Acclimation and Transcriptomic Responses of Bivalves to Climate Change Scenarios

After the acclimation period, four replicates of five animals each were subjected to each of the four combinations of temperature and oxygen concentration during 18 days in WAP and 21 days in SSA. The animals were randomly placed in 500 ml Kautex jars containing a 1 cm layer of sediment and water from the respective collection site. Each jar was then assigned to be exposed to treatment under one of the following experimental conditions: in WAP: In situ (1.5°C) and warming (4°C) in combination with normoxia (T_isOx_n, T_wOx_n) and hypoxia (T_isOx_hyp, T_wOx_hyp); in SSA: In situ (7°C) and South migration future scenario (4°C) in combination with normoxia (T_isOx_n, T_cOx_n) and hypoxia (T_isOx_hyp, T_cOx_hyp). The water temperature inside the jars was kept constant by submerging the jars in temperature-controlled water baths (Thermo Haake DC10-P21). Hypoxic conditions were created by providing the experimental units with a continuous water flow with 2% O₂ saturation, with flow velocity adjusted to achieve the exchange of the total water volume of the jars (500 ml) in the course of 1 day. The water was supplied from a 10 L tank bubbled with a gas mixture 2% O₂:98% N₂. This design could not be replicated in the normoxic treatment, since a test run with continuous water flow at 21% O₂ saturation through the hermetically sealed lids showed that animal and sedimentary/microbial respiration decreased oxygen to below 15% saturation within some hours, with uncontrollable variation between replicates and over time. Therefore, normoxic conditions were ensured by directly bubbling each experimental jar with air and manually replacing the total volume of water in each jar every day (without stirring up the sediment).
In both experiments, one animal per jar (i.e., four replicates per treatment) was collected at day 10 (t₁) for differential gene expression analysis. In the experiment in SSA, an additional step was conducted on day 18, by further decreasing the temperature in the T_cOx_n treatment from 4°C down to 2°C (T_c+Ox_n). On day 21 (t₂), one bivalve per jar was collected from T_isOx_n and T_c+Ox_n treatments for differential gene expression analysis (Figure 1). Animals were immediately dissected on ice under a stereomicroscope after collection, and mantle tissue was conserved in RNA later (SIGMA) and stored at −80°C.
During the total period of both experiments, temperature was recorded continuously (one measure per minute) in both experimental water baths, and measurements of oxygen were performed daily in each of the four replicates in the four treatments (Figure 1). Oxygen measurements were made approximately 1 cm away from the water-sediment interface, introducing the sensor through a small gate in the lid of the jars; outside the measurement periods these openings were kept sealed. For a graphical representation of the experimental design see Supplementary Figure S2.