Insect rearing and sampling
Pieris napi eggs were collected from wild plants in Skåne county
Sweden (Kullaberg; 56°18′N, 12°27′E and Vejbystrand; 56°18′N, 12°46′E),
then reared using a split brood design in the laboratory following
previously described methods (Lehmann et al., 2018; Lehmann et al.,
2016; Pruisscher et al., 2022). To induce divergent developmental
pathways, each cohort of larvae was placed in L:D 10 h:14 h at 20°C to
induce diapause or L:D 22 h:2 h at 20°C to induce direct development.
Upon pupation, individuals remained in their respective treatments for
the first ten days, after which temperatures for the diapausing
individuals were reduced to 10°C, and then to 2°C in total darkness
after day 17 of pupation. Diapausing pupae remained in 2°C 24 h darkness
until day 144, where temperatures were increased to 10°C, and then to
20°C on day 151. Direct developing pupae were sampled on the first day
of pupation (day 0), day 3, and day 6 for head tissue. Diapausing pupae
were sampled on 0, 3, 6, 24, 114, 144, and 155 after pupation (n = 3-4
individuals per time point for each treatment). When sampled,
individuals were flash frozen by being submerged in liquid nitrogen.
Only females were sampled to avoid the effect of sex on expression
patterns, and all individuals were sampled around the same time of day
(~12:00) to avoid any circadian effects.