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. (2020). 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. 2020). R-code for
calculations of all metabolic measures is available on the data
repository Zenodo (http://doi.org/10.5281/zenodo.4433723).