Appendix A. Respirometry details.
Respirometry setup information
The respirometer was kept in an insulated 70 × 70 × 70 cm plywood box with an open front. The temperature inside the box and respirometer was kept relatively constant with the help of a 700W electric heater and a fan. The room temperature in the cabin where the measurements were performed sometimes exceeded the preferred temperature for measurements, causing the measurement temperature to vary from 30.8 to 35.1ºC. We used two 20W fluorescent UV light bulbs and an 11W fluorescent lamp to produce flight-stimulating light without producing too much heat, which could have led to differences in body temperature between dark and pale individuals. The temperature inside the respirometer was measured with as Sable Systems NTC thermistor (Sable Systems, Las Vegas, NV, USA). Water vapor was removed from the air entering the respirometer using Drierite (W.A. Hammond, Xenia, OH, USA) and CO2 was scrubbed with Medisorb (GE Healthcare, UK) and Ascarite II (Thomas, Swedesboro, NJ, USA). The air was pumped through the 1 L respirometer at a rate of 1.0 L min-1 with a Sable Systems SS3 subsampler. The air was dried again after the respirometer using magnesium perchlorate (Alfa Aesar, Karlsruhe, Germany), before entering the CO2 analyzer (Li-Cor 6251, Li-Cor Biosciences, Lincoln, NE, USA). Analog data of the flow rate, respirometer temperature, CO2 concentration, and Li-Cor temperature were digitized using a Sable Systems UI-2 interface and recorded using a laptop PC with Sable Systems’ ExpeData software.
Respirometry protocol
Prior to the measurement butterflies were kept in a cage in shade. One hour before the measurement an individual was placed in a plastic cup with moist cotton wool on the bottom. The butterfly was allowed to ingest water ad libitum to thereby adjust its body water balance. The individual was transferred to the measurement chamber 25 mins prior to the measurement and left to adjust to the measurement temperature under a black cloth, while CO2-free air was pumped through the respirometer. When the CO2 level had reached a steady baseline the black cloth was removed and the butterfly was allowed to adjust to the light conditions for 30 secs. The chamber was then gently shaken to provoke the butterfly to fly. To keep the butterfly constantly active the chamber was then shaken or tapped every time the butterfly landed on the walls. The experiment was finished 10 mins after the first shake or first spontaneous flight burst, if the individual took off during the 30 secs adjusting period. All stimulation was terminated and the chamber was re-covered with a black cloth. The individual was removed from the chamber when the CO2 level had settled to the steady baseline and all the CO2 emitted during the experiment was thus washed out. Following the experiment individuals were allowed to ingest 20% honey-water solution ad libitum.