Samples and experimental design
The source population for thermal experiments was generated by pooling four isofemale lines of D. mojavensis originally collected in Santa Catalina Island, California. This population was maintained at 25°C, under a 12:12 h light:dark cycle, and controlled density conditions in 8-dram glass vials with banana-molasses media for four generations before experiments (Coleman et al., 2018). The experimental design to assess transcriptional within- (WGP) and transgenerational plasticity (TGP) was carried out as described in (Diaz et al., 2020) and involved acclimation treatments at 36 °C and a control treatment of 25 °C in the parental and offspring generations (Figure 1). The parental treatments were performed in adult samples, while the offspring treatments were performed independently for larvae and adults. For parental acclimation, 10-12 days-old adults were placed in cages with banana-molasses food plates at 36 °C or 25 °C for 24 h (Figure 1). Then, a new food plate was placed in each cage for flies to oviposit at 25°C for another 24 h. Half of these plates containing offspring eggs were immediately placed at 36 °C or 25 °C for 36 h to evaluate WGP in offspring larva. The second half of plates were transferred to vials at 25°C until adult flies eclosed to evaluate WGP by acclimating offspring adults at 36 °C or 25 °C for 24 h (Figure 1). The chosen temperatures and periods correspond to the maximum treatment that triggers a heat-shock response without killing individuals in the process. Groups of 50 individual larva or ten female adults were collected and pooled for each sample in the offspring generation. Three biological replicates were considered per each of the four combinations of parental and offspring acclimation treatments. This generated 12 pooled samples per stage and 24 samples overall. All samples were placed immediately in TRIzol and kept at -80 °C until RNA extractions.