Selecting an HPLC method for chemotaxonomic analysis of phytoplankton community in Mediterranean coastal lagoons

1 - Phytoplankton observations are commonly used to contribute to the assessment of aquatic ecosystem health and their trophic status. Compared to other methods, chemotaxonomic analysis based on High Pressure Liquid Chromatography (HPLC) presents many advantages (e.g., rapidity, reproducibility, and capacity to include pigments from all cell sizes), but its use in coastal lagoons studies is still not very common. The method of Wright et al., (1991) recommended by the UNESCO (Jeffrey et al., 1997) and most frequently used for phytoplankton analysis in coastal lagoons, so far, was selected and compared to the more novel method of Hagerthey et al. (2006). 2 - The two methods that differed slightly with respect to their solvent gradients during chromatography (mobile phase) and column (stationary phase), were tested using a pigment mix from DHI Water and Environment comprising 30 different pigments. Extraction methods were tested using replicates of 1l of sub-surface water from the Thau lagoon (South of France), sampled in June 2013. Optimization of the extraction was performed by testing different volumes of solvent (2 to 5 ml), different solvents based on a mix of methanol, acetone, dimethylformamide, water, compared to acetone 90% and pure methanol, as well as different extraction times (10 min to 2 h), and the addition of the ion-pairing agent tetrabutyl ammonium acetate hydroxide (TBAA). 3 - The second method of analysis allowed better separation and resolution of most of the pigments, especially of lutein and zeaxanthin. The early-eluting most polar pigments and the more hydrophobic pigments eluting in the end of the chromatogram (chlorophylls and carotenoids) showed also better separation and peak shapes. 5 mL of the mix of acetone/ methanol/ water (45:45:10) allowed the best extraction of the pigments. The use of TBAA showed negative effects. 4 - For pigment analysis in coastal lagoon, our final protocol used 1 h extraction with 5 mL of acetone/ methanol/ water, and analysis with the gradient from Hagerthey et al. (2006). On our analytical equipment it needed some adjustments. It uses a longer chromatography run and quantified the phytoplankton pigment markers better than the method of Wright et al. (1991)

Expression of SKP1 mRNA and protein in rat brain during postnatal development

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First steps of ecological restoration in Mediterranean lagoons: Shifts in phytoplankton communities

Along the French Mediterranean coast, a complex of eight lagoons underwent intensive eutrophication over four decades, mainly related to nutrient over-enrichment from continuous sewage discharges. The lagoon complex displayed a wide trophic gradient from mesotrophy to hypertrophy and primary production was dominated by phytoplankton communities. In 2005, the implementation of an 11 km offshore outfall system diverted the treated sewage effluents leading to a drastic reduction of anthropogenic inputs of nitrogen and phosphorus into the lagoons. Time series data have been examined from 2000 to 2013 for physical, chemical and biological (phytoplankton) variables of the water column during the summer period. Since 2006, total nitrogen and phosphorus concentrations as well as chlorophyll biomass strongly decreased revealing an improvement in lagoon water quality. In summertime, the decline in phytoplankton biomass was accompanied by shifts in community structure and composition that could be explained by adopting a functional approach by considering the common functional traits of the main algal groups. These phytoplankton communities were dominated by functional groups of small-sized and fast-growing algae (diatoms, cryptophytes and green algae). The trajectories of summer phytoplankton communities displayed a complex response to changing nutrient loads over time. While diatoms were the major group in 2006 in all the lagoons, the summer phytoplankton composition in hypertrophic lagoons has shifted towards green algae, which are particularly well adapted to summertime conditions. All lagoons showed increasing proportion and occurrence of peridinin-rich dinophytes over time, probably related to their capacity for mixotrophy. The diversity patterns were marked by a strong variability in eutrophic and hypertrophic lagoons whereas phytoplankton community structure reached the highest diversity and stability in mesotrophic lagoons. We observe that during the re-oligotrophication process in coastal lagoons, phytoplankton shows complex trajectories with similarities with those observed in freshwater lake systems

Ovogenèse chez l'Anguille (Anguilla anguilla L.) : ultrastructure de l'ovaire à différents stades de développement et implication des lipoprotéines au cours de la vitellogenèse

Dans les eaux douces, l'anguille est présente à deux stades de développement : le stade jaune où l'animal se nourrit et assure sa croissance et le stade argenté où il jeûne et se prépare à la migration de reproduction. Ce cycle est particulièrement intéressant pour l'étude des régulations hormonales de l'ovogénèse et en particulier celle de la vitellogenèse, étape pendant laquelle l'ovocyte incorpore la vitellogénine pour former le vitellus. Cet article décrit, d'une part, les différents critères de la vitellogenèse disponibles chez l'anguille (caractérisation des lipoprotéines, ultrastructure des ovocytes, teneurs plasmatiques des stéroïdes sexuels et de la vitellogénine, caractérisation des récepteurs hépatiques de l'oestradiol (RE2)) et, d'autre part, l'effet de traitements gonadotropes ou stéroïdiens sur ces critères pour dégager la nature des contrôles endocriniens impliqués au cours de la vitellogenèse. Les lipoprotéines présentes dans le plasma de l'anguille jaune ou argentée sont les VLDL (lipoprotéine de très faible densité) et les HDL (lipoprotéine de haute densité) ; les LDL (lipoprotéine de faible densité) ne sont pas trouvées. Des modifications des apolipoprotéines présentes dans les lipoprotéines de haute densité (HDL) sont observées au cours de l'argenture. Par des traitements à l'oestradiol, les VLDL augmentent considérablement ; il apparaît aussi une classe nouvelle de lipoprotéines, les VHDL (lipoprotéine de très haute densité) ou vitellogénine. L'analyse de l'ultrastructure ovocytaire montre que l'anguille jaune est prévitellogénique en raison de l'absence des structures ovocytaires spécifiques de l'endocytose ; elles sont présentes chez l'anguille argentée qui est donc vitellogénique. In vivo chez l'anguille jaune, après un mois de traitement gonadotrope, une augmentation de l'oestradiol (E2) plasmatique est observée sans apparition de vitellogénine circulante. Toutefois, si les traitements sont prolongés, la vitellogenèse s'amorce. Chez l'anguille argentée, la vitellogenèse est induite dès la deuxième semaine. La testostérone puis l'oestradiol augmente dans le plasma ainsi que la vitellogénine. Ce résultat suggère un rôle des androgènes dans l'induction hépatique de la vitellogenèse. Les études in vitro sur les hépatocytes d'anguille maintenus en culture primaire ont mis en évidence la compétence des oestrogènes et des androgènes à stimuler la synthèse de vitellogénine. Un effet amplificateur de l'hormone de croissance bovine (bGH) sur la synthèse de vitellogénine par des doses faibles de E2 a été mis en évidence in vivo et in vitro, suggérant une action directe de la GH sur le foie. Les récepteurs hépatiques de l'oestradiol (RE2) ont été caractérisés ; ils s'apparentent à la famille des récepteurs nucléaires des stéroïdes. Ils sont présents en quantité très faible dans le foie de l'anguille jaune ou argentée. Les premiers résultats obtenus suggèrent que l'action de la GH pourrait être associée à une synthèse de RE2 dans le foie. Des différences dans l'induction de la vitellogenèse par la gonadotropine de carpe sont mises en évidence entre les anguilles jaunes et argentées. Il semble que différentes lipoprotéines participent à l'élaboration du vitellus. L'induction et le contrôle de la vitellogenèse sont plurihormonaux

In vivo gene expression in granulosa cells during pig terminal follicular development.

International audienceOvarian antral follicular development is clearly dependent on pituitary gonadotrophins FSH and LH. Although the endocrine mechanism that controls ovarian folliculogenesis leading to ovulation is quite well understood, the detailed mechanisms and molecular determinants in the different follicular compartments remain to be clarified. The aim of this study was to identify the genes differentially expressed in pig granulosa cells along the terminal ovarian follicle growth, to gain a comprehensive view of these molecular mechanisms. First, we developed a specific micro-array using cDNAs from suppression subtractive hybridization libraries (345 contigs) obtained by comparison of three follicle size classes: small, medium and large antral healthy follicles. In a second step, a transcriptomic analysis using cDNA probes from these three follicle classes identified 79 differentially expressed transcripts along the terminal follicular growth and 26 predictive genes of size classes. The differential expression of 18 genes has been controlled using real-time PCR experiments validating the micro-array analysis. Finally, the integration of the data using Ingenuity Pathways Analysis identified five gene networks providing descriptive elements of the terminal follicular development. Specifically, we observed: (1) the down-expression of ribosomal protein genes, (2) the genes involved in lipid metabolism and (3) the down-expression of cell morphology and ion-binding genes. In conclusion, this study gives new insight into the gene expression during pig terminal follicular growth in vivo and suggested, in particular, a morphological change in pig granulosa cells accompanying terminal follicular growth