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

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For control and experimental individuals, hemolymph was withdrawn from the arthropodial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.