maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

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184 CHAPITRE 5. ADAPTATIONS RESPIRATOIRES DE S. MESATLANTICA creasing temperature (normal temperature effect). L-lactate, urate and thiosulfate are also well-known positive modulators of Hc affinity (triggered by anaerobic metabolism, stress, or sulfide detoxification for thiosulfate). Many crab species harbor Hc with original properties resulting from specific molecular adaptations to the environmental conditions they live in. For example, one terrestrial crab Hc has a reverse L-lactate effect (Adamczewska et Morris, 1998) ; Hc from the hydrothermal vent crab Bythograea thermydron is sensitive to thiosulfate (Sanders et Childress, 1992) whereas shore crab Hc is insensitive to it (Taylor et al., 1999). Some specific functional adaptations of Hc are known for hydrothermal crustaceans. The intrinsic affinity of their Hc is very high (2 to 5 Torr), as observed in others groups (annelids, molluscs) (Hourdez et Lallier, 2007). The temperature effect is usually negligible or slightly inverse, which is adaptive near hydrothermal vents since an increase in temperature is usually correlated with a decrease of the oxygen content of the mixed water due to a higher fraction of anoxic fluid. Such a specific temperature effect avoids a decrease of Hc affinity when animals are in the mixing zone which would result in an impaired oxygen loading at the gills in the depleted water. The Bohr effect is also often very strong for hydrothermal species, enabling an improved unloading of Hc near the active tissues. For the L-lactate, urate and thiosulfate effects, such general trends cannot be observed and the Hc sensitivity to each of them is rather species-specific. A previous study of Segonzacia mesatlantica showed that the affinity of its Hc was high, with a strong Bohr effect and a slightly reverse temperature effect (Chausson et al., 2004). Its hemolymph had a large buffer capacity and Hc accounted for approximatively 31 % of the total protein content. However, no other major protein was observed during Hc purification. Interestingly, structural studies made on hemolymph pools from crabs exposed to hypoxia and hyperoxia suggested that Hc composition could vary slightly between conditions (Chausson et al., 2004). This called for further study on hemolymph samples from single crabs in order to confirm this hypothesis. In this work, we used pressurized chambers coupled with a chemical regulation system in order to acclimate Segonzacia mesatlantica individuals to different conditions of temperature and oxygenation. Hemolymph composition and Hc structure and composition were determined for each condition. Mass spectrometry was used to determine subunit composition of the Hc complex. Multivariate analyses of variance were performed to test whether composition reacted to the experimental conditions experienced by the crabs. The effect of allosteric effectors (L-lactate, urate, Mg 2+ ) on Hc affinity was also determined.
5.4. MANUSCRIT : RESPIRATORY ADAPTATIONS OF S. MESATLANTICA 185 5.4.2 Materials and methods Animal collection, acclimation under pressure and hemolymph sampling Segonzacia mesatlantica individuals were collected during the Momareto 2006 oceanographic cruise on the Mid-Atlantic Ridge. Animals were captured with a slurp-gun manipulated by the remotely operated vehicle (ROV) Victor 6000. Collection sites were Lucky Strike (37°13.52’ N, 32°26.18’ W, 1600 m deep) for 58 animals and Rainbow (36°08.44’ N, 34°00.02’ W, 2200 m deep) for 5 animals. Animals were either used for experiments on the day they were caught or kept at sea-level pressure at 4°C for up to 3 days depending on the availability of the pressurized chambers. In each experiment, hemolymph from a control set of individuals was sampled at the beginning of acclimation for the experimental set. Acclimation lasted for approximately 16 h and took place in three 1-liter pressurized chambers DESEARES (Sarrazin et Sarradin, 2006) for which pressure can be set between 1 and 300 atm. These chambers were provided with surface seawater by the SYRENE system (described in Chausson et al. (2004)) enabling regulation of the gas content and pH. The chambers were thermostated by a water-jacket system and the pressure was set to 160-170 atm, corresponding to the Lucky Strike bottom pressure. The same pressure was used for all crabs to keep the acclimation results comparable between sites. Two temperatures and two oxygen levels were tested, leading to four experimental conditions : cool normoxia, hot normoxia, cool hypoxia, hot hypoxia. Temperature was comprised between 9 and 13°C in the cool conditions and 18 and 22°C in the hot conditions. These temperature levels are referred to as the 10°C and the 20°C conditions, respectively. O2 content was approximately 170 µM (range 140-200 µM) for normoxia and 60 µM (range 40-70 µM) for hypoxia. For control and experimental individuals, hemolymph was withdrawn from the arthropodial membrane of a walking leg with a syringe and immediately put on ice, then frozen at -80°C. Before analysis in the lab, samples were thawed on ice and centrifuged for 15 min at 10000 rpm to pellet coagulated proteins and cells ; the supernatant was kept on ice until analysis. Some samples were frozen and thawed twice since they were used in another study (oxidative stress). Hemolymph composition Protein content - Total protein content was determined using a Biuret assay with BSA standards (Sigma-Aldrich). Hemocyanin content - Hc concentration was determined with the method described by Sanders and Childress (Sanders et Childress, 1992). Briefly, hemolymph is diluted 1 :100 in a 50 mM Tris, 0.05 mM EDTA, pH 8.9 buffer and absorbance is read at 347 nm. The extinction coefficient used for Hc is 0.269 l.g-1.cm-1.
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iv SOMMAIRE 4.2 Objectifs de l’é
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FIG. 1.5 - Localisation des princip
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104 CHAPITRE 3. STRUCTURE DE L’HC
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Bibliographie Adair, G. S. The hemo
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BIBLIOGRAPHIE 239 Catalina, M. I.,
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BIBLIOGRAPHIE 241 Farmer, C. S., J.
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BIBLIOGRAPHIE 243 Hourdez, S. Adapt
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BIBLIOGRAPHIE 245 Mangum, C. P. Sub
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BIBLIOGRAPHIE 247 Rainer, J., C. P.
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BIBLIOGRAPHIE 249 Svedberg, T. et I
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BIBLIOGRAPHIE 251 Vanin, S., E. Neg
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Liste des figures 1.1 Cycle des mar
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LISTE DES FIGURES 255 4.17 Profils
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Table des matières Résumé Sommai
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TABLE DES MATIÈRES 261 4.3.5 Spect
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TABLE DES MATIÈRES 263 Table des m
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