysis acuminata, Dinophysis sacculus, Prorocentrum lima). These harmful species could threat the functioning of the Mellah lagoon and human health and require the establishment of a monitoring network. Finally, our study suggests that the observed decrease of the phytoplankton diversity between 2001 and 2016 could result from the reduction in water exchanges between the lagoon and the adjacent coast following the gradual clogging of the channel.

Oyster Farming, Temperature, and Plankton Influence the Dynamics of Pathogenic Vibrios in the Thau Lagoon.

Vibrio species have been associated with recurrent mass mortalities of juvenile oysters Crassostrea gigas threatening oyster farming worldwide. However, knowledge of the ecology of pathogens in affected oyster farming areas remains scarce. Specifically, there are no data regarding (i) the environmental reservoirs of Vibrio populations pathogenic to oysters, (ii) the environmental factors favoring their transmission, and (iii) the influence of oyster farming on the persistence of those pathogens. This knowledge gap limits our capacity to predict and mitigate disease occurrence. To address these issues, we monitored Vibrio species potentially pathogenic to C. gigas in 2013 and 2014 in the Thau Lagoon, a major oyster farming region in the coastal French Mediterranean. Sampling stations were chosen inside and outside oyster farms. Abundance and composition of phyto-, microzoo-, and mesozooplankton communities were measured monthly. The spatial and temporal dynamics of plankton and Vibrio species were compared, and positive correlations between plankton species and vibrios were verified by qPCR on isolated specimens of plankton. Vibrio crassostreae was present in the water column over both years, whereas Vibrio tasmaniensis was mostly found in 2013 and Vibrio aestuarianus was never detected. Moreover, V. tasmaniensis and V. crassostreae were found both as free-living or plankton-attached vibrios 1 month after spring mortalities of the oyster juveniles. Overall, V. crassostreae was associated with temperature and plankton composition, whereas V. tasmaniensis correlated with plankton composition only. The abundance of Vibrio species in the water column was similar inside and outside oyster farms, suggesting important spatial dispersion of pathogens in surrounding areas. Remarkably, a major increase in V. tasmaniensis and V. crassostreae was measured in the sediment of oyster farms during cold months. Thus, a winter reservoir of pathogenic vibrios could contribute to their ecology in this Mediterranean shellfish farming ecosystem.

What are the main environmental factors driving the development of the neurotoxic dinoflagellate Vulcanodinium rugosum in a Mediterranean ecosystem (Ingril lagoon, France)?

Vulcanodinium rugosum, a dinoflagellate developing in Ingril Lagoon (Mediterranean, France) is responsible for shellfish intoxications due to the neurotoxin pinnatoxin G. A one year survey (March 2012-April 2013) was conducted in this oligotrophic shallow lagoon and key environmental parameters were recorded (temperature, salinity and nutrients). The spatio-temporal distribution of V. rugosum in water column and on macrophytes was also determined. Planktonic cells of V. rugosum were observed at all sampling stations, but in relatively low concentrations (maximum of 1000 cell/L). The highest abundances were observed from June to September 2012. There was a positive correlation between cell densities and both temperature and salinity. Non-motile cells were detected on macrophytes, with a maximum concentration of 6300 cells/g wet weight. Nitrite and ammonium were negatively related to V. rugosum abundance whereas total nitrogen, total phosphorus and phosphates showed a positive correlation. Altogether, in situ results suggest that V. rugosum is rather thermophilic and that organic nutrients should be considered when studying the nutrition requirements for this noxious expanding dinoflagellate.

Effects of copper and butyltin compounds on the growth, photosynthetic activity and toxin production of two HAB dinoflagellates: The planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata.

Controlled laboratory experiments were conducted to test the effects of copper (Cu2+) and butyltins (BuT) on the growth, photosynthetic activity and toxin content of two HABs (Harmful Algal Blooms) dinoflagellates, the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata. Microalgae were exposed to increasing concentrations of Cu2+ (10-4 to 31 nM) or BuT (0.084 to 84 nM) for seven days. When considering the growth, EC50 values were 0.16 (±0.09) nM and 0.03 (±0.02) nM of Cu2+ for A. catenella and O. cf. ovata, respectively. Regarding BuT, EC50 was 14.2 (±6) nM for O. cf. ovata, while A. catenella growth inhibition appeared at BuT concentrations ≥27 nM. Photosynthetic activity of the studied dinoflagellates decreased with increasing Cu and BuT concentrations. For O. cf. ovata, the response of this physiological parameter to contamination was less sensitive than the biomass. Cu exposure induced the formation of temporary cysts in both organisms that could resist adverse conditions. The ovatoxin-a and -b concentrations in O. cf. ovata cells increased significantly in the presence of Cu. Altogether, the results suggest a better tolerance of the planktonic A. catenella to Cu and BuT. This could result in a differentiated selection pressure exerted by these metals on phytoplankton species in highly polluted waters. The over-production of toxins in response to Cu stress could pose supplementary health and socio-economic threats in the contaminated marine ecosystems where HABs develop.

New insights on the species-specific allelopathic interactions between macrophytes and marine HAB dinoflagellates.

Macrophytes are known to release allelochemicals that have the ability to inhibit the proliferation of their competitors. Here, we investigated the effects of the fresh leaves of two magnoliophytes (Zostera noltei and Cymodocea nodosa) and thalli of the macroalgae Ulva rigida on three HAB-forming benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima, and Coolia monotis). The effects of C. nodosa and U. rigida were also tested against the neurotoxic planktonic dinoflagellate Alexandrium pacificum Litaker sp. nov (former Alexandrium catenella). Co-culture experiments were conducted under controlled laboratory conditions and potential allelopathic effects of the macrophytes on the growth, photosynthesis and toxin production of the targeted dinoflagellates were evaluated. Results showed that U. rigida had the strongest algicidal effect and that the planktonic A. pacificum was the most vulnerable species. Benthic dinoflagellates seemed more tolerant to potential allelochemicals produced by macrophytes. Depending on the dinoflagellate/macrophyte pairs and the weight of leaves/thalli tested, the studied physiological processes were moderately to heavily altered. Our results suggest that the allelopathic activity of the macrophytes could influence the development of HAB species.