Fertility
Pupae were allocated to 3D-printed floating racks in pre-heated water-baths set to 23°C (‘no hardening’) or 36°C (‘hardening’) for 1h as above. Immediately following hardening, they were transferred into pre-heated water baths at 23°C (‘benign’) or 38°C (‘stress’), chosen as the highest temperature not resulting in significant mortality from a prior study (Walsh et al. 2020). After four hours at their treatment temperature, vials were subsequently removed from the water-baths and returned to benign temperatures (23°C). Emerging males were collected and immediately moved into individual vials with 4 sexually mature virgin female partners each. These groups were moved into new vials every 2 days for 7 times, giving a total of 8 vials across 16 days where fertility was recorded. Estimates ofDrosophila survival rates in nature suggest 16 days represents a substantial portion of their expected adult lifespan (Powell 1997). Males were deemed as qualitatively fertile at any given time-point if there was evidence of larvae present in the vial (either via direct observation of larvae or observing larval tracks in the food).