Experimental set-up
Behavioral changes including induction of oviposition have been observed to start approximately 4 - 5 days after eclosure, both in V. cardui (Stefanescu et al., 2021) and other nymphalid butterfly species (Wiklund & Friberg, 2022). This suggests that the ‘decision’ to invest in migration or reproduction, which can be influenced by, among other things, host plant availability for oviposition, should have been made at this time. To test how the response to host plant access affects the behaviour and investment into reproduction we exposed the females to presence or absence of host plants and sampled the females in the morning five days after eclosure to analyse differential histone mark enrichment at this time point.
Offspring to one painted lady female (3rd generation offspring of wild caught individuals captured in Catalonia in spring 2020) were reared under controlled conditions, including constant temperature at 23°C, constant day length at 16 hours (h) light, 4 h ambient light and 4 h darkness, and ad libitum access to host plants (Malva sylvestris , grown under controlled conditions in a common greenhouse). Upon pupation, the sex of each pupa was scored and when the imagines eclosed, individuals were immediately divided into two treatment cohorts (separate cages) using two replicate cages for each treatment. A first group of females was placed in one of two cages (40*40*40 cm) with five free-flying males and access to the host plantM. sylvestris . The female individuals in the other treatment group were placed in one of two cages with five free-flying males and no host plants. All butterflies had ad libitum access to energy sources (20% sugar water) and the climatic settings were again a stable temperature at 23°C, constant day length at 16 h light, 4 h ambient light and 4 h darkness. The females used for the experiment were sampled in the morning on the fifth day after eclosure and immediately snap frozen in liquid nitrogen for subsequent DNA-extraction. The host plants were exchanged every second day and the number of eggs were recorded in each specific cage. A total of 47 females were sampled, 12 from each of the four treatments/replicates, except for one treatment (with host plants) where only 11 females could be obtained.