Experimental design

In situ mitochondrial function was measured in 2 ml of respiration solution using a high-resolution respirometer (Oxygraph-2k with O2k-Fluorescence module; Oroboros Instruments, Innsbruck, Austria) at 12°C under continuous stirring. Gill fibres (8.0–24.9 mg wet mass) were allowed to sit for 5 min after being transferred to the chamber with the stirrer on. Respiration rate was measured from the rate of decline in O₂ concentration within the chamber. In the first step, we added malate (2 mmol l⁻¹) followed by pyruvate (5 mmol l⁻¹) to stimulate LEAK or state 4 respiration (L_N, LEAK respiration in the absence of ADP). ADP (5 mmol l⁻¹) was then added to stimulate respiration via complex I (P_PM, OXPHOS respiration with pyruvate and malate), reflecting the mitochondrial capacity for supporting oxidative phosphorylation (OXPHOS or state 3 respiration). Respiration was then measured after addition of glutamate (10 mmol l⁻¹) (P_PMG, OXPHOS respiration with pyruvate, malate and glutamate) and then succinate (25 mmol l⁻¹) (P_PMGS, OXPHOS respiration with pyruvate, malate, glutamate and succinate), to determine the maximal capacity for supporting OXPHOS via complex I and then complexes I+II (i.e. single and then convergent electron inputs to coenzyme Q), respectively. Cytochrome c (10 mmol l⁻¹) was then added to assess the viability of our mitochondrial preparations (increases in respiration following cytochrome c additions are often used as an index of poor outer mitochondrial membrane integrity; Rasmussen and Rasmussen, 1997; Kuznetsov et al., 2004). To ensure O₂ consumption rates were due to mitochondrial respiration and not to some other oxygen-consuming process, oligomycin [an F₀F₁ ATP synthase (ATPase) inhibitor] was added to inhibit respiration due to OXPHOS (so giving an alternative measure of LEAK respiration, L_Omy); followed by antimycin A; antimycin is a coenzyme Q–cytochrome c reductase (complex III) inhibitor which prevents the flow of electrons through the electron transport chain, inhibiting remaining mitochondrial O₂ consumption rates (state 5). Finally, ascorbate (0.5 mmol l⁻¹) followed by N,N,N,N-tetramethyl-p-phenylenediamine (TMPD; 0.5 mmol l⁻¹) was used to maximally stimulate complex IV (P_TM, OXPHOS respiration with TMPD and ascorbate). Respiration rates were measured for at least 3 min in each condition until a steady state was reached, with rates expressed relative to the wet mass of gills. The reagents used in this section, and elsewhere throughout the Materials and Methods, were obtained from Sigma-Aldrich (Gillingham, Dorset, UK) unless otherwise stated.

The system was washed 3 times before and after each experimental run with both 70% ethanol and ddH₂O. Calibration of the system was achieved by taking measurements of the background flux of the respiration buffer alone at 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 and 550 nmol O₂. We also explored respiration rates of gill tissues at the same O₂ concentrations. At concentrations below 200 nmol O₂, we observed decreases in gill O₂ consumption rates; therefore, to ensure oxygen limitation was not a factor, experimental runs were conducted at O₂ concentrations between 250 and 500 nmol O₂. All experiments were run in duplicate.