CGRP regulates the activity of mantle cells and hemocytes in abalone primary cell cultures (Haliotis tuberculata).

ABSTRACT Mollusc shell formation requires calcium ([Ca.sup.++]) and bicarbonate (C[O.sub.3]) ions, transported through the mantle via the hemolymph, and the secretion of an organic matrix that interacts with the mineral ions to form either the aragonite or calcite polymorph of calcium carbonate (CaC[O.sub.3]). The biomineralization process takes place in the extrapallial space, between inner shell and outer mantle epithelium, and is believed to be under the control of calcium-binding proteins and hormonal factors such as calcitonin-related molecules (CGRP-like peptides). Epithelial cells from the mantle are responsible from the secretion of matrix molecules whereas hemocytes are involved in ion transportation during shell formation and regeneration. To understand the biochemical and cellular events implied in the biomineralization process, we developed primary ceil cultures from the nacreous gastropod Haliotis tuberculata. Mantle cells as well as hemocyte cell cultures were successfully maintained in vitro and evaluated for their viability and proliferation using semiautomated assays. The effect of calcitonin-related molecules (h-CGRP) on cellular activity and proliferation was evaluated in those primary cell cultures. The results indicate that human-CGRP modulates the activity of both mantle cells and hemocytes and increases carbonic anhydrase activity in vitro. These results are consistent with those obtained in vivo, supporting the hypothesis that CGRP-like molecules control the activity of target cells involved in the biomineralization process.

KEY WORDS: Haliotis tuberculata, biomineralization, cell culture, mantle, hemolymph, CGRP-like peptides, carbonic anhydrase

INTRODUCTION

Molluscan shell biomineralization involves the uptake of mineral ions from the environment and their assembly into a functional organic matrix secreted by specialized cells of the outer mantle epithelium. This organic matrix is responsible for crystal nucleation, growth and orientation and its composition is specific to each shell layer (Belcher et al. 1996, Falini et al. 1996, Marxen & Becker 1997). According to the nature of organic matrix molecules, calcium carbonate will precipitate to form either calcite or aragonite polymorphs (Thompson et al. 2000, Marin & Luquet 2004). The extrapallial area, located between the mantle and the inner shell, is the preferential site for biomineralization where the organic components are secreted by mantle outer epithelial cells in ionic supersaturation conditions (Lin & Meyers 2005). In abalone, biomineralization is initiated by the deposition of an organic sheet at the inner shell surface, followed by the growth of calcite layer and finally by the growth of nacreous aragonite. The experimentally induced depositional sequence was shown to be identical to that observed in native shell and evidenced the role of mantle cells in the protein secretion activation (Fritz et al. 1994, Zaremba et al. 1996, Hawkes et al. 1996). But, the mechanisms of mantle cell regulation and their cooperation with hemocytes at the cell-mineral interface still remains unknown.

Two enzymes at least are involved in the biomineralization process, carbonic anhydrase, and alkaline phosphatase. Carbonic anhydrase catalyzes carbonate ion formation from carbon dioxide in solution in seawater and its activity has long been linked with shell formation (Wilbur & Jodrey, 1956, Freeman 1960, Medakovic & Lucu 1994, Medakovic 2000). In marine bivalves, carbonic anhydrase was previously identified in mantle and gills (Duvail et al. 1998, Medakovic 2000, Rousseau et al. 2003) and its activity was found to be correlated to larval shell development (Medakovic & Lucu 1994, Medakovic 2000). In the gastropod Haliotis tuberculata, the gill carbonic anhydrase increased during mollusc growth (Duvail & Fouchereau-Peron 2001). Moreover, the identification of a carbonic anhydrase domain in a nacreous protein from the oyster Pinctada Jucata suggested a role of this enzyme in the formation of the aragonite polymorph (Miyamoto et al. 1996).

There are only few studies on the regulation of the biomineralization process. Calcitonin-gene related peptides, derived from the calcitonin gene by alternative splicing, have been shown to control calcium metabolism in vertebrates at high concentration (Tippins et al. 1984). In primary culture of human osteoblast-like cells, CGRP increased cell proliferation, suggesting a role of the peptide in the local regulation of bone formation (Villa et al. 2000). CGRP-like molecules were identified in mantle and gills of the abalone Haliotis tuberculata (Duvail et al. 1997) and more recently, human-CGRP was found to increase carbonic anhydrase activity in oyster gills (Cudennec et al. 2006). In the same way, a Mollusk Insulin related Peptide, discovered in a gastropod species (Lymnea) regulates the concentration of calcium-binding protein (Ca-Bp) in the mantle (Joosse 1988). However, the role of these peptides in the control of calcification has not been clearly demonstrated.

Epithelial cells from the mantle are responsible from the secretion of matrix molecules whereas circulating cells (hemocytes) are involved in ion transportation during shell formation and regeneration (Wilbur 1964, Awaji & Suzuki 1998). To further understand the biochemical and cellular events orchestrated in the biomineralization process, we developed primary cell cultures from the mantle and hemolymph of the nacreous gastropod Haliotis tuberculata. Primary cultures derived from mollusc tissues have been successfully used for in vitro studies on cell growth, differentiation and induction of biomineralization (Lebel et al. 1996, Sud et al. 1998, 2001, Serpentini et al. 2000, Suja & Dharmaraj 2005). Because they provide heterogeneous models containing all the cell types that are present in the tissue of origin, explant primary cultures were suitable for further in vitro testing (Machii & Wada 1989, Auzoux et al. 1993, Domart-Coulon et al. 1994, Awaji & Suzuki 1998). We thus used the explants method, previously reported for cultivating mollusc tissues, to develop mantle cell primary cultures from the abalone Haliotis tuberculata. On the other hand, using the method previously described by Lebel et al. (1996), we established primary cultures of hemocytes that are known to participate in mollusc shell formation (Wilbur & Saleuddin 1983, Awaji & Suzuki 1998).

Our objectives focused on the identification and maintenance of cells, the evaluation of cell metabolism and cell-cycle status in vitro, and the use of primary cell cultures for in vitro testing of human-CGRP, a calciotropic hormone that is thought to participate in the control of biomineralization. Cell responses were compared in both mantle and hemolymph in vitro models and carbonic anhydrase was used as a biochemical marker of mineralizing activity.

MATERIALS AND METHODS

Source and Maintenance of Animals

Adult abalone (Haliotis tuberculata), 60-90 mm in shell length, were collected from the North coast of Britanny (Roscoff, France). They were maintained at the laboratory in tanks with aerated natural sea water at seasonal ambient temperature. Abalone were fed daily with Palmaria palmata algae and were fasted for two days before experiments.

Mantle Explants Cultures

Parts of the mantle, in contact with the shell edge, were dissected and placed overnight in an antiseptic solution containing 200 U/mL penicillin, 200 [micro]g/mL streptomycin, 250 [micro]g/ mL gentamycin and 2 [micro]g/mL amphotericin B. Mantle samples were minced into 2-3 [mm.sup.3] explants which were placed to adhere onto the bottom of plastic 6-well plates (10-12 explants/well). After one hour for adhesion, the explants were covered with 1 mL modified Leibovitz L-15 medium (L-15 adjusted to 1,100 mosmol/L by addition of mineral salts and supplemented with 100 U/ml penicillin, 100 [micro]g/mL streptomycin, 200 [micro]g/mL gentamycin and 1 [micro]g/mL amphotericin B). Explant primary cultures were incubated at 18[degrees]C in a humidified incubator and observed daily under an inverted phase contrast microscope. The medium was renewed every 3-4 days.

After 4-5 days of primary culture in the previously defined conditions, explants and mantle cells were removed and washed twice in a saline solution. The cell suspension was strained on a 70-[micro]m mesh filter (Cell strainer, VWR Internat.), resuspended in modified Leibovitz L-15 medium and assessed for cell density and viability. Cell viability was estimated on a hemocytometer using the Trypan blue exclusion dye test (Philips 1973) and cells were then subcultured in microplates to perform quantitative assays. For optimal cellular response, cell density was adjusted to [10.sup.5] cells/well in 96-wells microplates and to [9.10.sup.5] cells/wells in 6-well microplates.

Primary Cultures of Hemocytes

After an incision in the foot, hemolymph was diluted in a sterile anticoagulant solution (Alsever) according to the technique previously described by Lebel et al. (1996). Cell density was evaluated using a hemocytometer and cells were rapidly plated in multiwell plates for in vitro experiments. One hour after seeding, the anticoagulant solution was removed and replaced by 100 [micro]L/well of modified Leibovitz L-15 medium. Cell cultures were incubated at 18[degrees]C in a humidified incubator.

Human-CGRP Assay

Human-CGRP (h-CGRP) was purchased from Bachem and was stored in lmM acetic acid at-80[degrees]C. For in vitro assays, the stock solution was diluted in modified Leibovitz L-15 medium and added into wells at the final concentrations: 0.05, 0.5, 5, 50, and 500 ng/mL. Final acetic acid concentrations in all treated and control cultures were always maintained under 0.1% to prevent any cytotoxic effect. The effect of h-CGRP was tested on both mantle cells and hemocytes primary cultures. The evaluation of in vitro effects included (i) the use of the XTT reduction as a physiological marker of cell metabolism and (ii) the assessment of carbonic anhydrase activity as a biochemical marker of carbonate ion formation.

XTT Reduction Assay

Cellular activity was measured by the XTT assay (Roche Laboratory, France) based on the reduction of a tetrazolium salt (XTT) into yellow formazan salt by active mitochondria (Mosmann 1983). As for the MTT assays that we previously adapted to marine mollusc cells, the XTT assay provides a global evaluation of the number of viable cells and their in vitro mitochondrial activity (Domart-Coulon et al. 2000). For this assay, 50 [micro]L of a mixture of XTT/PMS was added to 100 [micro]l of cells in wells of a 96-well microplate. Plates were incubated for 6 h at 20[degrees]C. The reaction produced a colored product and the intensity of the color was measured by spectrophotometry at 490 nm with a 655 nm reference wave-length (in the Results, this is designed Optical Density: OD 490/655 nm and values were x 1,000).

Carbonic Anhydrase Activity

Cell suspensions were pelleted, homogenized in 0.01M PBS buffer, and centrifuged for 1 min at 10,000 g. The supernatant was fractionated in two aliquots for the measurement of carbonic anhydrase activity and the determination of protein content. The pH method was used to estimate the carbonic anhydrase activity at 4[degrees]C (Vitale et al. 1999). The reaction buffer was 225 mM mannitol, 75 mM sucrose, and 10 mM Tris-Phosphate, pH 7.4. For the enzymatic assay, 7.5 mL of buffer, 1 mL of CO2-saturated water and 0.1 mL of sample were used. The pH decrease was measured over 1 min with a pH-meter connected to a computer using Hanna Instruments software. A linear regression was fitted to pH data versus time, where the slope represented the catalyzed reaction rate (Ccr). The non-catalyzed reaction rate was determined from the pH decrease in the same reaction buffer using 0.1 mL of 0.01 M PBS as a control (Ncr). The protein content of each cell sample was measured by the bicinchoninic acid assay (Pierce, SIGMA) using serum albumine bovine (BSA) as a standard (Smith et al. 1985). Carbonic anhydrase activity was calculated using the following formula: (Ccr/Ncr)-1/mg of protein.

Flow Cytometric Analysis

Cellular DNA content was determined by staining cells with propidium iodide and measuring their fluorescence (FACScan, Becton Dickinson, Institut Jacques Monod, Paris, France). Mantle cells were resuspended and fixed at 20[degrees]C with 70% ethanol/PBS (10 mM [Na.sub.2]HP[O.sub.4], 138 mM NaC1, 2.7 mM KC1, pH 7.4). The fixed cells were incubated in a solution containing 100 [micro]g/mL RNase and 40 [micro]g/mL propidium iodide for 30 min at 37[degrees]C. For each cell population, 10,000 cells were analyzed by FACS and the percentage of cells in a specific stage of the cell cycle was determined with the propidium iodide DNA staining technique. Cells were classified in G0/G1, S and G2/M phases according to the intensity of the fluorescence peaks (Crissman et al. 1975).

Statistical Analysis

Statistical analysis was performed using the STATVIEW software (Biosoft). All data are expressed as means [+ or -] SD. Each graphic presents the mean values for one experiment calculated from 4-6 replicates. Each experiment was repeated at least three times. Differences between groups were assessed by ANOVA followed by a multiple comparison Tukey test. A probability of P < 0.05 was considered statistically significant.

RESULTS

Mantle Explants Primary Cultures

After one day of primary culture, 80% of the explants adhered to the flask bottom and an outgrowth of cells was observed out of the explant (Fig. 1). During the first 3-4 days, mantle cells migrated and spread on the flask bottom to form epithelial-like sheets (Fig. 2). Different cell types could be identified according to their morphology and behavior in vitro. Epithelial cells represented the main cell population and included round cells, 6-10 [micro]m in diameter, and cuboidal cells, 15-25 [micro]m in length, that are typical of the outer mantle epithelium. Fibroblast-like cells, 30-80 [micro]m in length, which are spindle-shaped adherent cells related to hemocytes, were identified. We also observed bigger round cells, 12 to 15-[micro]m in diameter, which were probably glandular mucous cells.

After 4-5 days primary culture, a large quantity of mantle cells was generated from the explants. At this time, approximately 90% of primary cultured cells were viable, according to the Trypan blue exclusion dye test and cells were transferred into 96-well microplates to perform quantitative assays.

Mantle Cells Metabolic Activity (XTT)

Figure 3 shows the XTT response (OD) as a function of mantle ceils number. The linear relationship demonstrated the correlation between mitochondrial activity and cell number (y = 0.0037x ; [R.sup.2] = 0.99). The XTT response of mantle cells appeared to be proportional to the number of viable cells and their mitochondrial activity.

[FIGURE 1 OMITTED]

To adjust the density in multiwell plates, and to measure the evolution of metabolic activity with time, we performed the XTT based assay on different series of subcultures seeded with cell densities from 25,000-100,000 cells per well. Figure 4 shows the evolution of the XTT response for a 14 day culture period. For each density, cellular metabolic activity remained quite constant during the 2 wk experiment. XTT response increased with cell density at any culture duration. At 100,000 cells/well, metabolic activity was increased slightly between 6 and 10 days and the largest value recorded was at day 14. For optimal cellular response, a minimum of 50,000 cells/well was used for further in vitro assays.

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Flow Cytometric Analysis of Mantle Cells

A flow cytometric analysis was performed on mantle cells from 5-day-old primary cultures to determine the position of cells in the cell-cycle. Cells were distributed in the different stages of their cell-cycle according to their fluorescent DNA content (Fig. 5). The major population was in the G0/G1 phase of the cell cycle (72%) and only a few cells were cycling as shown by the proportion in the G2/M phase (8%). The resting cells were either in synthesis (S) phase (10%) or apoptotic (10%).

Hemocyte Primary Cultures

During the first days of culture, hemocytes quickly adhered to, and displayed a great mobility on the bottom of culture flasks (Fig. 6). Two categories of hemocytes were recognized in vitro according to their morphology and behavior: amoeboidlike cells, 6-10 tam in diameter with a round shape, and fibroblast-like cells, 50 100-[micro]m in length, that firmly adhere to the flask and assemble together to form cellular networks (Fig. 6). Cell clusters were frequently observed at the beginning of culture and produced a lot of surrounding spindle-shaped cells that exhibited cytoplasmic expansions (Fig. 7). Further development of hemocyte cultures showed a decrease in fibroblast-like cells that were progressively replaced by round cells.

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Hemocytes Response to the XTT Assay

The XTT response of hemocytes also displayed a linear relationship with a good correlation between absorbances and cell density (y = 0.0215x; [R.sup.2] = 0.97, data not shown). When compared with mantle cells, the metabolic activity of hemocytes was found to be about 3-fold higher than that of epithelial mantle cells (Fig. 8).

[FIGURE 6 OMITTED]

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Effect of CGRP on Mantle Cell Activity

Figure 9 shows the metabolic activity of mantle cells, as a percent of control, in the presence of increasing concentrations of h-CGRP. An inhibition of cell metabolism occurred in the range 0.05 0.5 ng/mL, with a maximal inhibition at 0.05 ng/mL (58% of control), whereas a stimulation was observed at higher concentrations. Maximal activation was observed at 50 ng/mL (154% of control) and OD decreased afterwards.

[FIGURE 8 OMITTED]

[FIGURE 9 OMITTED]

Effect of CGRP on Hemocytes Metabolism

The change in hemocytes activity with the concentration of CGRP showed a similar sinusoidal curve, as hemocytes displayed a similar dose-dependant response to the peptide (Fig. 10). As observed for mantle cells, metabolic response was inhibited at low concentration (0.05 0.5 ng/mL) but was stimulated in the range 5-50 ng/mL with a maximal activation at 50 ng/mL (128% of control). At 500 ng/ml CGRP induced a decrease of hemocyte metabolic responses.

[FIGURE 10 OMITTED]

Effect of CGRP on Anhydrase Carbonic Activity

Figure 11 shows the evolution of carbonic anhydrase in mantle cells, normalized to protein content, in the presence of increasing concentrations of CGRP. A 2-fold increase of carbonic anhydrase activity was observed in the range 0.05-0.5 ng/mL and enzymatic activity reached a four times the control value between 5 and 500 ng/mL.

DISCUSSION

To better understand the biochemical and cellular events implied in the biomineralization process, we developed primary cell cultures from target tissues of the nacreous gastropod Haliotis tuberculata. Mantle cells as well as hemocyte cell cultures were successfully maintained in vitro and evaluated for their viability and proliferation using semiautomated assays and flow cytometry analysis. Primary explant cultures were established from parts of the mantle in contact with the shell edge. As previously observed in primary cultures of mollusc tissues, the explant method provided a suitable in vitro model that contained all cell types present in the tissue of origin (Kleinschuster et al. 1996). Two types of epithelial cells were related to typical cells from the outer mantle epithelium and circulating hemocytes were also present in vitro. The use of the XTT reduction assay allowed us to measure the metabolic activity of mantle cells. As for the MTT assay, that was previously adapted to marine mollusc cells (Domart-Coulon et al. 1994, Sud et al. 2001), the XTT assay provided a global evaluation of the number of viable cells and their in vitro mitochondrial activity. In our culture conditions, a minimum of 50,000 cells/wells was determined for an optimal cell response and mantle cell activity remained constant during 14 days after subculture.

[FIGURE 11 OMITTED]

A cytometric analysis was performed to assess the stage of mantle cells in the cell-cycle. Our results demonstrated that the majority of the cell population was in the G0/G1 phase of the cell cycle. As expected for somatic adult cells, the cytometric analysis revealed only a few percent (8%) of dividing cells. This result is not surprising and confirms previous studies that revealed only few dividing cells in primary cultures of adult tissues (Rinkevich 1999, Rinkevich 2005). As demonstrated in previous in vitro studies, the explant primary culture method provides an heterogeneous model where cell integrity is preserved and cell-to-cell interactions are maintained through the coexistence of epithelial mantle cells and circulating hemocytes (Machii & Wada 1989, Auzoux et al. 1993). Moreover, even in the absence of proliferation, our mantle primary cell culture displayed a basal metabolic activity that reflects cell functionality in vitro. When compared with previous dissociated cultures, explant primary cultures take more time to establish (4-5 days of cell migration) but provides a great diversity of healthy cells. Such explant cultures were recently used to study the mineralizing process in Haliotis varia abalone (Suja & Dharmaraj 2005). The authors reported an in vitro crystallization into specific granules elaborated by mantle epithelial cells that were related to granulocytes. Our primary explants cultures contained the main cell types involved in biomineralization process, epithelial cells and hemocytes, and were maintained in vitro for up to two weeks.

As hemocytes are known to participate in various physiological activities, such as immunity, shell regeneration and ion transportation, we also developed primary cultures of hemocytes and compared their metabolic activity to that of mantle cells. Our results are in accordance with previously reported hemocytes cultures and demonstrate the in vitro maintenance of circulating cells with a great metabolic activity. Typical circulating hemocytes (i.e., epithelial-like and fibroblast-like) were recognized in vitro, according to the classification from Auffret (1988). By contrast with previous reported cultures (Lebel et al. 1996) our primary cultures of hemocytes were maintained without any growth factor in the medium. As measured by the XTT reduction assay, metabolic activity of hemocytes was 3-fold higher than that of mantle cells. This is believed to be caused by their high "respiratory burst" a defense mechanism that has been described in various molluscs as an early response to parasite infection or pollutant contamination (Boulo et al. 1991, Wilson 1992, Lambert et al. 2003).

Primary cultures of hemocytes were used in parallel assays with mantle cells for in vitro testing of h-CGRP. In both in vitro models, we found an inhibition of cell activity at low concentration followed by an activation at higher concentration. Metabolic responses were inverted from 5 ng/mL CGRP; a concentration that corresponded to the physiological concentration of CGRP-like peptides found in mollusc tissues (Rousseau et al. 2003). Greater concentration of h-CGRP did not further increase cell responses and was believed to be cytotoxic. In vertebrates, it has been well established that many hormones and hormone disrupting chemicals exhibit a typical U-shaped dose-response curve (Almstrup et al. 2002). The cellular activation observed in our primary cell cultures may be because of an increase in cell metabolism and in cell number in response to the hormone. A stimulating effect of CGRP was previously observed on primary cultures of mammalian osteoblast-like cells and provided further evidence for the role of this peptide in bone formation (Villa et al. 2000). In marine molluscs, a variety of growth factors have been tested on mantle cells and hemocytes. Human IGF-1 was found to stimulate labeled-leucine incorporation in mantle cells of the scallop Pecten maximus and to increase protein synthesis in mantle cells of the oyster Crassostrea virginica (Gricourt et al. 2003). Molecules related to the insulin family (bovine insulin, EGF) also enhanced protein synthesis in hemocyte primary cultures from the abalone H. tuberculata (Lebel et al. 1996). Moreover, an EGF--like factor extracted from the mussel Mytilus edulis was shown to enhance mitogenic activity in dissociated mantle cells (Odintsova et al. 1993). Although they are widely distributed among invertebrate tissues, no reports have dealt with the effect of calcitonin-related molecules on mollusc cells. CGRP-like peptides were identified in mantle, gill, and hemolymph of molluscs and a decrease in CGRP level was correlated to an increase in circulating calcium in the oyster Pinctada margaritifera, suggesting a potential hypocalcemic effect (Rousseau et al. 2003). Our results demonstrate that human-CGRP modulates both epithelial cell and hemocyte activities in vitro. Further experiments are needed to identify CGRP receptors in cultured hemocytes and mantle cells.

To obtain more information on the mode of action of this peptide, we assayed its effect on the carbonic anhydrase activity, an enzyme involved in carbonate ion formation and previously identified in mollusc tissues (Duvail et al. 1998, Duvail & Fouchereau-Peron 2001, Rousseau et al. 2003). We found that mantle cells exhibited a carbonic anhydrase activity in vitro, and that human-CGRP significantly increased carbonic anhydrase activity in these cells. In addition to global mitochondrial activity, the presence of carbonic anhydrase activity in cultured mantle cells supports the maintenance of cell functionality in vitro. The mantle of molluscs is known to contain carbonic anhydrase activity (Freeman & Wilbur 1948, Wilbur & Jodrey 1956, Miyamoto et al. 1996, Medakovic 2000) and recent studies reported a stimulation of carbonic anhydrase activity by CGRP in oyster gill, supporting a role of the peptide in shell mineralization (Cudennec et al. 2006). Moreover, Miyamoto et al. (1996) suggested that carbonic anhydrase activity may result from matrix proteins secreted by epithelial cells from the mantle. Our results are in accordance with previously reported data and support the role of mantle cells in generating carbonic anhydrase activity.

In this study, we demonstrated that primary cell cultures of target tissues provide suitable models for further in vitro studies of the biomineralization process. The present data provide evidence for the role of CGRP-related molecules in the cell metabolism of the main cell types involved in the biomineralization process (epithelial cells as well as hemocytes). In addition to these in vitro metabolic effects, human-CGRP was shown to significantly increase the activity of carbonic anhydrase, which is known to catalyze carbonate ion formation. For the first time we demonstrate that the mantle, where carbonic anhydrase was identified, is also a target tissue for this enzyme. Taken together, our results support the hypothesis that CGRP-like molecules modulate the physiological activity of target cells in the process of mollusc shell biomineralization.

ACKNOWLEDGMENTS

This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS, Paris, France) and the Museum National d'Histoire Naturelle, (MNHN, Paris, France). The authors thank Dr Marie-Claude Gendron (Institut Jacques Monod, Paris, France) for fluorescent flow cytometry analysis and Dr Andrea Bullock (Ecole Pratique des Hautes Etudes, France) for her critical reading of the manuscript.

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STEPHANIE AUZOUX-BORDENAVE, (1) * MARTINE FOUCHEREAU-PERON, (1) MARIE-NOELLE HELLEOUET (1) AND DOMINIQUE DOUMENC (2)

UMR 5178 CNRS/UPMC/MNHN; (1) Station de Biologie Marine, BP 225 29182 Concarneau Cedex, France; (2) DMPA, Museum National d'Histoire Naturelle, 75231 Paris Cedex 05 France

* Corresponding author. E-mail: [e-mail]