Cardiac responses to TSA, and isoproterenol + atropine treatments which induced maximal heart rates. Example traces showing changes in heart rate and stroke volume with different levels of acute test temperature and A + I treatment (control and maximal) (A). The position of the laser Doppler probe is shown in the upper panel (B), and the transition from cool to warm acute test temperature can be seen in the example of a image from the thermal camera (B, lower panel; thermal range in photo = 17–28 °C), which we used to monitor acute test temperatures. We used a fine pipette tip inserted into the mouth of the fish to flush the gills and deliver drugs. Heart rate was determined by an interaction between acclimation temperature and A + I, and it increased with increasing acute test temperature (C). Relative stroke volume decreased when heart rate was maximal (i.e. with A + I treatment; D) and with increasing acclimation and acute test temperatures (E). Interestingly, there was a significant interaction between TSA treatment and acclimation temperature, and TSA increased relative stroke volume in cold acclimated fish, but had the opposite effect in warm acclimated fish (E). Marginal means (±s.e.) are shown, asterisks indicate significant differences between individual bars, horizontal lines with different letters indicate significant differences between treatment groups, and significant interactions are indicted below the title in individual panels. N = 8–11 fish for each treatment group, and the complete data set is shown in Supplementary Fig. S3.