Fertility
Virgin males were allocated to vials (10 per treatment) and treated in pre-heated water-baths at 23°C (‘non-hardening’) or 33°C (‘hardening’) for 1h as above. Immediately following heat-hardening, flies were transferred into pre-heated water baths at 34°C for a further 4 hours (‘stress’, chosen as the lowest whole-degree Celsius temperature at which D. virilis are sterilised (Parratt et al. 2021)). Vials were subsequently removed from the water-baths and males were placed in new individual vials with 4 virgin female partners each. Previous experiments have shown that, when stressed as adults, maleD. virilis initially retain fertility for several days and then become sterilised (Walsh et al. 2019). Hence, unlike our assay with pupal-stress flies, we did not passage males to new vials every 2 days immediately. Instead, we gave males an initial 7-day period in a single vial with 4 females. We then gave each male 4 new virgin females and passaged each group every 2 days for 4 times.