Materials and Methods
In overview, we test if heat-shocks experienced during pupal and adult life-history stages result in male sterility. We also test if a brief period of heat-hardening can ameliorate these effects. In a series of experiments, adult and pupal male D. virilis were exposed to a 1 hour heat hardening treatment followed immediately by a 4 hour heat stress, and assayed for survival and fertility over 1-2 weeks to reveal temporal patterns in fertility loss and restoration. We chose a 4 hour stress because midday rises to high temperature are relatively common (Geletič, Lehnert & Jurek 2020), and we think it is ecologically reasonable that a fly in nature might be exposed to these conditions for a few hours. Moreover, it is an experimentally tractable time period, and previous work has demonstrated this method can create male sterility in many Drosophila species, including D. virilis (Walshet al. 2020; Parratt et al. 2021).