Estudio ultraestructural de la ovogénesis en Marphysa sanguinea (Annelida: Polychaeta: Eunicida) en la Laguna de Túnez

Ultrastructural features of oogenesis of the iteroparous and long lived eunicid polychaete Marphysa sanguinea (Montagu, 1815) from the Lagoon of Tunis were studied using both light and transmission electron microscopy methods. The ovaries consist of coelomic germ-cell clusters surrounded by a thin envelope of follicle cells derived from the peritoneum. They are attached to the genital blood vessels. Oogenesis is asynchronous; therefore, germ cells in premeiotic and previtellogenic phases are observed in one cluster. The oogenesis is of the extraovarian type. In each cluster the more differentiated oocytes detach and float free in the coelomic cavity where they undergo vitellogenesis as solitary cells. Inside the ovary, follicle cells are connected to young oocytes by intercellular bridges and data suggest that in the early stages of vitellogenesis, ribosomes, multivesicular bodies and dense bodies are transferred from the follicle cells to the oocytes through the intercellular bridges. Different morphological evidence supports the heterosynthetic (presence of endocytotic vesicles, abundant microvilli adorning the surface of the egg) and autosynthetic (proteosynthetic organelles well developed, formation of blebs by the external membrane of the nuclear envelope) origin of yolk in M. sanguinea. The cytoplasmic material of the mature oocytes is asymmetrically distributed; large lipid droplets (1.5 μm in size) and large yolk spheres (6 μm in size) occupy the vegetal pole of the oocyte while small yolk spheres (2-3 μm in size) and numerous membranous organelles (mitochondria, cisternae of endoplasmic reticulum) occupy the animal hemisphere. Spherical to elongated cortical granules up to 1.8 μm in size are located in the cortex of the mature oocytes. Finally, fibrogranular aggregates (“nuage”) similar to that within the nucleus are observed in the cortical ooplasm located in the animal pole of mature oocytes. These aggregates probably come from the nucleus and represent maternal cytoplasmic determinants of embryonic cell fate. Oocytes that have completed vitellogenesis measure 250–300 μm in diameter.Se estudiaron las características ultraestructurales de la ovogénesis del poliqueto de la familia Eunicidae, Marphysa sanguínea (Montagu, 1815), caracterizado por ser iteróparo y presentar una largo periodo de vida vital. Los ejemplares proceden de la Laguna de Túnez y se estudiaron tanto mediante microscopía óptica como mediante microscopía electrónica de transmisión. Esta especie presenta ovarios consistentes en agrupaciones de células germinales celómicas rodeadas por una envoltura delgada de células foliculares derivado del peritoneo y unidos a los vasos sanguíneos genitales. La ovogénesis es asincrónica, por lo que se observan células germinales en fases premeióticas y previtelogénicas en cada clúster, y es de tipo extraovárico. En cada clúster, los ovocitos más diferenciados se desprenden y flotan libres en la cavidad abdominal donde experimentan la vitelogénesis como celdas aisladas. En el interior del ovario, los ovocitos jóvenes están conectados con células foliculares mediante puentes intercelulares, los cuales según los datos obtenidos, permiten la transferencia de ribosomas, cuerpos multivesiculares y cuerpos densos desde las células foliculares a los ovocitos en las primeras etapas de vitelogénesis. Diferentes pruebas morfológicas apoyan el origen heterosintético (presencia de vesículas endocitíticas, abundantes microvellosidades adornando la superficie del huevo) y autosintético (orgánulos proteosintéticos bien desarrollados, la formación de vesículas a partir de la membrana externa de la envoltura nuclear) del vitelo en M. sanguinea. El material citoplasmático de los ovocitos maduros se distribuye asimétricamente; grandes gotas de lípidos (1,5 μm) y grandes esferas de vitelo (6 μm) ocupan el polo vegetal del ovocito, mientras que esferas pequeñas de vitelo (2-3 μm) y numerosos orgánulos membranosos (mitocondrias, cisternas del retículo endoplásmatico) ocupan el hemisferio animal. Los ovocitos maduros presentan gránulos corticales (entre esféricos y alargados y de hasta 1,8 μm) en el córtex, mientras que en el ooplasma cortical del polo animal se presentan agregados fibrogranulares (“nuage”) similares a los del núcleo. Estos agregados probablemente provienen del núcleo y representan determinantes citoplasmáticos maternos del destino de las células embrionarias. Una vez completada la vitelogénesis, los ovocitos miden entre 250-300 μm de diámetro

Subcellular localization of microcystin in the liver and the gonads of medaka fish acutely exposed to microcystin-LR

International audienceAmong the diverse toxic components produced by cyanobacteria, microcystins (MCs) are one of the most toxic and notorious cyanotoxin groups. Besides their potent hepatotoxicity, MCs have been revealed to induce potential reproductive toxicity in various animal studies. However, little is still known regarding the distribution of MCs in the reproductive organ, which could directly affect reproductive cells. In order to respond to this question, an acute study was conducted in adult medaka fish (model animal) gavaged with 10 μg.g −1 body weight of pure MC-LR. The histological and immunohistochemical examinations reveal an intense distribution of MC-LR within hepatocytes along with a severe liver lesion in the toxin-treated female and male fish. Besides being accumulated in the hepatocytes, MC-LR was also found in the connective tissue of the ovary and the testis, as well as in oocytes and degenerative spermatocyte-like structures but not spermatocytes. Both liver and gonad play important roles in the reproductive process of oviparous vertebrates. This observation constitutes the first observation of the presence of MC-LR in reproductive cells (female, oocytes) of a vertebrate model with in vivo study. Our results, which provide intracellular localization of MC-LR in the gonad, advance our understanding of the potential reproductive toxicity of MC-LR in fish

Global Metabolomic Characterizations of Microcystis spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity

Cyanobacteria are photosynthetic prokaryotes capable of synthesizing a large variety of secondary metabolites that exhibit significant bioactivity or toxicity. Microcystis constitutes one of the most common cyanobacterial genera, forming the intensive blooms that nowadays arise in freshwater ecosystems worldwide. Species in this genus can produce numerous cyanotoxins (i.e., toxic cyanobacterial metabolites), which can be harmful to human health and aquatic organisms. To better understand variations in cyanotoxin production between clones of Microcystis species, we investigated the diversity of 24 strains isolated from the same blooms or from different populations in various geographical areas. Strains were compared by genotyping with 16S-ITS fragment sequencing and metabolite chemotyping using LC ESI-qTOF mass spectrometry. While genotyping can help to discriminate among different species, the global metabolome analysis revealed clearly discriminating molecular profiles among strains. These profiles could be clustered primarily according to their global metabolite content, then according to their genotype, and finally according to their sampling location. A global molecular network of all metabolites produced by Microcystis species highlights the production of a wide set of chemically diverse metabolites, including a few microcystins, many aeruginosins, microginins, cyanopeptolins, and anabaenopeptins, together with a large set of unknown molecules. These components, which constitute the molecular biodiversity of Microcystis species, still need to be investigated in terms of their structure and potential bioactivites (e.g., toxicity)

Les contaminations chroniques de poissons par les cyanobactéries

Les écosystèmes aquatiques subissent des altérations croissantes, dont l’apparition d’efflorescences à cyanobactéries produisant des cyanotoxines. Ces efflorescences mettent en péril le fonctionnement des milieux aquatiques. Le but de ce projet était l’étude des contaminations chroniques des poissons par les cyanobactéries

Capsular polysaccharides secreted by building façade colonisers: characterisation and adsorption to surfaces.

International audienceExopolymers secreted by algal and cyanobacterial strains isolated from building façades were imaged by microscopy techniques. They were extracted and characterised to investigate their possible contribution to interactions with solid surfaces. The polymers were polysaccharides, with anionic and hydrophobic properties varying between the various strains. Capsular polysaccharides extracted from a strain of Klebsormidium flaccidum adsorbed in higher amounts on hydrophobic than on hydrophilic surfaces. These results tend to confirm the hypothesis that exopolymers are important in the colonisation process of microorganisms to surfaces

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

International audienceThe decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments

Bacterial communities associated with Microcystis colonies differ from free-living communities living in the same ecosystem

International audienceThe search for a better understanding of why cyanobacteria often dominate phytoplankton communities in eutrophic freshwater ecosystems has led to a growing interest in the interactions between cyanobacteria and bacteria. Against this background, we studied the location of bacteria within Microcystis colonies, and compared the structural and phylogenetic diversity of Microcystis-attached and free-living bacterial communities living in the same French lake, the Villerest reservoir. Using transmission electron microscopy, we show that most of the bacteria inside the colonies were located close to detrital materials that probably resulted from lysis of Microcystis cells. The 16S rRNA sequencing approach revealed a clear distinction between the attached and free-living communities at the levels of both their general structure and their operational taxonomic unit (OTU) composition. In particular, Microcystis colonies appeared to be depleted of Actinobacteria, but conversely enriched in Gammaproteobacteria, in particular when the bloom was declining. At the OTU level, a clear distinction was also found between attached and free-living bacteria, and new clades were identified among our sequences. All these findings suggest that Microcystis colonies constitute a distinct habitat for bacteria living in freshwater ecosystems, and that direct and indirect interactions (cell lysis, nutrient recycling, etc.) may occur between them inside these colonies

Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a nonpathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments

Structures of 1–6 isolated from <i>P. variabile.</i>

<p>All structures were assigned by detailed analysis (NMR spectroscopic and mass data).</p