Illumina HiSeq 2500 paired end sequencing; Gene expression profiling across ontogenetic stages in wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation.
Gene expression profiling across ontogenetic stages in wood white (Leptidea sinapis) reveals pathways linked to butterfly diapause regulation.
Three mated female L. sinapis (Swedish ecotype) were collected in south central Sweden (Uppsala region) during spring 2016 and kept in captivity in separate 50*50*50 cm cages in a common garden with constant day light (18 hours light, 6 hours dark) and access to sugar water and stands of a commonly utilized host plant, birdsfoot trefoil (Lotus corniculatus), for egg laying. Eggs collected from each of the three unrelated females (families, from here on) were divided into two cohorts and reared in growth chambers with constant temperature (23degreesC), under either short-day (12 hours daylight + 12 hours dark) or long-day (22 hours daylight + 2 hours dark) light conditions (Supplementary Figure 1). Lights-off switch time was synchronized between treatments. Based on previous data in other butterfly species with a pupal overwintering stage, including the related pierid, green-veined white (Pieris napi), the critical point for diapause or direct development induction takes place during larval instar-IV (Friberg et al. 2011). Developing individuals were therefore monitored daily and samples were harvested the day after molting into larval stages instar-III and instar-V, and one day after pupation. Adult samples were also collected for the long-day light treatment the day after eclosion. Apart from instar-III, six samples (three males and three females, one from each family) were collected for each developmental stage and light treatment. Only three samples were collected for instar-III larvae (one from each family) since sexing based on morphology is impracticable at that stage (Friberg & Wiklund, personal observation). Hence, in total 36 samples were harvested representing three (short-day treatment) or four (long-day treatment) developmental stages (Supplementary Figure 1). At least ten additional individuals from the same families were raised under each light treatment to control that short-day and long-day treatments triggered diapause induction and direct development, respectively. All ten individuals raised under long-day light conditions went into direct development and eclosed after 7-10 days as pupae. All ten individuals reared under short-day light conditions entered diapause upon arriving to the pupal stage (and remained in diapause for several months). whole body of the butterfly at the particular stage was used for the analysis.  Sacrificed at instar 3 sacrificed at pupal stage  Sacrificed at adult stage long-day (22 hours daylight + 2 hours dark) Total RNA was extracted from the whole body using a modified acid phenol-chloroform protocol (Chomczynski & Mackey 1995; Chomczynski & Sacchi 1987). Individual libraries were generated using the Illumina TruSeq Stranded mRNA protocol (Illumina Inc.) with Poly-A selection and using dUTP for second strand cDNA synthesis.