Supplementary material from "Energetics of free- and tethered-flight in bumblebees (<i>Bombus terrestris</i>, Linnaeus 1758)"

Many insect species are reluctant to fly freely in wind tunnels, limiting direct free-flight energetics measurements to just two species. More commonly tethered flight energetics have been investigated, though the effects of tethering on metabolic rate are unclear. Here, we report mass-specific gross metabolic rate (assessed as the rate of carbon dioxide production; V ̇_(〖CO〗_2)^*) across a speed range (0–4.1 m s-1) in bumblebee (Bombus terrestris) workers during tethered and free-flight in a closed-circuit wind tunnel. V ̇_(〖CO〗_2)^* followed a U-shaped relationship with airspeed during both free- (P=0.009) and tethered- (P<0.001) flights. Bees were anaesthetised with isoflurane during tethering, which had no subsequent effect on their metabolic rate (P>0.05), avoiding issues reported during with immobilisation with CO2 or cold exposure. Tethered V ̇_(〖CO〗_2)^* was 45% lower than during free-flight (P<0.001), but the minimum power speed and the trajectory of the metabolic power-speed relationship (P>0.8) were similar. Overall flight efficiency ranged from 7.3–14.7% and did not vary with airspeed. These findings confirm a U-shaped metabolic power-speed relationship in insects, and suggest that tethered-flight may approximate free-flight energetics. However, a shift in the maximum range speed to slower speeds during tethered flight warrants caution against using this variable to predict behaviour.