Metal bioaccumulation and physiological condition of the Pacific oyster (Crassostrea gigas) reared in two shellfish basins and a marina in Normandy (northwest France).

A 5-month experiment combining a geochemical survey of metals with a bioaccumulation study in batches of Crassostrea gigas was conducted in two shellfish farming areas and a marina in Normandy (France). Various endpoints at different levels of biological organization were studied. ROCCH data showed differences in biota contamination between the two shellfish areas but the present study revealed only slight differences in metallic contamination and biomarkers. By contrast, significantly different values were recorded in the marina in comparison with the two other sites. Indeed, higher levels of Cd, Cu and Zn were measured in the oysters from the marina, and these oysters also showed a poorer physiological condition (e.g., condition index, histopathological alterations and neutral lipid content). For coastal monitoring, the multi-biomarker approach coupled with an assessment of metallic contamination in biota appeared to be suitable for discriminating spatial differences in environmental quality after only a few months of exposure.

Toxicity of five antidepressant drugs on embryo-larval development and metamorphosis success in the Pacific oyster, Crassostrea gigas.

Unlike conventional pollutants, pharmaceutical residues are continuously discharged at low levels (low to mid ng l(-1) concentrations), which results in the chronic contamination of non-target organisms, but little is known about the effects of these residues. The purpose of this study was to provide the first assessment of the ecotoxicity of five antidepressants (selective serotonin reuptake inhibitors [SSRIs] fluoxetine and sertraline, tricyclic antidepressants [TCAs] clomipramine and amitriptyline, and serotonin norepinephrine reuptake inhibitor [SNRI] duloxetine) at a wide range of concentrations from 0.1 to 100,000 µg l(-1) on two early life stages in the Pacific oyster. The toxicity was quantified in D-shaped larvae after 36 h of exposure, and in 21-day-old pediveliger larvae after 24 h of exposure using the percentage of normal larval development and the metamorphosis rate as endpoints, respectively. The embryotoxicity assays reported that the EC50 values were within the same range of concentrations (67 to 192 µg l(-1)) for all of the tested molecules. The metamorphosis tests revealed that the antidepressants can be ranked along an increasing severity gradient: clomipramine < amitriptyline < duloxetine ~ fluoxetine. Sertraline appeared to be the less toxic molecule on this endpoint; however, a different concentration range was used. The embryotoxicity test was more sensitive than the metamorphosis bioassay for three of the five molecules tested, but the latter test showed more practical benefits. Overall, the obtained toxicity values were at least 10,000-fold higher than the reported environmental concentrations.

Shell repair process in the green ormer Haliotis tuberculata: a histological and microstructural study.

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata ('Oreille de Saint-Pierre') were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297-300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous (i.e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.

Preliminary study of the effects of commercial lactobacilli preparations on digestive metabolism of juvenile sea bass (Dicentrarchus labrax).

Two forms of the same commercial product (SORBIAL, Allonnes, France), one with live bacteria (PSA) and the other with heat-inactivated bacteria (PSI), containing a mixture of 2 strains of lactobacilli and their growth medium were tested as a diet complement for juvenile sea bass (Dicentrarchus labrax) during a 103-day experiment. In addition to zootechnical parameters (survival, growth, conformation), some effects on digestive metabolism were studied, including enzymatic, ultrastructural and microbial aspects. Microbial preparations improved survival rate. The ventral, dorsal and operculum malformations which usually occur in juveniles did not appear in those receiving PSA and PSI. Furthermore, they stimulated, but not constantly, trypsin and acid phosphatase activities. Intestinal ultrastructure showed an increase in the number of endocytosis vesicles at the apical pole of enterocytes in fishes receiving enrichments. Bacterial flora was not modified in terms of quantity, especially the lactic acid bacteria counts, which were not changed in fishes receiving live lactobacilli (PSA). The mode of action of these multiple beneficial effects appears complex and could be caused by different molecules inside the bacterial cell or excreted into their medium.

Cryopreservation of mantle dissociated cells from Haliotis tuberculata (Gastropoda) and postthawed primary cell cultures.

Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me(2)SO) or 10% glycerol (Gly) as a cryoprotector. The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells. Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities. Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells. In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors. These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure. Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology.

In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix.

In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [(3)H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [(3)H]-d-glucosamine macromolecules synthesized, [(3)H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% +/- 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.

Collagen study and regulation of the de novo synthesis by IGF-I in hemocytes from the gastropod mollusc, Haliotis tuberculata.

To evidence a collagen synthesis and identify which type(s) of collagen is present in hemocytes from the mollusc Haliotis tuberculata, we have performed three separate approaches, namely, de novo synthesis by cultured cells, immunological approaches, and northern blot analysis. We demonstrated first that after 40-hr labeling, the de novo synthesis of collagen in the cell layer of cultured hemocytes represents 9.48 +/- 1.25% with respect to the total [(3)H]proline-labeled protein synthesis. In addition, IGF-I elicited a significant stimulation of collagen synthesis in cultured hemocytes in a dose-dependent manner from 10(-10) to 10(-8) M. The maximal stimulation (10(-9) M) induced an increase of 286 +/- 56% with respect to 100% control. By immunocytochemistry and immunoblotting, we showed that hemocytes present immunoreactive molecules to antibodies directed against the type I fibrillar collagen. In addition, using as a probe Hf 677 corresponding to a human pro alpha1(I) collagen cDNA and which encompasses the (Gly-X-Y) repeated sequence found in all Metazoa, four collagen transcripts of approximately 6.4, 5, 2.2, and 2 kb in length have been detected. These data suggest the presence of fibrillar type I collagen in hemocytes and are compatible with the concept that these cells are involved in the extracellular matrix deposition, a cardinal function in tissue repair as well as in developmental processes. Our model may appear as an excellent system to study the role of growth factors on the regulation of collagen synthesis by molluscan hemocytes. J. Exp. Zool. 287:275-284, 2000.

[Cyclospora cayetanensis: review of an emerging intestinal pathogen]. / Cyclospora cayetanensis, un agent pathogène intestinal émergent.

Cyclospora cayetanensis is an emerging pathogen. It is a new human coccidian agent of intestinal disease. Twenty years ago, the first known human cases of cyclosporiasis were reported in the medical literature. Cyclosporiasis occurs in persons of all ages and either in immunocompetent or immunocompromised hosts. The most characteristic feature of this infection is a syndrome of acute or chronic diarrhea. This parasite has a world-wide distribution. In previous reports, Cyclospora cayetanensis was associated with prolonged diarrhea in travellers, returning from developing countries. However, Cyclospora infection has recently been reported in non travellers in the United States and Canada. Cyclospora can be transmitted by ingestion of water or food contaminated with oocysts. The life cycle of Cyclospora cayetanensis is not fully known. Diagnosis of cyclosporiasis is made by direct examination of stool samples. To date, oral trimethoprim-sulfamethoxazole is the only effective treatment for Cyclospora infection.