Unraveling the Gonyaulax baltica Species Complex: Cyst-theca Relationship of Impagidinium variaseptum, Spiniferites pseudodelicatus sp. nov. and S. ristingensis (Gonyaulacaceae, Dinophyceae), With Descriptions of Gonyaulax bohaiensis sp. nov, G. amoyensis sp. nov. and G. portimonensis sp. nov.

The taxonomy of the extant dinoflagellate genus Gonyaulax is challenging since its thecate morphology is rather conservative. In contrast, cysts of Gonyaulax are varied in morphology and have been related with the fossil-based genera Spiniferites and Impagidinium. To better understand the systematics of Gonyaulax species, we performed germination experiments on cysts that can be identified as S. ristingensis, an unidentified Spiniferites with petaloid processes here described as Spiniferites pseudodelicatus sp. nov. and Impagidinium variaseptum from Chinese and Portuguese waters. Despite marked differences in cyst morphology, motile cells of S. pseudodelicatus and I. variaseptum are indistinguishable from Gonyaulax baltica. Motile cells hatched from S. ristingensis are morphologically similar to G. baltica as well but differ in the presence of one pronounced antapical spine. Three new species, Gonyaulax amoyensis (cyst equivalent S. pseudodelicatus), Gonyaulax bohaiensis (cyst equivalent I. variaseptum), and Gonyaulax portimonensis (cyst equivalent S. ristingensis), were erected. In addition, a new ribotype (B) of G. baltica was reported from South Korea and a bloom of G. baltica ribotype B is reported from New Zealand. Molecular phylogeny based on LSU and SSU rRNA gene sequences revealed that Gonyaulax species with minute or short antapical spines formed a well-resolved clade, whereas species with two pronounced antapical spines or lack of antapical spines formed the sister clade. Six strains of four above species were examined for yessotoxin production by liquid chromatography coupled with tandem mass spectrometry, and very low concentrations of yessotoxin were detected for one G. bohaiensis strain.

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.

Combined Effects of Temperature, Irradiance, and pH on Teleaulax amphioxeia (Cryptophyceae) Physiology and Feeding Ratio For Its Predator Mesodinium rubrum (Ciliophora)1.

The cryptophyte Teleaulax amphioxeia is a source of plastids for the ciliate Mesodinium rubrum and both organisms are members of the trophic chain of several species of Dinophysis. It is important to better understand the ecology of organisms at the first trophic levels before assessing the impact of principal factors of global change on Dinophysis spp. Therefore, combined effects of temperature, irradiance, and pH on growth rate, photosynthetic activity, and pigment content of a temperate strain of T. amphioxeia were studied using a full factorial design (central composite design 23 *) in 17 individually controlled bioreactors. The derived model predicted an optimal growth rate of T. amphioxeia at a light intensity of 400 µmol photons · m-2 · s-1 , more acidic pH (7.6) than the current average and a temperature of 17.6°C. An interaction between temperature and irradiance on growth was also found, while pH did not have any significant effect. Subsequently, to investigate potential impacts of prey quality and quantity on the physiology of the predator, M. rubrum was fed two separate prey: predator ratios with cultures of T. amphioxeia previously acclimated at two different light intensities (100 and 400 µmol photons · m-2 s-1 ). M. rubrum growth appeared to be significantly dependent on prey quantity while effect of prey quality was not observed. This multi-parametric study indicated a high potential for a significant increase of T. amphioxeia in future climate conditions but to what extent this would lead to increased occurrences of Mesodinium spp. and Dinophysis spp. should be further investigated.

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.

Effect of Nitrate, Ammonium and Urea on Growth and Pinnatoxin G Production of Vulcanodinium rugosum.

Vulcanodinium rugosum, a recently described dinoflagellate species producing a potent neurotoxin (pinnatoxin G), has been identified in French Mediterranean lagoons and was responsible for recurrent episodes of shellfish toxicity detected by mouse bioassay. Until now, the biology and physiology of V. rugosum have not been fully investigated. We studied the growth characteristics and toxicity of a V. rugosum strain (IFR-VRU-01), isolated in the Ingril lagoon in June 2009 (North-Western French Mediterranean Sea). It was cultivated in Enriched Natural Sea Water (ENSW) with organic (urea) and inorganic (ammonium and nitrate) nitrogen, at a temperature of 25 °C and irradiance of 100 µmol/m²·s(-1). Results showed that ammonium was assimilated by cells more rapidly than nitrate and urea. V. rugosum is thus an osmotrophic species using urea. Consequently, this nitrogen form could contribute to the growth of this dinoflagellate species in the natural environment. There was no significant difference (Anova, p = 0.856) between the growth rate of V. rugosum cultivated with ammonium (0.28 ± 0.11 day(-1)), urea (0.26 ± 0.08 day(-1)) and nitrate (0.24 ± 0.01 day(-1)). However, the production of chlorophyll a and pinnatoxin G was significantly lower with urea as a nitrogen source (Anova, p < 0.027), suggesting that nutritional conditions prevailing at the moment of the bloom could determine the cellular toxicity of V. rugosum and therefore the toxicity measured in contaminated mollusks. The relatively low growth rate (≤0.28 day(-1)) and the capacity of this species to continuously produce temporary cysts could explain why cell densities of this species in the water column are typically low (≤20,000 cells/L).

Beta-N-methylamino-L-alanine: LC-MS/MS optimization, screening of cyanobacterial strains and occurrence in shellfish from Thau, a French Mediterranean lagoon.

ß-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 µg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 µg/g DW), while only several samples contained quantifiable free BMAA.

Complex toxin profile of French Mediterranean Ostreopsis cf. ovata strains, seafood accumulation and ovatoxins prepurification.

Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 µg·kg-1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 µg·kg-1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell-1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Cytotoxicity, fractionation and dereplication of extracts of the dinoflagellate Vulcanodinium rugosum, a producer of pinnatoxin G.

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of "fast-acting toxins", its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL⁻¹ and 0.19 µg mL⁻¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL⁻¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

An unprecedented bloom of Lingulodinium polyedra on the French Atlantic coast during summer 2021.

At the end of July 2021, a bloom of Lingulodinium polyedra developed along the French Atlantic coast and lasted six weeks. The REPHY monitoring network and the citizen participation project PHENOMER contributed to its observation. A maximum concentration of 3,600,000 cells/L was reached on the 6th of September, a level never recorded on French coastlines. Satellite observation confirmed that the bloom reached its highest abundance and spatial extension early September, covering about 3200 km2 on the 4th of September. Cultures were established, and morphology and ITS-LSU sequencing identified the species as L. polyedra. The thecae displayed the characteristic tabulation and sometimes a ventral pore. The pigment composition of the bloom was similar to that of cultured L. polyedra, confirming that phytoplankton biomass was dominated by this species. The bloom was preceded by Leptocylindrus sp., developed over Lepidodinium chlorophorum, and was succeeded by elevated Noctiluca scintillans concentrations. Afterwards, relatively high abundance of Alexandrium tamarense were observed in the embayment where the bloom started. Unusually high precipitation during mid-July increased river discharges from the Loire and Vilaine rivers, which likely fueled phytoplankton growth by providing nutrients. Water masses with high numbers of dinoflagellates were characterized by high sea surface temperature and thermohaline stratification. The wind was low during the bloom development, before drifting it offshore. Cysts were observed in the plankton towards the end of the bloom, with concentrations up to 30,000 cysts/L and relative abundances up to 99%. The bloom deposited a seed bank, with cyst concentrations up to 100,000 cysts/g dried sediment, particularly in fine-grained sediments. The bloom caused hypoxia events, and concentrations of yessotoxins up to 747 µg/kg were recorded in mussels, below the safety threshold of 3,750 µg/kg. Oysters, clams and cockles also were contaminated with yessotoxins, but at lower concentrations. The established cultures did not produce yessotoxins at detectable levels, although yessotoxins were detected in the sediment. The unusual environmental summertime conditions that triggered the bloom, as well as the establishment of considerable seed banks, provide important findings to understand future harmful algal blooms along the French coastline.

Quantitative analysis of azaspiracids in Azadinium spinosum cultures.

Azaspiracids (AZAs) are secondary metabolites of Azadinium spinosum that can accumulate in shellfish and cause food poisoning when consumed. We describe here an analytical procedure for the determination of AZAs in cultures of A. spinosum with a focus on the formation of AZA methyl esters as artefacts during extraction and sample pre-treatment. A. spinosum cells were collected from bioreactor cultures using centrifugation or filtration. Different extraction procedures were evaluated for formation of methyl ester artefacts, yield, and matrix effects. Filtration of cultures using glass-fibre filters led to increased formation of methyl esters, and centrifugation is recommended for recovery of cells. The extraction solvent (methanol (MeOH), acetone, and acetonitrile (MeCN)) did not significantly affect the yield of AZAs as long as the organic content was 80% or higher. However, the use of MeOH as extraction solvent led to increased formation of methyl esters. AZA1 recovery over two successive extractions was 100% at the 95% confidence level for acetone and MeOH. In standard-addition experiments, no significant matrix effects were observed in extracts of A. spinosum or Azadinium obesum up to a sample size of 4.5 × 10(9) µm(3). Moreover, experiments carried out to clarify the formation and structure of methylated AZA analogues led to the description of two AZA methyl esters and to the correction of the chemical structures of AZAs29-32.

Ovatoxin-a and palytoxin accumulation in seafood in relation to Ostreopsis cf. ovata blooms on the French Mediterranean coast.

Dinoflagellates of the genus Ostreopsis are known to cause (often fatal) food poisoning in tropical coastal areas following the accumulation of palytoxin (PLTX) and/or its analogues (PLTX group) in crabs, sea urchins or fish. Ostreopsis spp. occurrence is presently increasing in the northern to north western Mediterranean Sea (Italy, Spain, Greece and France), probably in response to climate change. In France, Ostreopsis. cf. ovata has been associated with toxic events during summer 2006, at Morgiret, off the coast of Marseille, and a specific monitoring has been designed and implemented since 2007. Results from 2008 and 2009 showed that there is a real danger of human poisoning, as these demonstrated bioaccumulation of the PLTX group (PLTX and ovatoxin-a) in both filter-feeding bivalve molluscs (mussels) and herbivorous echinoderms (sea urchins). The total content accumulated in urchins reached 450 µg PLTX eq/kg total flesh (summer 2008). In mussels, the maximum was 230 µg eq PLTX/kg (summer 2009) compared with a maximum of 360 µg found in sea urchins during the same period at the same site. This publication brings together scientific knowledge obtained about the summer development of Ostreopsis spp. in France during 2007, 2008 and 2009.

Production and isolation of azaspiracid-1 and -2 from Azadinium spinosum culture in pilot scale photobioreactors.

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell · mL(-1) at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day(-1), with optimum toxin production at 0.25 day(-1). After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

Combined effects of temperature and light intensity on growth, metabolome and ovatoxin content of a Mediterranean Ostreopsis cf. ovata strain.

Ostreopsis cf. ovata is a benthic and ovatoxin-producing dinoflagellate proliferating yearly along the Mediterranean coasts where blooms have been related to human illness and unusual mortality of marine organisms. The spreading of O. cf. ovata in this temperate area has been linked to global changes and its consequences such as the increase of temperature or light intensities. In the present study, an experimental design using batch cultures of pre-acclimated cells of a strain of O. cf. ovata isolated from Villefranche-sur-Mer (NW Mediterranean Sea, France), was implemented to investigate the combined effect of temperature (23, 27 and 30 °C) and light intensity (200, 400 and 600 µmol m-2s-1) on the growth, metabolome and OVTX content. Both light intensity and temperature affected the growth as significantly higher growth rates were obtained under 400 and 600 µmol m-2s-1 while the maximum values were obtained at 27 °C (0.48 d-1). Metabolomic analyses highlighted a clear effect only for temperature that may correspond to two different strategies of acclimation to suboptimal temperatures. Significant features (such as carotenoid and lipids) modified by the temperature and/or light conditions were annotated. Only temperature induced a significant change of OVTX content with higher values measured at the lowest temperature of 23 °C (29 - 36 pg cell-1). In a context of global changes, these results obtained after acclimation suggest that the increase of temperature might favor the proliferation of less toxic cells. However, in the light of the intraspecific variability of O. cf. ovata, further studies will be necessary to test this hypothesis. This study also highlighted the lack of knowledge about the metabolome composition of such non-model organisms that impairs data interpretation. There is a need to study more deeply the metabolome of toxic dinoflagellates to better understand how they can acclimate to a changing environment.

Molecular networking as a novel approach to unravel toxin diversity of four strains of the dominant Dinophysis species from French coastal waters.

Some species of the genus Dinophysis contain Diarrhetic shellfish Poisoning (DSP) toxins and are the main threat to shellfish farming in Europe including France. Dinophysis species are known to produce two families of bioactive lipophilic toxins: (i) okadaic acid (OA) and their analogues dinophysistoxins (DTXs) and (ii) pectenotoxins (PTXs). Only six toxins (OA, DTX1, DTX2, DTX3, PTX1 and PTX2) regulated by the European Union Legislation (EC No. 15/2011; 3) are routinely monitored using targeted chemical analysis by liquid chromatography coupled to mass spectrometry (LC-MS/MS) while toxic species of Dinophysis produce many other analogues. To tentatively identify unknown toxin analogues, a recent approach (Molecular Networking, MN) was used based on fragmentation data obtained by untargeted high resolution mass spectrometry (HRMS). An optimization of the data-dependent LC-HRMS/MS acquisition conditions was conducted to obtain more informative networks. The MN was applied to provide an overview of the chemical diversity of four strains belonging to three major Dinophysis species isolated from French coastal waters (D. acuta, D. caudata and the "D. acuminata complex" species D. acuminata and D. sacculus). This approach highlighted species-specific chemical patterns and also that Dinophysis chemical diversity is largely unexplored. Using MN allowed to identify directly known toxins and their relationship between species of Dinophysis, leading to the discovery of five new putative PTX analogues.

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.

Characterization of toxin-producing strains of Dinophysis spp. (Dinophyceae) isolated from French coastal waters, with a particular focus on the D. acuminata-complex.

Dinoflagellates of the genus Dinophysis are the most prominent producers of Diarrhetic Shellfish Poisoning (DSP) toxins which have an impact on public health and on marine aquaculture worldwide. In particular, Dinophysis acuminata has been reported as the major DSP agent in Western Europe. Still, its contribution to DSP events in the regions of the English Channel and the Atlantic coast of France, and the role of the others species of the Dinophysis community in these areas are not as clear. In addition, species identification within the D. acuminata complex has proven difficult due to their highly similar morphological features. In the present study, 30 clonal strains of the dominant Dinophysis species have been isolated from French coasts including the English Channel (3 sites), the Atlantic Ocean (11 sites) and the Mediterranean Sea (6 sites). Morphologically, strains were identified as three species: D. acuta, D. caudata, D. tripos, as well as the D. acuminata-complex. Sequences of the ITS and LSU rDNA regions confirmed these identifications and revealed no genetic difference within the D. acuminata-complex. Using the mitochondrial gene cox1, two groups of strains differing by only one substitution were found in the D. acuminata-complex, but SEM analysis of various strains showed a large range of morphological variations. Based on geographical origin and morphology, strains of the subclade A were ascribed to 'D. acuminata' while those of the subclade B were ascribed to 'D. sacculus'. Nevertheless, the distinction into two separate species remains questionable and was not supported by our genetic data. The considerable variations observed in cultured strains suggest that physiological factors might influence cell contour and bias identification. Analyses of Dinophysis cultures from French coastal waters using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed species-conserved toxin profiles for D. acuta (dinophysistoxin 2 (DTX2), okadaic acid (OA), pectenotoxin 2 (PTX2)), D. caudata (PTX2) and D. tripos (PTX2), irrespective of geographical origin (Atlantic Ocean or Mediterranean Sea). Within the D. acuminata-complex, two different toxin profiles were observed: the strains of 'D. acuminata' (subclade A) from the English Channel and the Atlantic Ocean contained only OA while strains of 'D. sacculus' (subclade B) from Mediterranean Sea/Atlantic Ocean contained PTX2 as the dominant toxin, with OA and C9-esters also being present, albeit in lower proportions. The same difference in toxin profiles between 'D. sacculus' and 'D. acuminata' was reported in several studies from Galicia (NW- Spain). This difference in toxin profiles has consequences in terms of public health, and consequently for monitoring programs. While toxin profile could appear as a reliable feature separating 'D. acuminata' from 'D. sacculus' on both French and Spanish coasts, this does not seem consistent with observations on a broader geographical scale for the D. acuminata complex, possibly due to the frequent lack of genetic characterization.

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).

Cyclic imine toxins survey in coastal european shellfish samples: Bioaccumulation and mode of action of 28-O-palmitoyl ester of pinnatoxin-G. first report of portimine-A bioaccumulation.

Cyclic imine toxins exhibit fast acting neurotoxicity and lethality by respiratory arrest in mice explained by their potent antagonistic activity against muscular nicotinic acetylcholine receptors. We performed a survey of gymnodimine-A, 13-desmethyl spirolide-C, 13,19-didesmethyl spirolide-C, 20-methyl spirolide-G, pinnatoxin-A, pinnatoxin-G, portimine-A and 28-O-palmitoyl ester of pinnatoxin-G in 36 shellfish samples collected in coastal areas of 8 European countries using a microplate receptor binding assay and UPLC-MS/MS for toxin identification and quantification. The major toxins found in these samples were pinnatoxin-G, 20-methyl spirolide-G, 13-desmethyl spirolide-C, gymnodimine-A and portimine-A. Traces of 13,19-didesmethyl spirolide-C, pinnatoxin-A and 28-O-palmitoyl ester of pinnatoxin-G were also detected. The rapid death of mice was correlated with higher pinnatoxin-G concentrations in mussel digestive gland extracts injected intraperitoneally. Our survey included nontoxic control samples that were found to contain moderate to trace amounts of several cyclic imine toxins. Shellfish may bioaccumulate not only cyclic imine toxins but also a large number of acyl derivatives as a product of metabolic transformation of these neurotoxins. This is the first report in which portimine-A and 28-O-palmitoyl ester of pinnatoxin-G were detected in shellfish extracts from digestive glands of mussels collected in Ingril lagoon. The bioaccumulation of portimine-A is particularly of concern because it is cytotoxic and is able to induce apotosis. The mode of action of 28-O-palmitoyl ester of pinnatoxin-G was studied by receptor binding-assay and by two-electrode voltage clamp electrophysiology. The antagonistic behavior of the acylated pinnatoxin-G towards nicotinic acetylcholine receptor of muscle type is shown here for the first time. Since cyclic imine toxins are not regulated further monitoring of these emerging toxins is needed to improve evidence gathering of their occurrence in shellfish commercialized for human consumption in Europe given their potent antagonism against muscle and neuronal nicotinic acetylcholine receptors.

Cultures of Dinophysis sacculus, D. acuminata and pectenotoxin 2 affect gametes and fertilization success of the Pacific oyster, Crassostrea gigas.

Harmful algal blooms (HABs) of toxic species of the dinoflagellate genus Dinophysis are a threat to human health as they are mainly responsible for diarrheic shellfish poisoning (DSP) in the consumers of contaminated shellfish. Such contamination leads to shellfish farm closures causing major economic and social issues. The direct effects of numerous HAB species have been demonstrated on adult bivalves, whereas the effects on critical early life stages remain relatively unexplored. The present study aimed to determine the in vitro effects of either cultivated strains of D. sacculus and D. acuminata isolated from France or their associated toxins (i.e. okadaic acid (OA) and pectenotoxin 2 (PTX2)) on the quality of the gametes of the Pacific oyster Crassostrea gigas. This was performed by assessing the ROS production and viability of the gametes using flow cytometry, and fertilization success using microscopic counts. Oocytes were more affected than spermatozoa and their mortality and ROS production increased in the presence of D. sacculus and PTX2, respectively. A decrease in fertilization success was observed at concentrations as low as 0.5 cell mL-1 of Dinophysis spp. and 5 nM of PTX2, whereas no effect of OA could be observed. The effect on fertilization success was higher when both gamete types were concomitantly exposed compared to separate exposures, suggesting a synergistic effect. Our results also suggest that the effects could be due to cell-to-cell contact. These results highlight a potential effect of Dinophysis spp. and PTX2 on reproduction and recruitment of the Pacific oyster.

Effect of filtration rate on coal-sand dual-media filter performances for microalgae removal.

This study tested the efficiency of granular filtration using a bilayer sand filter for microalgae removal from culture dilutions ranging from 10,000 to 17,000 cells/mL. The objective is to evaluate the removal capacity of the filter without chemical coagulation. Two filter media, sand and anthracite, with mean grain sizes of 0.395 and 1.2 mm, respectively, were used in constant-flow-rate experiments (down-flow mode) with suspensions containing Heterocapsa triquetra microalga. The conventional rapid filtration which usually operates at a constant rate of approximately 5 m3/m2 h is compared to high-rate filtration. Two filtration velocities (5 and 10 m/h) were investigated with bed depth of 1100 mm. Average microalgal cell removal rates were 90% at 5 m/h and 68% at 10 m/h. Turbidity removal was more than 71% at 5 m/h but just 57% at 10 m/h. Head losses did not increase significantly, and values measured at process end were 32 mbar at 5 m/h and 78 mbar at 10 m/h. Retention probabilities were calculated from experimental data. A theoretical model was used to evaluate the contributions of the different drivers of microalgae removal. Hypotheses are developed on the understanding of change in the mechanisms of retention as a function of filtration velocity.