Measurements of standard metabolic rate (SMR)
SMR was measured using intermittent flow respirometry system (Loligo Systems, Viborg, Denmark).. A detailed description of the respirometry set-up is described in Andersson et al. (early view). Briefly, our set-up was comprised of four acrylic respirometry chambers, submerged in two aquaria (two chambers per tank) which contained water maintained at 18.1 ± 0.07 °C (average ± SD) and air-stones to maintain oxygen at air-saturation levels. Oxygen concentration in each chamber was measured using fiber-optic optodes connected to flow-through oxygen cells in conjunction with a Wiltrox4 oxygen meter (Loligo Systems). Chambers were size matched to each fish. Measurement loops for the trials consisted of a 180 sec flush phase, a 30 sec wait phase and a 210 sec measurement phase. Measurement loops for calculating background oxygen consumption rate (ṀO2 (m gO2 h−1)) consisted of a 180 sec flush phase, a 30 sec wait phase and a 900 sec measurement phase. The entire system was drained and flushed with bleach between trials to prevent the build-up of microbes over the course of the experiment. At the beginning of each trial, the system was refilled with aerated tap water that had been maintained at 18 ± 0.5°C for a minimum of 24h.
Fish were fasted for approximately 24 hours before the start of the respirometry trial. Individuals were removed from their husbandry tank using a mesh net and transferred to the respirometer in a 9-liter bucket filled with aerated 18 ± 0.5°C tap water. Fish were placed in the respirometer in the afternoon and remained in the respirometer for a minimum of 19 hours and 43 minutes. Following the trial, fish were killed with an overdose of benzocaine and frozen for stable isotope analysis. Measurements for background respiration were taken directly before and after each trial. AutoResp software (Loligo Systems, version 2.2.0), was used to calculate ṀO2 (mgO2h−1) from the linear decrease in dissolved oxygen over each measure phase. Chamber specific background respiration was calculated by fitting a linear regression between all of the starting and ending background values with R2 > 0.1 grouped by chamber. The linear regressions between all values, as opposed to just values specific to each trial, were used so that measures with R2 < 0.1 could be excluded, while still allowing for chamber specific background, which accounts for slight differences in calibration between each chamber. Each trial was adjusted for background respiration by subtracting fitted values estimating background respiration from measures of ṀO2for each fish at each timepoint. Background corrected measures were divided by fish weight to calculate mass-specific ṀO2(mgO2 kg−1 h−1) and any estimate of ṀO2 with an R2< 0.95 was removed prior to calculating SMR. SMR was calculated as the mean of the lowest 10% of ṀO2measures (Baktoft et al. 2016, Andersson et al. early view). R-code for calculations of all metabolic measures is available on the data repository Zenodo (http://doi.org/10.5281/zenodo.4433723).