Mussel as a Tool to Define Continental Watershed Quality

Bivalves appear as relevant sentinel species in aquatic ecotoxicology and water quality assessment. This is particularly true in marine ecosystems. In fact, several biomonitoring frameworks in the world used mollusks since several decades on the base of contaminant accumulation (Mussel Watch, ROCCH) and/or biological responses called biomarker (OSPAR) measurements. In freshwater systems, zebra and quagga mussels could represent alternative sentinels, which could be seen as the counterparts of mussel marine species. This chapter presents original studies and projects underlying the interest of these freshwater mussels for water quality monitoring based on contaminant accumulation and biomarker development measurements. These sentinel species could be used as a tool for chemical/biological monitoring of biota under the European water framework directive and for the development of effect-based monitoring tools

Cell motility and immunocompetence of Mytilus edulis hemocytes : applications to environmental diagnostics

La moule bleue Mytilus edulis est un mollusque bivalve filtreur et sessile. Cet organisme présente un intérêt majeur d'une part en conchyliculture et d'autre part dans de nombreux programmes de surveillance environnementale comme espèce indicatrice du niveau de contamination par les xénobiotiques. Ce manuscrit propose une revue de la compréhension actuelle des mécanismes de l'immunité exclusivement innée des invertébrés, en s'attachant plus particulièrement au rôle prévalent des cellules immunocompétentes chez Mytilus : les hémocytes. Nos résultats permettent de proposer une classification des sous-populations hémocytaires de Mytilus edulis. A partir de colorations cytologiques classiques combinées à la cytométrie en flux et au volume Coulter ainsi que d'une caractérisation originale des motilités cellulaires par videomicroscopie d'intervalle (time-lapse), nos résultats proposent une description des activités et des interactions d'au moins trois sous-populations hémocytaires : les basophiles, les hyalinocytes et les granulocytes éosinophiles. La vitesse de migration des hémocytes a été mesurée in vitro par une nouvelle méthode, le tracking des noyaux. Cette approche permet de quantifier les activités migratoires de ces cellules ainsi que leurs perturbations, notamment par les stress environnementaux. Le second axe développé consiste en une analyse du phénotype de Multi Xenobiotic Resistance (MXR) au sein des sous-populations hémocytaires. Ce travail indique également que les transporteurs ABCC/MR Prégulent la motilité des cellules, ce qui ouvre des perspectives importantes.Mytilus edulis is a bivalve mollusk and sessile filter feeder. This organism has a major advantage, both in aquaculture and in many environmental monitoring programs as an indicator species of the level of contamination by xenobiotics. This manuscript provides a review of the current understanding of the mechanisms of innate immunity exclusively invertebrates, focusing particularly prevalent role of immunocompetent cells in M. edulis : hemocytes . Our results allow us to propose an hemocyte subpopulations classification. By conventional cytological staining, flow cytometry equipped with a Coulter volume and characterization of cell motilities by videomicroscopy (time-lapse), our results provide a description of the activities and interactions at least three hemocyte subpopulations: basophils, and eosinophils hyalinocytes . The rate of of hemocytes migration was measured in vitro by a new method, the tracking of nuclei in time-lapse. This approach allows quantifying the migratory activity of these cells and their perturbations, including environmental stress. The second axis consists of an analysis of the phenotype Multi Xenobiotic Resistance (MXR) in the hemocyte subpopulations. This work also indicates that carriers ABCC / MRP regulate cell motility, which opens important perspectives in terms of biology immunity and evaluation of the immunocompetence of these animals

Motilité cellulaire et immunocompétence des hémocytes de Mytilus edulis (applications aux diagnostics environnementaux)

LE HAVRE-BU Centrale (763512101) / SudocSudocFranceF

Cell tracking and velocimetric parameters analysis as an approach to assess activity of mussel (Mytilus edulis) hemocytes in vitro

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A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes

International audienceImmunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 16 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity andphagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations

Molecular analysis of differential antiproliferative activity of resveratrol, epsilon viniferin and labruscol on melanoma cells and normal dermal cells

IF 3.778 (2016)International audienceVery recently, we have produced new resveratrol derived compounds, especially labruscol by culture of elicited grapevine cell suspensions (Vitis labrusca L.). This new polyphenolic oligomer could function as cancer chemopreventive agent in similar manner of resveratrol. In this study, we have determined the efficiency of resveratrol, ε-viniferin and the labruscol on human melanoma cell with or without metastatic phenotype. Our results show a differential activity of the three compounds where the resveratrol remains the polyphenolic compound with the most effective action compared to other oligomers. These three compounds block cell cycle of melanoma cells in S phase by modulating key regulators of cell cycle i.e. cyclins A, E, D1 and their cyclin-dependent kinases 1 and 2. These effects are associated with an increase of cell death while these compounds have no cytotoxic action on normal human dermal fibroblasts

Characterisation of <i>Mytilus edulis</i> hemocyte subpopulations by single cell time-lapse motility imaging

International audienceIn bivalve molluscs, defence against pathogens mainly relies on fast tissue infiltration by immunocompetent hemocytes that migrate from circulating hemolymph to sites of infection, in order to deliver, in situ, an effective immune response. In the present work, we have investigated dynamics of hemocyte subpopulations motility by combining flow cytometry coupled to Coulter-type cell volume determination, Hoffman modulation contrast microscopy, time-lapse imaging and off-line analysis of cell shape changes. Our results revealed fast modifications of hemocyte aspect in vitro, with bidirectional transitions from spread outlines to condensed cell body morphologies, in the minute range. Amoeboid or non-amoeboid types of locomotion were observed, depending on the cell shapes and on the cell subtypes, with velocities reaching up to 30 μm min−1. Correlations between motion profiles, Hemacolor staining and flow cytometry analysis on living cells help to propose a functional mussel hemocyte classification including the motile properties of these cells. In particular, basophils were shown to be involved in dynamic hemocyte–hemocyte interactions and in the constitution of aggregation cores. Physiological implications, in terms of immune response in organisms devoid of endothelium-closed vascular system, and potential applications of hemocyte motility studies for the development and the interpretation of experiments involving hemocytes in the field of marine ecotoxicology are discussed

Xylanase production by Thermobacillus xylanilyticus is impaired by population diversification but can be mitigated based on the management of cheating behavior

International audienceBackground The microbial production of hemicellulasic cocktails is still a challenge for the biorefineries sector and agro-waste valorization. In this work, the production of hemicellulolytic enzymes by Thermobacillus xylanilyticus has been considered. This microorganism is of interest since it is able to produce an original set of thermostable hemicellulolytic enzymes, notably a xylanase GH11, Tx-xyn11. However, cell-to-cell heterogeneity impairs the production capability of the whole microbial population. Results Sequential cultivations of the strain on xylan as a carbon source has been considered in order to highlight and better understand this cell-to-cell heterogeneity. Successive cultivations pointed out a fast decrease of xylanase activity (loss of ~ 75%) and Tx-xyn11 gene expression after 23.5 generations. During serial cultivations on xylan, flow cytometry analyses pointed out that two subpopulations, differing at their light-scattering properties, were present. An increase of the recurrence of the subpopulation exhibiting low forward scatter (FSC) signal was correlated with a progressive loss of xylanase activity over several generations. Cell sorting and direct observation of the sorted subpopulations revealed that the low-FSC subpopulation was not sporulating, whereas the high-FSC subpopulation contained cells at the onset of the sporulation stage. The subpopulation differences (growth and xylanase activity) were assessed during independent growth. The low-FSC subpopulation exhibited a lag phase of 10 h of cultivation (and xylanase activities from 0.15 ± 0.21 to 3.89 ± 0.14 IU/mL along the cultivation) and the high-FSC subpopulation exhibited a lag phase of 5 h (and xylanase activities from 0.52 ± 0.00 to 4.43 ± 0.61 over subcultivations). Serial cultivations on glucose, followed by a switch to xylan led to a ~ 1.5-fold to ~ 15-fold improvement of xylanase activity, suggesting that alternating cultivation conditions could lead to an efficient population management strategy for the production of xylanase. Conclusions Taken altogether, the data from this study point out that a cheating behavior is responsible for the progressive reduction in xylanase activity during serial cultivations of T. xylanilyticus . Alternating cultivation conditions between glucose and xylan could be used as an efficient strategy for promoting population stability and higher enzymatic productivity from this bacterium

Ex vivo immune responses in freshwater fish leucocytes exposed to environmental serotypes of lipopolysaccharides

In aquatic ecosystems, detected endotoxins or lipopolysaccharide (LPS) contamination can be linked to cyanobacteria blooms or enterobacterial contaminations. Water contaminations by endotoxins is of particular interest as these microbiological molecules may be released in freshwater often in combination with other chemicals (pharmaceutics…). Their immunomodulatory properties rise interest towards their pro-inflammatory action on mammalians and aquatic vertebrates. Despite the fact that some E.coli LPS serotypes such as O55:B5 are often used as immunostimulant in experimentations, no data are available concerning some other serotypes as O157:H7 even if they may significantly contaminate freshwater ecosystems, particularly in agricultural field contexts. We aimed to compare here the immunomodulation patterns potentially induced by two E.coli LPS serotypes (O55:B5 and O157:H7) alone or in combination with diclofenac, an anti-inflammatory drugs which chronically contaminate European freshwaters. Using an ex vivo approach, we studied immune parameters (Oxidative activity, Phagocytosis and cytotoxicity) of roach (Rutilus rutilus) leucocytes populations (lymphocytes, monocytes/macrophages and granulocytes) isolated from blood, head-kidney and spleen in response to endotoxins exposure. Leucocytes were exposed from 6 to 12 hours to sub-lethal concentrations of LPS and/or diclofenac (1μg/mL LPS, 0,1μM diclofenac) determined from preliminary results. Like diclofenac, endotoxins induced significant decrease in intracellular ROS production by leucocytes at time 0. After 12 hours of exposure to the E.coli O55:B5 LPS / diclofenac combination, the oxidative activity was stimulated in spleen leucocytes and, simultaneously, they showed a significant decrease in phagocytosis. Moreover, while the E.coli O157:H7 LPS alone showed tendencies to decrease phagocytic activity, the two endotoxin / diclofenac combinations significantly decreased it in head-kidney leucocytes after 6 hours. All these results will be set versus an ongoing in vivo experiment which would allow us to study more inflammation aspects like cytokines and immune-related factors production. Whereas environmental monitoring and studies of endotoxins presently concern only human health like in wastewater units, our results rise the questioning on environmental reality of endotoxins and their potential immunomodulations in fish