First insights into the distribution and diversity of toxic dinoflagellate cysts in the surface sediments of Dakhla Bay (African Atlantic coast): relationships with environmental factors and mollusk intoxication events.

Dakhla Bay, situated on the African Atlantic coast, has witnessed sporadic harmful algal blooms (HABs) caused by toxic dinoflagellate species over the past two decades. In this study, we investigated the distribution, abundance, and diversity of dinoflagellate cysts, with a focus on potentially toxic species that develop in this ecosystem where such data are lacking. Sediment samples were collected in April 2018 through coring at 49 stations distributed across the bay. The highest abundance of dinoflagellate cysts was recorded at 304 cysts/g dry sediment, observed at the inner part of the bay, indicating that this area is the preferential zone for cyst accumulation. Pearson's tests revealed significant positive correlations (P < 0.05) between cyst abundance and the water content, organic matter, and fine fraction (< 63 µm) of the sediment. Cyst morphotypes of potentially toxic dinoflagellate species known to produce saxitoxins, such as Alexandrium minutum, Alexandrium tamarense species complex, Gymnodinium catenatum, and yessotoxins, such as Lingulodinium polyedrum and Gonyaulax cf. spinifera, were identified in the sediment of Dakhla Bay. These findings were further supported by our long-term monitoring period (2005-2018), underscoring the presence of these HAB species in Dakhla Bay. During our survey, sporadic mollusk intoxication events were recorded at station PK25 for the grooved razor shell Solen marginatus and at station Boutelha for the oyster Crassostrea gigas. Paralytic shellfish toxin concentrations exceeded the sanitary threshold (80 µg STX di-HCl eq/100 g of shellfish meat) only twice, in December 2006 and January 2007 at station PK25. Contamination by amnesic shellfish toxins occurred sporadically but never reached the sanitary threshold of 20 µg/g of shellfish meat. Lipophilic shellfish intoxication occurred multiple times in the two investigated areas. These observations suggest that the cysts of the identified HAB species germinated and inoculated the water column, resulting in the observed intoxication events. Relatively low levels of intoxication could be attributed to the moderate abundances of cysts, which may reduce the seeding capacity. This could be explained by the significant interaction of Dakhla Bay with the Atlantic Ocean, characterized by hydrological dynamics that impede the deposition and accumulation of cysts in the bay's sediments. This study reaffirms the importance of investigating dinoflagellate cysts in assessing the diversity of HAB species and evaluating associated sanitary risks.

Effect of temperature, salinity and nutrients on the growth and toxin content of the dinoflagellate Gymnodinium catenatum from the southwestern Mediterranean.

The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 µM to 1764 µM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 µM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell-1 to 22.4 pg STXeq.cell-1) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell-1 at 441 µM to 22.4 pg STXeq.cell-1 at 1764 µM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell-1 and 16.4 pg STXeq.cell-1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication.

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.

Estimation of the introduction risk of non-indigenous species through ship ballast water in the Port of Douala (Cameroon).

The transport of non-indigenous species in ship's ballast water represents a threat to marine biodiversity. This study is the first on marine bioinvasion in Sub-Saharan Africa. The Port of Douala (PoD), located in the Gulf of Guinea, is experiencing increasing maritime traffic, hence the importance of preventing biological invasions. PoD received ballast water from 41 ports and 20 ecoregions during the study period (2018-2021). We used a biological invasion model and showed that ships from the ports of Antwerp, Durban, Dar es Salaam, Pointe-Noire (Southern Gulf of Guinea) and Dakar (Sahelian Upwelling), with their associated ecoregions present a major invasion risk. Treating ballast water from these ships to IMO D-2 standards could reduce their probability of biological invasion by 97.18, 98.43, 98.80, 98.77 and 98.84 %, respectively. Climate change may also mitigate the risk of biological invasion, particularly for ships in the North Sea ecoregion from the port of Antwerp.

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.

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.

Four new species of Hysterocinetida (Protozoa: Ciliophora) from the digestive tract of earthworms collected to the lower Nyong estuary (South Coast, Cameroon).

Hysterocinetida are a large assemblage of ciliates characterized by sucker and buccal apparatus. They have a wide distribution in the digestive tract of oligochaete annelids of the genus Alma that inhabit hydromorphic parts of non-anthropic hydrosystems. On the basis of morphological, morphometric and infraciliature characteristics revealed by ammoniacal silver carbonate and 4',6-Diamidino-2-Phenyl Indole (DAPI) staining techniques, four forms were identified as new members belonging to three genera: Preptychostomum, Proptychostomum and Thurstonia. Preptychostomum donendaensis n. sp. is ovoid with a slightly narrowed anterior part and is commensal to the digestive tract of Alma emini and A. nilotica. There are 20-23 kineties on the lower side and 42-45 on the upper side. Proptychostomum gigas n. sp. is pear-shaped, with a narrowed anterior part and a wide posterior part (125-138 µm × 80-85 µm). There are 40 to 44 kineties, equally distributed on both sides of the ciliate. Its sucker is circular and measures 31-34 µm in diameter. The peristome follows the contour of the cell and the infundibulum has a turn of spire. Thurstonia emini n. sp. is identified in the digestive tract of A. emini. The cell is elongate, with the anterior pole narrower than the truncate posterior pole (90-115 µm × 40-55 µm). The kineties are equally distributed on both sides of the ciliate. The sucker is in the shape of an inverted U. Thurtonia nilotica n. sp. is commensal of the digestive tract of A. nilotica. The cell is pear-shaped, with the anterior part narrowed and the posterior part swollen (110-114 µm × 85-92 µm). 46 to 50 kineties are unevenly distributed on both sides of the ciliate. The sucker is shaped like an inverted lunar crescent.

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.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

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).

In silico prediction of the secretome from the invasive neurotoxic marine dinoflagellate Alexandrium catenella.

Alexandrium catenella, a marine dinoflagellate responsible for harmful algal blooms (HABs), proliferates with greater frequency, distribution and intensity, in disturbed marine coastal ecosystems. The proteins secreted into seawater may play a crucial role in maintaining this dinoflagellate in these ecosystems, but this possibility has never been investigated before. In this study, the A. catenella secretome was predicted from its transcriptome by combining several bioinformatics tools. Our results predict a secretome of 2 779 proteins, among which 79% contain less than 500 amino acids, suggesting that most secreted proteins are short in length. The predicted secretome includes 963 proteins (35%) with Pfam domains: 773 proteins with one Pfam domain and 190 proteins with two or more Pfam domains. Their functional annotation showed that they are mainly involved in (i) proteolysis, (ii) stress responses and (iii) primary metabolism. In addition, 47% of the secreted proteins appear to be enzymes, primarily peptidases, known to be biologically active in the extracellular medium during stress responses. Finally, this study provides a wealth of candidates of proteins secreted by A. catenella, which may interact with the marine environment and help this dinoflagellate develop in various environmental conditions.

Resting Cyst Distribution and Molecular Identification of the Harmful Dinoflagellate Margalefidinium polykrikoides (Gymnodiniales, Dinophyceae) in Lampung Bay, Sumatra, Indonesia.

Margalefidinium polykrikoides, an unarmored dinoflagellate, was suspected to be the causative agent of the harmful algal blooms - associated with massive fish mortalities - that have occurred continually in Lampung Bay, Indonesia, since the first bloom event in October 2012. In this study, after examination of the morphology of putative M. polykrikoides-like cysts sampled in bottom sediments, cyst bed distribution of this harmful species was explored in the inner bay. Sediment samples showed that resting cysts, including several morphotypes previously reported as M. polykrikoides, were most abundant on the northern coast of Lampung Bay, ranging from 20.6 to 645.6 cysts g-1 dry sediment. Molecular phylogeny inferred from LSU rDNA revealed that the so-called Mediterranean ribotype was detected in the sediment while M. polykrikoides motile cells, four-cell chain forming in bloom conditions, belonged to the American-Malaysian ribotype. Moreover, hyaline cysts, exclusively in the form of four-cell chains, were also recorded. Overall, these results unequivocally show that the species M. polykrikoides is abundantly present, in the form of vegetative cells, hyaline and resting cysts in an Indonesian area.

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.

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.

Modifications of the soluble proteome of a mediterranean strain of the invasive neurotoxic dinoflagellate Alexandrium catenella under metal stress conditions.

The soluble proteome of the mediterranean strain ACT03 of the invasive neurotoxic dinoflagellate Alexandrium catenella exposed to lead or zinc at 6, 12 or 18µM (total concentrations), or under control conditions, was characterized by two-dimensional gel electrophoresis (2-DE). Zinc reduced (P<0.05) the total number of protein spots (-41%, -52% and -60%, at 6, 12 or 18µM, respectively). Besides, most of the proteins constituting the soluble proteome were down-regulated in response to lead or zinc stresses. These proteins were involved mainly in photosynthesis (20-37% for lead; 36-50% for zinc) (ribulose-1,5-bisphosphate carboxylase/oxygenase: RUBISCO; ferredoxin-NADP+ reductase: FNR; peridinin-chlorophyll a-protein: PCP), and in the oxidative stress response (29-34% for lead; 17-36% for zinc) (superoxide dismutase: SOD; proteasome α/ß subunits). These negative effects could be partly compensated by the up-regulation of specific proteins such as ATP-synthase ß subunit (+16.3 fold after exposure to lead at 12µM). Indeed, an increase in the abundance of ATP-synthase could enrich the ATP pool and provide more energy available for the cells to survive under metal stress, and make the ATP-synthase transport of metal cations out of the cells more efficient. Finally, this study shows that exposure to lead or zinc have a harmful effect on the soluble proteome of A. catenella ACT03, but also suggests the existence of an adaptative proteomic response to metal stresses, which could contribute to maintaining the development of this dinoflagellate in trace metal-contaminated ecosystems.

Toxicity and Growth Assessments of Three Thermophilic Benthic Dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) Developing in the Southern Mediterranean Basin.

Harmful benthic dinoflagellates, usually developing in tropical areas, are expanding to temperate ecosystems facing water warming. Reports on harmful benthic species are particularly scarce in the Southern Mediterranean Sea. For the first time, three thermophilic benthic dinoflagellates (Ostreopsis cf. ovata, Prorocentrum lima and Coolia monotis) were isolated from Bizerte Bay (Tunisia, Mediterranean) and monoclonal cultures established. The ribotyping confirmed the morphological identification of the three species. Maximum growth rates were 0.59 ± 0.08 d-1 for O. cf. ovata, 0.35 ± 0.01 d-1 for C. monotis and 0.33 ± 0.04 d-1 for P. lima. Toxin analyses revealed the presence of ovatoxin-a and ovatoxin-b in O. cf. ovata cells. Okadaic acid and dinophysistoxin-1 were detected in P. lima cultures. For C. monotis, a chromatographic peak at 5.6 min with a mass m/z = 1061.768 was observed, but did not correspond to a mono-sulfated analogue of the yessotoxin. A comparison of the toxicity and growth characteristics of these dinoflagellates, distributed worldwide, is proposed.

Toxin and Growth Responses of the Neurotoxic Dinoflagellate Vulcanodinium rugosum to Varying Temperature and Salinity.

Vulcanodinium rugosum, a recently described species, produces pinnatoxins. The IFR-VRU-01 strain, isolated from a French Mediterranean lagoon in 2010 and identified as the causative dinoflagellate contaminating mussels in the Ingril Lagoon (French Mediterranean) with pinnatoxin-G, was grown in an enriched natural seawater medium. We tested the effect of temperature and salinity on growth, pinnatoxin-G production and chlorophyll a levels of this dinoflagellate. These factors were tested in combinations of five temperatures (15, 20, 25, 30 and 35 °C) and five salinities (20, 25, 30, 35 and 40) at an irradiance of 100 µmol photon m(-2) s(-1). V. rugosum can grow at temperatures and salinities ranging from 20 °C to 30 °C and 20 to 40, respectively. The optimal combination for growth (0.39 ± 0.11 d(-1)) was a temperature of 25 °C and a salinity of 40. Results suggest that V. rugosum is euryhaline and thermophile which could explain why this dinoflagellate develops in situ only from June to September. V. rugosum growth rate and pinnatoxin-G production were highest at temperatures ranging between 25 and 30 °C. This suggests that the dinoflagellate may give rise to extensive blooms in the coming decades caused by the climate change-related increases in temperature expected in the Mediterranean coasts.

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).

ß-N-methylamino-l-alanine (BMAA) and isomers: Distribution in different food web compartments of Thau lagoon, French Mediterranean Sea.

The neurotoxin BMAA (ß-N-methylamino-l-alanine) and its isomer DAB (2,4-diaminobutyric acid) have been detected in seafood worldwide, including in Thau lagoon (French Mediterranean Sea). A cluster of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with BMAA, has also been observed in this region. Mussels, periphyton (i.e. biofilms attached to mussels) and plankton were sampled between July 2013 and October 2014, and analyzed using HILIC-MS/MS. BMAA, DAB and AEG (N-(2-aminoethyl)glycine) were found in almost all the samples of the lagoon. BMAA and DAB were present at 0.58 and 0.83, 2.6 and 3.3, 4.0 and 7.2 µg g(-1) dry weight in plankton collected with nets, periphyton and mussels, respectively. Synechococcus sp., Ostreococcus tauri, Alexandrium catenella and eight species of diatoms were cultured and screened for BMAA and analogs. While Synechococcus sp., O. tauri and A. catenella did not produce BMAA under our culture conditions, four diatoms species contained both BMAA and DAB. Hence, diatoms may be a source of BMAA for mussels. Unlike other toxins produced by microalgae, BMAA and DAB were detected in significant amounts in tissues other than digestive glands in mussels.