Survival
Pupae were collected from stock vials within 24 hours of pupation, allocated to vials of 20 pupal flies. Three vials were allocated to each treatment (giving 60 flies total per treatment, ~30 males, as sex cannot be determined in young pupae). These vials were randomly assigned to 3D-printed floating racks into pre-heated water baths (Grant TXF200) for 1 hour at either a control non-hardening temperature at 23°C (‘no hardening’) or a range of hardening temperatures (‘hardening’: 34, 35 & 36°C). These are non-lethal pupal temperatures that also do not significantly sterilise males (Walshet al. 2020). After this hardening treatment, they were immediately moved into different pre-heated water-baths for 4-hours at either 23°C (‘benign’) or at a range of sub-lethal to lethal temperatures between 37 - 41°C (‘stress’). Immediately following treatment, vials were returned to benign conditions (23°C) and emerging individuals were collected and sexed. This allowed us to assess survival of pupae at extreme temperatures, and gave us an idea of whether survival may be sex specific. However, as we were unable to determine the sex of the pupae prior to stress, we could not explicitly test for sex differences in survival thermal tolerance.