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

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194 CHAPITRE 5. ADAPTATIONS RESPIRATOIRES DE S. MESATLANTICA levels. The observed R2 values for the main factors are small but a large part of the variance was explained by the covariables used (cumulative R2 for the three covariables : 0.40) ; so, the R2 values of 0.12 to 0.16 for the main factors represent 20 to 26 % of the remaining unexplained variance. The four resulting biplot graphs are depicted in Figure 5.7 ; for these representations, analyses were rerun without the covariables in order to produce clear graphs, without distortion of the explanatory variables by the covariables. Each graph has four explanatory arrows : the control or acclimated status of the crabs, and the two states of the tested factor for acclimated crabs. The relationships between the response variables and the explanatory factors can be described considering three main orientations, as shown in Figure 5.7e : the α variable increases in acclimated crabs in condition "a", the β variable increases in acclimated crabs in condition "b" and the γ variable decreases in all acclimated crabs. The opposite directions correspond to a decrease in condition "a", in condition "b", and an increase in acclimated crabs in both conditions, respectively. Some general trends can be observed in all graphs : urate and sodium contents tend to increase in all acclimated crabs regardless of the experimental conditions, and lactate content only slightly correlates with the explanatory factors. It is also clear on the graphs that the dodecamer proportion increases at 10°C and decreases at 20°C, independently of oxygenation (Figure 5.7a,b). Sodium is more correlated with high temperature. Magnesium content decreases at low temperature and increases at high temperature ; under high temperature it is more correlated with hypoxia. Calcium content increases under high oxygenation, whatever the temperature is. Potassium content increases under simultaneous high oxygenation and high temperature. Hemocyanin content increases under low temperature and under high oxygenation. Hemocyanin structure Segonzacia mesatlantica Hc presents the classical dodecameric and hexameric assemblies, along with a small amount of 18-meric complexes (Figure 5.6a,b). As already pointed out above, hexamer proportion is high for a brachyuran crab, with a mean value of 54 % compared to typical values of 5-20 % for the shore crab Carcinus maenas (personal observation). Statistical analysis of the effect of experimental conditions on complex proportions is presented above. The MALLS estimated masses fit well with the expected masses of 1350 kDa, 900 kDa and 450 kDa for the 18-meric, dodecameric and hexameric fractions, respectively. Native hemolymph samples were analyzed in supramolecular ESI-MS (non-covalent conditions) (Figure 5.8). Hexamer and dodecamer are also the main complexes detected by this method ; a lower amount of 18-mer and monomer are detected. Masses obtained by supramolecular ESI-MS also fit unambiguously with the estimated masses of the complexes. ESI-MS analysis in denaturing conditions gives access to the subunit composition of the Hc com-

5.4. MANUSCRIT : RESPIRATORY ADAPTATIONS OF S. MESATLANTICA 195 FIG. 5.8 – Examples of supramolecular ESI-MS spectra of Segonzacia mesatlantica native Hc. Whole hemolymph was desalted in 10 mM AcNH 4 and analyzed in non-covalent conditions. (a) example of a dodecamer-rich sample (9 % 18-mer, 48 % dodecamer, 43 % hexamer, proportions in mass as measured by SEC), (b) example of an hexamer-rich sample.

5.4. MANUSCRIT : RESPIRATORY ADAPTATIONS OF S. MESATLANTICA 195<br />

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FIG. 5.8 – Examples of supramolecular ESI-MS spectra of <strong>Segonzacia</strong> <strong>mesatlantica</strong> native Hc. Whole<br />

hemolymph was desalted in 10 mM AcNH 4 <strong>and</strong> analyzed in non-covalent conditions. (a) example of<br />

a dodecamer-rich sample (9 % 18-mer, 48 % dodecamer, 43 % hexamer, proportions in mass as<br />

measured by SEC), (b) example of an hexamer-rich sample.

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