Survival
Virgin males and females (all 7 days old) were separated and allocated to vials of 10 flies per vial of their respective sex. These vials were randomly allocated to 3D-printed floating racks in pre-heated water-baths for one hour at a hardening temperature at 23°C (‘no hardening’) or 33°C (‘hardening’, determined as the highest temperature in which no sterility is observed (Parratt et al. 2021)). After this hardening treatment, vials were immediately moved into different pre-heated water-baths for four hours at either 23°C (‘benign’) or 38°C (‘stress’, determined as lowest lethal temperature from (Parrattet al. 2021)). Immediately following treatment, vials were returned to benign conditions (23°C) and left for 24 hours to ensure that any flies that were immobilised by heat but not killed could recover. After 24 hours, the number of surviving males and females from each treatment was assessed.