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.

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.

First Characterization of Ostreopsis cf. ovata (Dinophyceae) and Detection of Ovatoxins during a Multispecific and Toxic Ostreopsis Bloom on French Atlantic Coast.

Blooms of the benthic toxic dinoflagellate genus Ostreopsis have been recorded more frequently during the last two decades, particularly in warm temperate areas such as the Mediterranean Sea. The proliferation of Ostreopsis species may cause deleterious effects on ecosystems and can impact human health through skin contact or aerosol inhalation. In the eastern Atlantic Ocean, the toxic O. cf. ovata has not yet been reported to the north of Portugal, and the only species present further north was O. cf. siamensis, for which the toxic risk is considered low. During summer blooms of unidentified Ostreopsis species on the French Basque coast (Atlantic) in 2020 and 2021, people suffered from irritations and respiratory disorders, and the number of analyzed cases reached 674 in 2021. In order to investigate the causes, sampling was carried out during summer 2021 to (i) taxonomically identify Ostreopsis species present using a molecular approach, (ii) isolate strains from the bloom and culture them, and (iii) characterize the presence of known toxins which may be involved. For the first time, this study reports the presence of both O. cf. siamensis and O. cf. ovata, for which the French Basque coast is a new upper distribution limit. Furthermore, the presence of ovatoxins a, b, c, and d in the environmental sample and in a cultivated strain in culture confirmed the toxic nature of the bloom and allowed identifying O. cf. ovata as the producer. The present data identify a new health risk in the area and highlight the extended distribution of some harmful dinoflagellates, presumably in relation to climate change.

Toxicity of palytoxin, purified ovatoxin-a, ovatoxin-d and extracts of Ostreopsis cf. ovata on the Caco-2 intestinal barrier model.

Human intoxications in the Mediterranean Sea have been linked to blooms of the dinoflagellate Ostreopsis cf. ovata, producer of palytoxin (PlTX)-like toxins called ovatoxins (OVTXs). Exposure routes include only inhalation and contact, although PlTX-poisoning by seafood has been described in tropical regions. To address the impact of OVTXs on the intestinal barrier, dinoflagellate extracts, purified OVTX-a and -d and PlTX were tested on differentiated Caco-2 cells. Viability, inflammatory response and barrier integrity were recorded after 24 h treatment. OVTX-a and -d were not cytotoxic up to 20 ng/mL but increased IL-8 release, although to a lesser extent compared to PlTX. While PlTX and OVTX-a (at 0.5 and 5 ng/mL respectively) affected intestinal barrier integrity, OVTX-d up to 5 ng/mL did not. Overall, OVTX-d was shown to be less toxic than OVTX-a and PlTX. Therefore, oral exposure to OVTX-a and -d could provoked lower acute toxicity than PlTX.

Two toxigenic Ostreopsis species, O. cf. ovata and O. siamensis (Dinophyceae), from the South China Sea, tropical Western Pacific.

In the dinophyte genus Ostreopsis, seven out of 11 described species are known to produce various toxic compounds that were characterized in the palytoxins family. Species in the genus shared identical thecal plate patterns but differed in size, shape, and thecal plate ornamentation. Two species, O. cf. ovata and O. siamensis, have been reported from the Western Pacific, but information on toxin production is scarce. Here, we established nine strains of Ostreopsis from six localities in the South China Sea (SCS), covering the Gulf of Thailand, northern SCS (Hainan Island, Beibu Bay), and southern SCS (Peninsular Malaysia). Their morphology was examined by light and electron microscopy and the molecular phylogeny was inferred based on the LSU rDNA (D1-D3) and ITS rDNA sequences using maximum likelihood and Bayesian inference. Both O. cf. ovata and O. siamensis, albeit morphologically closely related, can be distinguished by a feature of the thecal pores with pronounced ridges in the latter. Molecular data further supported their species identity. Toxin production in the strains was examined by LC-MS/MS. O. cf. ovata strain T5PRBost02 was observed to produce Ovatoxin-k and Ovatoxin-j2 only; while Ostreocin-B and Ostreocin-D was produced by O. siamensis strain T10PRBost04. This is the first report confirming the production of palytoxins analogs in Ostreopsis species from the region.

Effect of a short-term salinity stress on the growth, biovolume, toxins, osmolytes and metabolite profiles on three strains of the Dinophysis acuminata-complex (Dinophysis cf. sacculus).

Dinophysis is the main dinoflagellate genus responsible for diarrheic shellfish poisoning (DSP) in human consumers of filter feeding bivalves contaminated with lipophilic diarrheic toxins. Species of this genus have a worldwide distribution driven by environmental conditions (temperature, irradiance, salinity, nutrients etc.), and these factors are sensitive to climate change. The D. acuminata-complex may contain several species, including D. sacculus. The latter has been found in estuaries and semi-enclosed areas, water bodies subjected to quick salinity variations and its natural repartition suggests some tolerance to salinity changes. However, the response of strains of D. acuminata-complex (D. cf. sacculus) subjected to salinity stress and the underlying mechanisms have never been studied in the laboratory. Here, a 24 h hypoosmotic (25) and hyperosmotic (42) stress was performed in vitro in a metabolomic study carried out with three cultivated strains of D. cf. sacculus isolated from the French Atlantic and Mediterranean coasts. Growth rate, biovolume and osmolyte (proline, glycine betaine and dimethylsulfoniopropionate (DMSP)) and toxin contents were measured. Osmolyte contents were higher at the highest salinity, but only a significant increase in glycine betaine was observed between the control (35) and the hyperosmotic treatment. Metabolomics revealed significant and strain-dependent differences in metabolite profiles for different salinities. These results, as well as the absence of effects on growth rate, biovolume, okadaic acid (OA) and pectenotoxin (PTXs) cellular contents, suggest that the D. cf. sacculus strains studied are highly tolerant to salinity variations.

Physiological changes induced by sodium chloride stress in Aphanizomenon gracile, Cylindrospermopsis raciborskii and Dolichospermum sp.

Due to anthropogenic activities, associated with climate change, many freshwater ecosystems are expected to experience an increase in salinity. This phenomenon is predicted to favor the development and expansion of freshwater cyanobacteria towards brackish waters due to their transfer along the estuarine freshwater-marine continuum. Since freshwater cyanobacteria are known to produce toxins, this represents a serious threat for animal and human health. Saxitoxins (STXs) are classified among the most powerful cyanotoxins. It becomes thus critical to evaluate the capacity of cyanobacteria producing STXs to face variations in salinity and to better understand the physiological consequences of sodium chloride (NaCl) exposure, in particular on their toxicity. Laboratory experiments were conducted on three filamentous cyanobacteria species isolated from brackish (Dolichospermum sp.) and fresh waters (Aphanizomenon gracile and Cylindrospermopsis raciborskii) to determine how salinity variations affect their growth, photosynthetic activity, pigment composition, production of reactive oxygen species (ROS), synthesis of compatible solutes and STXs intracellular quotas. Salinity tolerance was found to be species-specific. Dolichospermum sp. was more resistant to salinity variations than A. gracile and C. raciborskii. NaCl variations reduced growth in all species. In A. gracile, carotenoids content was dose-dependently reduced by NaCl. By contrast, in C. raciborskii and Dolichospermum sp., variations in carotenoids content did not show obvious relationships with NaCl concentration. While in Dolichospermum sp. phycocyanin and phycoerythrin increased within the first 24 h exposure to NaCl, in both A. gracile and C. raciborskii, these pigments decreased proportionally to NaCl concentration. Low changes in salinity did not impact STXs production in A. gracile and C. raciborskii while higher increase in salinity could modify the toxin profile and content of C. raciborskii (intracellular STX decreased while dc-GTX2 increased). In estuaries, A. gracile and C. raciborskii would not be able to survive beyond the oligohaline area (i.e. salinity > 5). Conversely, in part due to its ability to accumulate compatible solutes, Dolichospermum sp. has the potential to face consequent salinity variations and to survive in the polyhaline area (at least up to salinity = 24).

Summer bloom of Vulcanodinium rugosum in Cienfuegos Bay (Cuba) associated to dermatitis in swimmers.

The marine dinoflagellate Vulcanodinium rugosum produces powerful paralyzing and cytotoxic compounds named pinnatoxins (PnTX) and portimines. Even though, no related human intoxication episodes following direct exposure in seawater or the ingestion of contaminated seafood have been documented so far. This study aimed at investigating a dinoflagellate bloom linked to acute dermatitis cases in two recreational beaches in Cienfuegos Bay, Cuba. We used epidemiological and clinical data from 60 dermatitis cases consisting of individuals in close contact with the bloom. Seawater physical-chemical properties were described, and the microorganism causing the bloom was identified by means of light and scanning electron microscopy. Morphological identification was confirmed genetically by sequencing the internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA region. Toxic compounds were identified from a bloom extract using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS), and their concentrations were estimated based on low-resolution tandem mass spectrometry (LC-MS/MS). Sixty people who had prolonged contact with the dinoflagellate bloom suffered acute dermal irritation. Most patients (79.2%) were children and had to be treated with antibiotics; some required >5-day hospitalization. Combined morphological and genetic characters indicated V. rugosum as the causative agent of the bloom. rDNA sequences of the V. rugosum genotype found in the bloom aligned with others from Asia, including material found in the ballast tank of a ship in Florida. The predominant toxins in the bloom were portimine, PnTX-F and PnTX-E, similar to strains originating from the Pacific Ocean. This bloom was associated with unusual weather conditions such as frequent and prolonged droughts. Our findings indicate a close link between the V. rugosum bloom and a dermatitis outbreak among swimmers in Cienfuegos Bay. Phylogenetic evidence suggests a recent introduction of V. rugosum from the Pacific Ocean into Caribbean waters, possibly via ballast water.

Taxonomy and toxicity of a bloom-forming Ostreopsis species (Dinophyceae, Gonyaulacales) in Tahiti island (South Pacific Ocean): one step further towards resolving the identity of O. siamensis.

Among dinoflagellates responsible for benthic harmful algal blooms, the genus Ostreopsis primarily described from tropical areas has been increasingly reported from subtropical and temperate areas worldwide. Several species of this toxigenic genus produce analogs of palytoxin, thus representing a major threat to human and environmental health. The taxonomy of several species needs to be clarified as it was based mostly on morphological descriptions leading in some cases to ambiguous interpretations and misidentifications. The present study aims at reporting a benthic bloom that occurred in April 2019 in Tahiti island, French Polynesia. A complete taxonomic investigation of the blooming Ostreopsis species was realized using light, epifluorescence and field emission electron microscopy and phylogenetic analyses inferred from LSU rDNA and ITS-5.8S rDNA regions. Toxicity of a natural sample and strains isolated from the bloom was assessed using both neuroblastoma cell-based assay and LC-MS/MS analyses. Morphological observations showed that cells were round to oval, large, 58.0-82.5 µm deep (dorso-ventral length) and 45.7-61.2 µm wide. The cingulum was conspicuously undulated, forming a 'V' in ventral view. Thecal plates possessed large pores in depressions, with a collar rim. Detailed observation also revealed the presence of small thecal pores invisible in LM. Phylogenetic analyses were congruent and all sequences clustered within the genotype Ostreopsis sp. 6, in a subclade closely related to sequences from the Gulf of Thailand and Malaysia. No toxicity was found on the field sample but all the strains isolated from the bloom were found to be cytotoxic and produced ostreocin D, a lower amount of ostreocins A and B and putatively other compounds. Phylogenetic data demonstrate the presence of this species in the Gulf of Thailand, at the type locality of O. siamensis, and morphological data are congruent with the original description and support this identification.

Alexandrium pacificum and Alexandrium minutum: Harmful or environmentally friendly?

Alexandrium minutum and Alexandrium pacificum are representatives of the dinoflagellate genus that regularly proliferate on the French coasts and other global coastlines. These harmful species may threaten shellfish harvest and human health due to their ability to synthesize neurotoxic alkaloids of the saxitoxin group. However, some dinoflagellates such as A. minutum, and as reported here A. pacificum as well, may also have a beneficial impact on the environment by producing dimethylsulfoniopropionate-DMSP, the precursor of dimethylsulfur-DMS and sulfate aerosols involved in climate balance. However, environmental conditions might influence Alexandrium physiology towards the production of harmful or environmentally friendly compounds. After assessing the influence of two salinity regimes (33 and 38) relative to each species origin (Atlantic French coast and Mediterranean Lagoon respectively), it appears that DMSP and toxin content was variable between the three experimented strains and that higher salinity disadvantages toxin production and tends to favor the production of the osmolytes DMSP and glycine betaine. Hence, this key metabolite production is strain and species-dependent and is influenced by environmental conditions of salinity which in turn, can diversely affect the environment. Widespread coastal blooms of A. minutum and A. pacificum, although being a risk for seafood contamination with toxins, are also a DMSP and DMS source that potentially contribute to the ecosystem structuration and climate. Regarding recent advances in DMSP biosynthesis pathway, 3 dsyB homologs were found in A. minutum but no homolog of the diatom sequence TpMMT.

Toxin content of Ostreopsis cf. ovata depends on bloom phases, depth and macroalgal substrate in the NW Mediterranean Sea.

Over the last fifteen years, blooms of the genus Ostreopsis have been reported more frequently and at higher abundances in the Mediterranean area. Ostreopsis cf. ovata is known to produce ovatoxins (OVTXs), structural analogues of palytoxin, which is one of the most potent non-polymeric toxins. However, the production of OVTXs is poorly characterized in situ. The present study focuses on toxin content and profile according to the bloom phase during summer 2017 in Villefranche-sur-Mer, France (NW Mediterranean Sea), depth (from 0.5 to 5 m) and three different macroalgal substrates of this epiphytic dinoflagellate (Padina pavonica, Dictyota spp. and Halopteris scoparia). Ovatoxin quantification of all samples was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The bloom started at the end of June and declined in mid-July, showing the typical seasonal pattern of the NW Mediterranean Sea area. The peak was observed on the 10 July with 1.8 × 106 cells/g FW and 1.7 × 104 cells/L for benthic and planktonic cells, respectively. Total toxin content of cells, collected using artificial substrates, increased during the exponential and stationary growth phases. After reaching a maximum concentration of 9.2 pg/cell on 18 July, toxin concentration decreased and remained stable from 25 July until the end of monitoring. A decreasing trend of the abundance and of the associated total toxin content was noted with depth. Finally, the decreasing order of maximal epiphytic concentration of O. cf. ovata was: Dictyota spp. (8.3 × 105 cells/g FW), H. scoparia (3.1 × 105 cells/g FW) and P. pavonica (1.6 × 105 cells/g FW). Interestingly, the highest OVTX quota was obtained in cells present on Halopteris scoparia, then on Dictyota spp. and Padina pavonica. This suggests that the nature of the macroalgal substrate influences both growth and toxin production of O. cf. ovata and further work will be required to understand the underlying mechanisms (e.g., competition for nutrition, pH or allelopathic interaction). However, the toxin profiles (i.e., the proportion of each ovatoxin analogue) were not affected by any of the studied parameters (bloom phase, depth, macroalgae or artificial substrates).

Acute Oral Toxicity of Pinnatoxin G in Mice.

Pinnatoxin G (PnTx-G) is a marine cyclic imine toxin produced by the dinoflagellate Vulcanodiniumrugosum, frequently detected in edible shellfish from Ingril Lagoon (France). As other pinnatoxins, to date, no human poisonings ascribed to consumption of PnTx-G contaminated seafood have been reported, despite its potent antagonism at nicotinic acetylcholine receptors and its high and fast-acting toxicity after intraperitoneal or oral administration in mice. The hazard characterization of PnTx-G by oral exposure is limited to a single acute toxicity study recording lethality and clinical signs in non-fasted mice treated by gavage or through voluntary food ingestion, which showed differences in PnTx-G toxic potency. Thus, an acute toxicity study was carried out using 3 h-fasted CD-1 female mice, administered by gavage with PnTx-G (8-450 µg kg-1). At the dose of 220 µg kg-1 and above, the toxin induced a rapid onset of clinical signs (piloerection, prostration, hypothermia, abdominal breathing, paralysis of the hind limbs, and cyanosis), leading to the death of mice within 30 min. Except for moderate mucosal degeneration in the small intestine recorded at doses of 300 µg kg-1, the toxin did not induce significant morphological changes in the other main organs and tissues, or alterations in blood chemistry parameters. This acute oral toxicity study allowed to calculate an oral LD50 for PnTx-G equal to 208 g kg-1 (95% confidence limits: 155-281 µg kg-1) and to estimate a provisional NOEL of 120 µg kg-1.

Acclimation of the Marine Diatom Pseudo-nitzschia australis to Different Salinity Conditions: Effects on Growth, Photosynthetic Activity, and Domoic Acid Content1.

Toxic Pseudo-nitzschia australis strains isolated from French coastal waters were studied to investigate their capacity to adapt to different salinities. Their acclimation to different salinity conditions (10, 20, 30, 35, and 40) was studied on growth, photosynthetic capacity, cell biovolume, and domoic acid (DA) content. The strains showed an ability to acclimate to a salinity range from 20 to 40, with optimal growth rates between salinities 30 and 40. The highest cell biovolume was observed at the lowest salinity 20 and was associated with the lowest growth rate. Salinity did not affect the photosynthetic activity; Fv /Fm values and the pigment contents remained high with no significant difference among salinities. An enhanced production of zeaxanthin was, however, observed in the late stationary and decline phases in all cultures except for those acclimated to salinity 20. In terms of cellular toxin content, DA concentrations were 2 to 3-fold higher at the lowest salinity (20) than at the other salinities and were combined with a low amount of dissolved DA. The fact that P. australis accumulate more DA per cell in less saline waters, illustrates that climate-related changes in salinity may affect Pseudo-nitzschia physiology through direct effects on growth, physiology, and toxin content.

Physiological and Metabolic Responses of Freshwater and Brackish-Water Strains of Microcystis aeruginosa Acclimated to a Salinity Gradient: Insight into Salt Tolerance.

Proliferation of microcystin (MC)-producing Microcystis aeruginosa in brackish waters has been described in several locations and represents a new concern for public and environmental health. While the impact of a sudden salinity increase on M. aeruginosa physiology has been studied, less is known about the mechanisms involved in salt tolerance after acclimation. This study aims to compare the physiological responses of two strains of M. aeruginosa (PCC 7820 and PCC 7806), which were isolated from contrasted environments, to increasing salinities. After acclimation, growth and MC production rates were determined and metabolomic analyses were conducted. For both strains, salinity decreased the biovolume, growth, and MC production rates and induced the accumulation of polyunsaturated lipids identified as monogalactosyldiacylglycerol. The distinct salt tolerances (7.5 and 16.9) obtained between the freshwater (PCC 7820) and the brackish-water (PCC 7806) strains suggested different strategies to cope with the osmotic pressure, as revealed by targeted and untargeted metabolomic analyses. An accumulation of trehalose as the main compatible solute was obtained in the freshwater strain, while sucrose was mainly accumulated in the brackish one. Moreover, distinct levels of glycine betaine and proline accumulation were noted. Altogether, metabolomic analysis illustrated a strain-specific response to salt tolerance, involving compatible solute production.IMPORTANCE Blooms of Microcystis aeruginosa and the production of microcystins are major issues in eutrophic freshwater bodies. Recently, an increasing number of proliferations of M. aeruginosa in brackish water has been documented. The occurrence of both M. aeruginosa and microcystins in coastal areas represents a new threat for human and environmental health. In order to better describe the mechanisms involved in Microcystis sp. proliferation in brackish water, this study used two M. aeruginosa strains isolated from fresh and brackish waters. High salinity reduced the growth rate and microcystin production rate of M. aeruginosa In order to cope with higher salinities, the strains accumulated different cyanobacterial compatible solutes, as well as unsaturated lipids, explaining their distinct salt tolerance.

Ostreopsis cf. ovata Bloom in Currais, Brazil: Phylogeny, Toxin Profile and Contamination of Mussels and Marine Plastic Litter.

Ostreopsis cf. ovata is a toxic marine benthic dinoflagellate responsible for harmful blooms affecting ecosystem and human health, mostly in the Mediterranean Sea. In this study we report the occurrence of a summer O. cf. ovata bloom in Currais, a coastal archipelago located on the subtropical Brazilian coast (~25° S). This bloom was very similar to Mediterranean episodes in many aspects: (a) field-sampled and cultivated O. cf. ovata cells aligned phylogenetically (ITS and LSU regions) along with Mediterranean strains; (b) the bloom occurred at increasing temperature and irradiance, and decreasing wind speed; (c) cell densities reached up to 8.0 × 104 cell cm-2 on fiberglass screen and 5.6 × 105 cell g-1 fresh weight on seaweeds; (d) and toxin profiles were composed mostly of ovatoxin-a (58%) and ovatoxin-b (32%), up to 35.5 pg PLTX-eq. cell-1 in total. Mussels were contaminated during the bloom with unsafe toxin levels (up to 131 µg PLTX-eq. kg-1). Ostreopsis cells attached to different plastic litter, indicating an alternate route for toxin transfer to marine fauna via ingestion of biofilm-coated plastic debris.

Ostreopsis lenticularis Y. Fukuyo (Dinophyceae, Gonyaulacales) from French Polynesia (South Pacific Ocean): A revisit of its morphology, molecular phylogeny and toxicity.

To date, the genus Ostreopsis comprises eleven described species, of which seven are toxigenic and produce various compounds presenting a major threat to human and environmental health. The taxonomy of several of these species however remains controversial, as it was based mostly on morphological descriptions leading, in some cases, to ambiguous interpretations and even possible misidentifications. The species Ostreopsis lenticularis was first described by Y. Fukuyo from French Polynesia using light microscopy observations, but without genetic information associated. The present study aims at revisiting the morphology, molecular phylogeny and toxicity of O. lenticularis based on the analysis of 47 strains isolated from 4 distinct locales of French Polynesia, namely the Society, Australes, Marquesas and Gambier archipelagos. Observations in light, epifluorescence and field emission scanning electron microscopy of several of these strains analyzed revealed morphological features in perfect agreement with the original description of O. lenticularis. Cells were oval, not undulated, 60.5-94.4 µm in dorso-ventral length, 56.1-78.2 µm in width, and possessed a typical plate pattern with thecal plates showing two sizes of pores. Phylogenetic analyses inferred from the LSU rDNA and ITS-5.8S sequences revealed that the 47 strains correspond to a single genotype, clustering with a strong support with sequences previously ascribed to Ostreopsis sp. 5. Clonal cultures of O. lenticularis were also established and further tested for their toxicity using the neuroblastoma cell-based assay and LCMS/MS analyses. None of the 19 strains tested showed toxic activity on neuroblastoma cells, while LCMS/MS analyses performed on the strains from Tahiti Island (i.e. type locality) confirmed that palytoxin and related structural analogs were below the detection limit. These findings allow to clarify unambiguously the genetic identity of O. lenticularis while confirming previous results from the Western Pacific which indicate that this species shows no toxicity, thus stressing the need to reconsider its current classification within the group of toxic species.

Influence of sudden salinity variation on the physiology and domoic acid production by two strains of Pseudo-nitzschia australis.

Several coastal countries including France have experienced serious and increasing problems related to Pseudo-nitzschia toxic blooms. These toxic blooms occur in estuarine and coastal waters potentially subject to fluctuations in salinity. In this study, we document for the first time the viability, growth, photosynthetic efficiency, and toxin production of two strains of Pseudo-nitzschia australis grown under conditions with sudden salinity changes. Following salinity variation, the two strains survived over a restricted salinity range of 30-35, with favorable physiological responses, as the growth, effective quantum yield and toxin content were high compared to the other conditions. In addition, high cellular quotas of domoic acid (DA) were observed at a salinity of 40 for the strain IFR-PAU-16.1 in comparison with the other strain IFR-PAU-16.2 where the cell DA content was directly released into the medium. On the other hand, the osmotic stress imposed at lower salinities, 20 and 10, resulted in cell lysis and a sudden DA leakage in the medium. Intra-specific variability was observed in growth and toxin production, with the strain IFR-PAU-16.1 apparently able to withstand higher salinities than the strain IFR-PAU-16.2. On the whole, DA does not appear to act as an osmolyte in response to sudden salinity changes. Since most of the shellfish harvesting areas of bivalve molluscs in France are located in areas where the salinity generally varies between 30 and 35, Pseudo-nitzschia australis blooms might potentially impact public health and commercial shellfish resources in these places.

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.