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.