To measure oxygen consumption, individual larvae were placed within chambers of a closed microplate reader system that uses optical fluorescence to measure oxygen concentration (PreSens, Germany). After a 5-minute acclimation period, the change in dissolved oxygen was measured every 30 seconds over 20 minutess. The microplate reader had 2 blocks of 24 wells.
In Experiment 1, 20 fish larvae were measured per run. In Experiment 2 (see Related Datasets), 40 fish were measured per run. For all chambers, the investigators ran a linear regression of oxygen concentration on time (n = 40 measurements). The respiration rate for each fish (VO2, expressed in milligrams of O2 per individual per hour (mg O2 ind-1 h-1)) was calculated as:
VO2 = V (S – B)
where S is the slope describing the change in O2 concentration for individual chambers with the fish,
B is the average slope for the four chambers with no fish (both in units of milligrams O2 per liter per hour (mg O2 L-1 h-1) and inferred to be per individual since each well held only one larvae), and
V is the volume of water in the chamber (1.500 × 10-3 liters (L)).
Note the displacement volume of grunion larvae in this study was <2.5 × 10-6 L and thus negligible in these calculations. Fish were assigned to each chamber at random, and larvae were used once in the respiration measurements and then humanely euthanized.
To describe the duration and magnitude of the Specific Dynamic Action (SDA) response, the investigators first calculated delta.VO2, the difference between the mean rates of oxygen consumption for the pair of fed and non-fed larvae.
These data were summarized by Siegfried and Johnson (2023) and plotted in figures 1, 3, and 5.