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International audience; Studies on phytoplankton and in particular Harmful Algal Blooms (HABs) species in southern Mediterranean waters are scarce. We performed from April 2008 to June 2009 weekly investigations on microphytoplankton community structure and abundance in two contrasted marine ecosystems located in the western Moroccan Mediterranean coast, M'diq Bay and Oued Laou Estuary. Simultaneously, we measured the main physico-chemical parameters. Globally, the two studied areas showed comparable values of the assessed abiotic environmental factors. Temperature and salinity followed seasonal variation with values ranging from 13.5°C to 21.4°C and 31 to 36.8, respectively. Average nutrient values in surface water ranged from 0.7 to 45.76 μM for dissolved inorganic nitrogen, 0.02-2.10 μM for PO 4 and 0.23-17.46 μM for SiO 4 in the study areas. A total of 92 taxa belonging to 8 taxonomic classes were found. The highest number of microphytoplankton abundance reached 1.2 × 10 6 cells L −1 with diatoms being the most abundant taxa. Factorial Discriminant Analysis (FDA) and Spearman correlation test showed a significant seasonal discrimination of dominant microphytoplankton species. These microorganisms were associated with different environmental variables, in particular temperature and salinity. Numerous HABs species were encountered regularly along the year. Although Dinophysis species and Prorocentrum lima were present in both sites, no Lipophilic Shellfish Poisoning was detected for the analyzed bivalve mollusks. Domoic acid (DA), produced by toxic species of Pseudo-nitzschia was found with concentrations up to 18 µg DA g −1 in the smooth clam Callista chione. Data showed that the observed persistent and dramatic Paralytic Shellfish Poisoning (PSP) intoxication of mollusks resulted probably of Gymnodinium catenatum pro-liferations in both studied areas. Contrary to C. chione, the cockle Achanthocardia tuberculatum showed a permanent and extremely high toxicity level during the 15 months survey with up to 7545 µg Equivalent Saxitoxin kg −1 flesh (ten times higher than the sanitary threshold of 800 µg eqSTX Kg −1 flesh). The present work highlights for the first time the dynamic of microphytoplankton including HABs species and their associated toxin accumulation in the commercially exploited shellfish in the southern western Mediterranean waters of Morocco. Furthermore, the acquired data will help us to improve the monitoring of HABs species and related toxins in these coastal marine systems.

International audience; Atoll lagoons display a high diversity of trophic states due mainly to their specific geomorphology, and probably to their level and mode of human exploitation. We investigated the functioning of the Ahe atoll lagoon, utilized for pearl oyster farming, through estimations of photosynthetic parameters (pulse amplitude modulation fluorometry) and primary production (13C incorporation) measurements of the size structured phytoplankton biomass (2 μm). Spatial and temporal scales of variability were surveyed during four seasons, over 16 months, at four sites within the lagoon. While primary production (P) was dominated by the picophytoplankton, its biomass specific primary productivity (PB) was lower than in other atoll lagoons. The variables size fraction of the phytoplankton, water temperature, season, the interaction term station * fraction and site, explained significantly the variance of the data set using redundancy analysis. No significant trends over depth were observed in the range of 0-20 m. A clear spatial pattern was found which was persistent over the seasons: south and north sites were different from the two central stations for most of the measured variables. This pattern could possibly be explained by the existence of water cells showing different water residence time within the lagoon. Photoacclimation strategies of the two size fractions differed through their light saturation coefficient (higher for picophytoplankton), but not through their maximum photosynthetic capacity (ETRmax). Positive linear relationships between photosynthetic parameters indicated that their dynamic was independent of light availability in this ecosystem, but most probably dependent on nutrient availability and/or rapid changes in the community structure. Spatial and temporal patterns of the measured processes are then further discussed in the context of nutrient availability and the possible role of cultured oysters in nutrient recycling

Coccolithus braarudii and Calcidiscus leptoporus are 2 coccolithophores (Prymnesio- phyceae: Haptophyta) known to possess a complex heteromorphic life cycle, with alternation between a motile holococcolith-bearing haploid stage and a non-motile heterococcolith-bearing diploid stage. The ecological implications of this type of life cycle in coccolithophores are currently poorly known. The nutritional preferences of each stage of both species, and their growth response to conditions of turbulence were investigated by varying their growth conditions. Of the different cul- ture media tested, only the synthetic seawater medium did not support the growth of both stages of C. braarudii and C. leptoporus. With natural seawater-based media, the growth rate of the haploid phase of both coccolithophores was stimulated by the addition of soil extract (K/2: 0.23 ± 0.02 d -1 and K/2 with soil extract 0.35 ± 0.01 d -1 for the C. braarudii haploid stage), while the diploid phase was not, indicating that the motile stage is capable of utilizing compounds present in soil extract or ingest- ing bacteria that are activated in enriched media. The addition of sodium acetate to the medium also stimulated the haploid phase of C. braarudii, and further experiments using labeled bacteria demon- strated the capacity for phagotrophy of this motile stage. The effect of nutrient concentrations on the growth rates of both species was evaluated, showing clear differences between the 2 phases of the life cycle, with higher growth rates for the diploid stage in nutrient-rich media (K/2: 0.34 ± 0.04 d -1 , K/25: 0.29 ± 0.01 d -1 , K/100: 0.16 ± 0.01 d -1 for the haploid stage of C. leptoporus). Responses of the 2 phases of both coccolithophores to physical turbulence were also different, with a haploid flagellate stage sensitive to mixing and a more resistant non-motile diploid stage. The results of these experi- ments strongly indicate that each morphological stage of C. braarudii and C. leptoporus corresponds to a different ecological niche, the motile haploid stage exploiting a more stable oligotrophic niche than the diploid non-motile stage, in accordance with field observations. The 2-dimensional phyto- plankton niche space model of Margalef (1978; Oceanol Acta 1:493-509), defined by nutrients and turbulence, was amended to integrate this hypothesis.

The number of microalgae of high nutritional value actually available to mollusc hatcheries is limited to a few species and this represents a constraint for the industry. Fifteen microalgal species belonging to seven different taxonomic classes were selected from the Algobank-Caen microalgal culture collection of the University of Caen. An initial screening of these microalgae, based on their cytomorphological characteristics and growth performances, led to the selection of four species (Imantonia rotunda, Emiliania huxleyi, Pseudoisochrysis paradoxa and Diacronema vlkianum), which were then tested as food for Crassostrea gigas larvae. Two species (I. rotunda and E. huxleyi) were of poor food value for oyster larvae, while P. paradoxa and D. vlkianum resulted in high growth rate and low mortality. These two microalgae were then characterised (dry weight, ash, gross composition, fatty acids) at different stages of growth and their productivity in standard hatchery conditions (2-l glass carboys, 300-l cylinders) was assessed in order to evaluate the potential of these species for use in commercial hatcheries.

Microscopical and molecular analyses were used to investigate the diversity and spatial community structure of spring phytoplankton all along the estuarine gradient in a macrotidal ecosystem, the Baie des Veys (eastern English Channel). Taxa distribution at high tide in the water column appeared to be mainly driven by the tidal force which superimposed on the natural salinity gradient, resulting in a two-layer flow within the channel. Lowest taxa richness and abundance were found in the bay where Teleaulax-like cryptophytes dominated. A shift in species composition occurred towards the mouth of the river, with the diatom Asterionellopsis glacialis dramatically accumulating in the bottom waters of the upstream brackish reach. Small thalassiosiroid diatoms dominated the upper layer river community, where taxa richness was higher. Through the construction of partial 18S rDNA clone libraries, the microeukaryotic diversity was further explored for three samples selected along the surface salinity gradient (freshwater - brackish - marine). Clone libraries revealed a high diversity among heterotrophic and/or small-sized protists which were undetected by microscopy. Among them, a rich variety of Chrysophyceae and other lineages (e.g. novel marine stramenopiles) are reported here for the first time in this transition area. However, conventional microscopy remains more efficient in revealing the high diversity of phototrophic taxa, low in abundances but morphologically distinct, that is overlooked by the molecular approach. The differences between microscopical and molecular analyses and their limitations are discussed here, pointing out the complementarities of both approaches, for a thorough phytoplankton community description. peerReviewed

International audience; Estuaries and coastal lagoons are included within the transitional waters category, according to the Water Framework Directive. However, criteria for their differentiation and characterisation are still under discussion and require more research. In particular, detailed observations of biodiversity in more complex transitional and coastal waters are lacking. Microscopic and molecular analyses were therefore used to investigate phytoplankton diversity and spatial community structure, in early spring, along the freshwater-to-marine continuum of the Segura River (Spain), an intensively regulated semiarid basin discharging into the Mediterranean Sea. In addition to the salinity gradient as the major factor determining taxa distribution, influence of multiple anthropogenic and climatic impacting factors (drought, confined waters, irrigation canal) leads to a significant spatial heterogeneity of the aquatic habitat types associated with variations in community composition. Several shifts within the phytoplankton distribution pattern along the continuum are revealed using multivariate analyses. An impressive bloom of the cryptophyte Plagioselmis prolonga occurred in the mixing zone, associated with a typical euryhaline community indicative of eutrophication. The 18S rDNA diversity revealed a microeukaryotic richness including several little-known groups, heterotrophic representatives, and potential parasites. By combining morphological and molecular approaches we revealed the presence of a ‘hidden’ diversity often neglected in traditional surveys.

The diversity of Pseudo-nitzschia (Bacillariophyceae) and accumulation of the neurotoxin domoic acid (DA) in two types of shellfish; tuberculate cockles (Acanthocardia tuberculata) and sweet clams (Challista chione) was explored in M’diq Bay,Morocco during 2007. The highest abundances of Pseudo-nitzschia were found during the period from March to October, with peaks occurring in May and September. Toxin analysis showed an accumulation of domoic acid in shellfish sampled during spring and autumn. The maximum toxin concentration was 4.9 mg DAg-1 of the whole tissue recorded in sweet clam during spring. Using transmission electron microscopy, thirteen Pseudo-nitzschia species were identified, eight of which are known as producers of domoic acid: P. multistriata, P. cuspidata, P. galaxiae, P. multiseries, P. pseudodelicatissima, P. pungens var. aveirensis, P. calliantha and P. fraudulenta. The five non- toxic species observed were P. subpacifica, P. arenysensis, P. dolorosa, P. subfraudulenta, and P. cf. caciantha

International audience; Background: DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates. Methodology/Principal Findings: The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name. Conclusions/Significance: The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.

International audience; Time series of soluble extracellular polymeric substance (S-EPS) and transparent exopolymer particle (TEP) fractions, as well as biological, physical and chemical parameters, were studied in natural phytoplankton assemblages at 2 macrotidal sites located in the English Channel for 3 yr. The first site, the Bay des Veys (BDV), is sheltered from prevailing winds and influenced by a high river discharge, whereas the second, Lingreville-sur Mer (LGV), is exposed to the open ocean and to dominant winds. At both sites, the highest TEP concentrations were measured in spring and summer (1735 and 3604 μg equiv X l-1 at BDV and LGV, respectively) and were correlated with phytoplankton biomass and nitrogen concentrations. During the autumn and winter, TEP dynamics were not related to phytoplankton dynamics but appeared controlled by hydrodynamics. In contrast to TEP concentrations, S-EPS did not present any seasonal dynamics and was not correlated with TEP variations. The highest amount of S-EPS was recorded in spring at LGV (25.8 mg equiv. glucose l-1) and in summer at BDV (26.4 mg equiv. glucose l-1). The S-EPS pool was separated in 2 fractions according to the molecular weight: low (LW) and high (HW). The LW fraction was produced in higher quantity than the HW fraction, and with larger temporal fluctuations. Therefore, both S-EPS fractions seem controlled by different environmental parameters depending on the season and on the studied ecosystem. The different dynamics observed for TEP and S-EPS confirm the complexity of carbon excretion processes in phytoplankton, and the results reveal different metabolic pathways and different origins for these carbon excretions.

The brown seaweed Sargassum vulgare is abundant along the coast of Rio de Janeiro state. An investigation of the spatial variation of antifouling activity was conducted, in which algae were collected at five locations along the coast of Rio de Janeiro during October 2008. Hexane, dichloromethane, methanol and acetone/water extracts were prepared and screened for their bioactivities against the growth of five strains of marine fouling bacteria, five biofilm-associated microphytobenthic strains and attachment of the mussel Perna perna. The most active were the hexane extracts from Bananal algae that inhibited the growth of all microalgae tested; the methanol and dichloromethane extracts from Mar do Norte, which inhibited Vibrio aestuarianus and Pseudoalteromonas elyakovii and the polyphenol extracts from Ilha de Itacuruca and Bananal that inhibited mussel attachment, respectively, by 64% and 71% compared to controls.

Four unidentified new strains of Pavlovophyceae [Pavlova sp. AC 250 (Pfl), Pavlova sp. AC 248 (Psh), Pavlova sp. AC 251 (Pth), Pavlova sp. AC 538 (Psm)] and two known species [Pavlova pinguis (Ppi), Rebecca salina (ex Pavlova salina, Rsa)] were characterized (productivity, size, dry weight, ash, gross composition, fatty acids, sterols) and their nutritional value in bispecific diets were evaluated both on Crassostrea gigas and Pecten maximus larvae. All microalgae exhibited poor food value for C. gigas larvae. P. pinguis and R. salina did not sustain any growth, like the control starved larvae. No exo-toxicity of P. pinguis and R. salina was detected. These two species were weakly ingested by C. gigas larvae, while the four other pavlovophytes were effectively grazed. When used as food for P. maximus larvae, P. pinguis and Psh led to poor development. In contrast, a diet with Pfl resulted in significantly better growth than the control.

Background: Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known ‘‘species’’, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings: In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance: COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.