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.