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

5.4. MANUSCRIT : RESPIRATORY ADAPTATIONS OF S. MESATLANTICA 191
hexamer is visible in all samples compared to shallow water brachyurans (average hexamer proportion
of 54 % for S. mesatlantica). This proportion varies from one crab to another, with values ranging from
33 to 66 % of Hc content. For Carcinus maenas fresh hemolymph, hexamer content is typically 5 to
20 % (personal observation). Figure 5.6 shows an example of a "high-hexamer" and a "low-hexamer"
sample analyzed by FPLC.
Canonical redundancy analysis of experimental effects
The effect of experimental conditions was tested using RDA, a non-parametric method enabling
to test if the multivariate response data from different experimental groups are significantly different,
without a priori assumptions about normality. It also permits to produce graphical representations of
the relationships between the response variables (in our case, 11 hemolymph variables) and the explanatory
variables representing the experimental conditions (Legendre et Legendre, 1998, Legendre
et Anderson, 1999).
The analysis was performed on the table of 11 measured response variables for 44 crabs for which
information without missing values was available (total protein, Hc, sodium, potassium, calcium, magnesium,
L-lactate and urate concentrations, dodecamer, hexamer and 18-mer proportions). For each
crab, the explanatory variables are the collection site, sex, size, number of thawings (some hemolymph
samples were thawed once before analysis for another study on oxidative stress), experimental
groups (one group for each of the 4 experiments ; in each group, the control crabs were dissected prior
to acclimating the others), and experimental conditions (control or acclimated crab, temperature and
oxygen levels if the crab was acclimated).
The interaction between oxygen and temperature effects must be tested first. If it is significant,
the main factors cannot be tested in a 2-way anova ; factor 1 has to be tested separately in each of
the classes of factor 2 and conversely. If the interaction is not significant, we do not have to take it
into account as a covariable in the tests involving the environmental variables. Coding variables were
created to represent the oxygen and temperature treatments and control/acclimated status of each
crab. A variable Control with mean 0 separated the 18 control crabs (coding value = 1/18) from the
26 acclimated animals (coding value = -1/26) ; a variable Oxygenation with mean 0 represented the
17 crabs submitted to hypoxia (coding value = -1/17), the 18 control crabs (coding value = 0), and
the 9 animals subjected to normoxia (coding value = 1/9) ; and a variable Temperature with mean 0
represented the 10 crabs subjected to 10°C (coding value = -1/10), the 18 control crabs (coding value
= 0), and the 16 animals subjected to 20°C (coding value = 1/16). An Interaction variable was created
by multiplying the corresponding values of the oxygen and temperature treatment variables. Control
was orthogonal (correlation r = 0) to the two variables representing the main factors, but because of