A First Set of Freshly-Collected Mussels Was Conditioned in Experimental 70-L PVC Tanks

Experimental design and sample collection

Heat shock experiments. A first set of freshly-collected mussels was conditioned in experimental 70-L PVC tanks for 72 h at atmospheric pressure in normoxic sea-water at 5°C before experimentation on board during the cruise (July 2001). A second set of individuals was transferred in situ into retrievable cages (Dixon et al. 2001) that allowed their acoustic recovery by the Portuguese R/V Arquipélago, operated from Horta, one week after the end of the ATOS cruise (August 2001). Individuals were kept inside insulated boxes at 5°C for their 24-h travel back to Horta, and were then acclimatised at our field laboratory (LabHorta, FaialIsland, Azores) in the same tanks and following the same experimental design as used during the ATOS cruise. Because the LabHorta mussels had sustained more environmental variation during their capture, they were subjected to a supplementary 24-h acclimatisation period at atmospheric pressure, compared to the first set of experimental mussels.

The shell length of both sets of individuals was measured to the nearest millimeter using a caliper and randomly size-distributed into three groups. During the two sets of experiments, tank water was renewed every day with freshly chilled sea water (5°C) and temperature, O2 and salinity were measured before and after the heat shock. For both sets of experiments, oxygen concentrations ranged from 6.5 to 6.8 mg.L-1 in acclimatisation tanks and fell to 4.3 mg.L-1 during heat exposure, whereas salinity increased from 36.5 to 38.3 g.L-1, a result of evaporation.

Gills and adductor muscle were dissected from each mussel (n=5 for each sampling time) and immediately frozen in liquid nitrogen until further analysis. During the experiment, mussels were not subjected to methane or sulphide. Animals were manually checked for survival prior to dissection. No mortality has been observed during the course of the experiments.

Differential mortalities under a pressure of one atmosphere. During the experimental period, individuals were checked every 3-4 hours for survival and dead individuals were removed, dissected straightforward and stored in liquid nitrogen for further electrophoretic analyses until the DL50 was reached. The remaining surviving individuals were also checked, dissected and stored in liquid nitrogen for genotyping. Batches containing mussels from Rainbow acclimated at 20 and 25°C were not considered in the subsequent analyses, because all individuals dead after 6h of exposure. During all experiments, oxygen concentration, temperature and salinity were monitored using a multiparameter electrode. Over the course of the experiment, the oxygen and salinity values ranged from 4.3 to 6.1 mg.L-1 and from 36.6 to 38.2 g.L-1, respectively. Temperature means were 11.5°C (± 1.9), 15.8°C (± 1.2), 21.4°C (± 1.1) and 25.1°C (± 0.9) for each of the 4 thermal conditions tested.

References

Dixon, D. R., Dando, P. R., Santos, R. S., Gwynn, J. P. & the VENTOX Consortium. 2001 Retrievable cages open up new era in deep-sea vent research. InterRidge News, 10, 21-23.