The world's oceans are acidifying and warming as a result of increasing atmospheric CO2 concentrations. The thermal tolerance of fish greatly depends on the cardiovascular ability to supply the tissues with oxygen. The highly oxygen-dependent heart mitochondria thus might play a key role in shaping an organism's tolerance to temperature. The present study aimed to investigate the effects of acute and chronic warming on the respiratory capacity of European sea bass (Dicentrarchus labrax L.) heart mitochondria.
Broodstock fish were caught in the Gulf of Morbihan, France. Larvae were raised at the aquaculture facility Aquastream (Ploemeur-Lorient, France) and obtained at 2 dph (20 January 2016).
European sea bass were reared in the laboratory in six ocean acidification and warming (OAW) conditions: two temperatures (warm and cold life condition) and three PCO2 conditions (control, Δ500 and Δ1000). Conditions were chosen to follow the predictions of the IPCC for the next 130 years: ΔT = 5°C and ΔPCO2 = 500 and 1000 µatm, following RCP 6.0 and RCP 8.5 respectively. The fish were reared under these conditions from 3 dph (days post hatch) until mitochondrial respiration measurements at 3700 to 4100 dd (degree days, 183–199 dph and 234–249 dph in warm and cold life conditioned fish, respectively).
During the experimental period, fish of all three PCO2 conditions of the respective temperature were used for mitochondrial respiration measurements on permeabilized heart fibres. Fish were not fed for 2 days prior to the experiments. Two batches of eight fish each were processed per day. Juveniles were randomly caught from their tanks and anesthetized with MS-222. Mass, fork length and body length were directly determined with a precision balance (Mettler, Columbus, OH, USA) and a calliper, to the nearest 0.01 g and 0.01 mm, respectively. Afterwards, fish were killed by a cut through the neck, and the heart was completely dissected from the fish, followed by excavation and permeabilization of the ventricle. Tissue from a whole ventricle was used for respiration measurements in each respiration chamber of the oxygraphs and respiration rates were normalized to ventricle mass. During the permeabilization step, the livers and the carcasses of the fish were weighed to calculate the hepatosomatic index (HSI) and condition factor (K).
Mitochondrial respiration of the permeabilized heart fibres was measured using four Oroboros Oxygraph-2K respirometers with DatLab 6 software (Oroboros Instruments, Innsbruck, Austria). Permeabilized fibers have the advantage of resembling the living state as closely as possible, while still allowing control of the supply of substrates and inhibitors to the mitochondria (Saks et al., 1998; Pesta and Gnaiger, 2012). Measurements were conducted at 15 and 20°C for all treatments to determine the effect of acute temperature changes on mitochondrial metabolism in vitro. A standard substrate–uncoupler–inhibitor titration protocol was employed to measure the respiration rates of the different complexes. Residual respiration after antimycin A addition was used to correct all mitochondrial respiration rates.
