The metabolic rate (ṀO2) of eurythermal fishes changes in response to temperature, yet it is unclear how changes in mitochondrial function contribute to changes in ṀO2. We hypothesized that ṀO2 would increase with acclimation temperature in the threespine stickleback (Gasterosteus aculeatus) in parallel with metabolic remodeling at the cellular level but that changes in metabolism in some tissues and organs, such as liver, would contribute more to changes in ṀO2 than others. Threespine stickleback were acclimated to 5, 12 and 20°C for 21 weeks. At each temperature, standard and maximum metabolic rate (SMR and MMR, respectively), and aerobic scope (AS) were quantified, along with mitochondrial respiration rates in liver, oxidative skeletal, and cardiac muscles, and the maximal activity of citrate synthase (CS) and lactate dehydrogenase (LDH) in liver, and oxidative and glycolytic skeletal muscles. SMR, MMR and AS increased with acclimation temperature, along with rates of mitochondrial phosphorylating respiration in all tissues. Low SMR and MMR at 5°C were associated with low or undetectable rates of mitochondrial complex II activity and a greater reliance on complex I activity in liver, oxidative skeletal muscle, and heart. SMR was positively correlated with cytochrome c oxidase (CCO) activity in liver and oxidative muscle but not mitochondrial proton leak, while MMR was positively correlated with CCO in liver. Overall, the results suggest that changes in ṀO2 in response to temperature are driven by changes in some aspects of mitochondrial function in some, but not all tissues of threespine stickleback.

Threespine stickleback were collected using minnow traps. Animals were transported to the University of Alaska Fairbanks where they were maintained between 7 to 21 weeks at three different acclimation temperatures (5, 12 or 20°C). Standard and maximum metabolic rates (SMR and MMR, respectively) were measured using a 170 mL glass Loligo swim tunnel respirometer (Loligo Systems, Viborg, Denmark). Mitochondrial respiration rates were measured in liver, oxidative muscle and heart at the acclimation temperature of the animal (5, 12 or 20°C) using an Oroboros O2k high resolution respirometer (Oroboros Instruments, Innsbruck, Austria) following a substrate-uncoupler-inhibitor titration (SUIT) protocol. Values were normalized per g tissue. Maximal activity of citrate synthase and lactate dehydrogenase were measured following a modified method by Srere et al. (1963) and by Hansen and Sidell (1983), respectively, in liver, oxidative and glycolytic muscle in fish acclimated to the three temperature groups.

Metabolic rates data were collected using AutoResp version 2.3.0 (Loligo Systems, Viborg, Denmark) and processed using RStudio version 1.3.1093 (R Studio Team 2016). Code was modified from the “FishResp” package version 1.0.3 (Morozov et al., 2019) to correct raw data for background ṀO2. To obtain SMR values, dissolved oxygen (DO) values were smoothed by removing cycles with r2 < 0.95. The final SMR estimate for each animal was calculated using the mean of the lowest normal distribution (MLND) when the coefficient of variation of MLND (CVMLND) ≥ 5.4, and the 20% quantile method when CVMLND ≤ 5.4. To obtain MMR values, we either 1) fitted DO values to a linear model to estimate the slope of the line. In this case, the entire (5 min) measurement period that yielded the highest estimate was chosen as MMR (hereafter referred to as “traditional MMR”). And 2) employed a second approach (hereafter referred to as “truncated MMR”), in which each measurement period was edited to retain only the steepest declines in DO as long as there was a minimum of 100 s (33% of the measurement period) of data remaining (mean ± s.d. = 225 ± 78 s, range = 100 – 300 s) and processed as described above for SMR. Finally, Absolute aerobic scope (AAS) was calculated as MMR – SMR.

The temperature coefficient (Q10) were calculated for each mitochondrial function and for metabolic rate between 5 and 12°C and between 12 and 20°C using the following formula: Q10 = (R2/R1)^(10/(T2-T1)), where R1 and R2 are the mean values after removing outliers at temperature T1 or T2, respectively.

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