Seasonal phenotype‐specific transcriptional reprogramming during metamorphosis in the European map butterfly Araschnia levana

Abstract

The European map butterfly (Araschnia levana) is a classic example of seasonal polyphenism because the spring and summer imagoes display two distinct morphological phenotypes. The light regime and temperature during larval and prepupal development determine whether or not the pupae commit to diapause and overwintering and thus whether spring or summer imagoes emerge. We used suppression subtractive hybridization to experimentally screen for genes that are differentially expressed in prepupae committed either to accelerated metamorphosis and egg production or diapause and overwintering. The range and ontology of the differentially expressed genes in prepupae developing from larvae exposed either to long‐day (LD) or short‐day (SD) conditions revealed fundamental differences. The SD prepupae preferentially expressed genes related to cuticle formation and immunity, reflecting the formation of a robust pupal exoskeleton and the upregulation of antimicrobial peptides as preparations for overwintering. One protein preferentially expressed in SD prepupae has a counterpart in Bombyx mori that functions as a diapause duration clock. The differentially expressed genes in LD prepupae included several members of the dusky and osiris families. We also observed the strong induction of different yellow‐like genes under SD and LD conditions which suggest a role in the developmental choice between seasonal phenotypes. Our transcriptomic data will facilitate the more detailed analysis of molecular mechanisms underlying seasonal polyphenism.

Immunological larval polyphenism in the map butterfly Araschnia levana reveals the photoperiodic modulation of immunity

“…The expression levels of examined AMP genes were higher in the short‐day larvae than the long‐day larvae across all sampling points (Figure ). AMP genes were selected as markers based on their relative expression levels in a recent transcriptome analysis (Vilcinskas & Vogel, ) but also the ability of the corresponding AMPs to kill Gram‐negative bacteria such as P. entomophila . Interestingly, in short‐day larvae, the four tested AMP genes clustered into two distinct response phases—namely early and delayed expression (Figure a,c vs b,d).…”

“…Primers for real‐time PCR were designed using Primer3 software (Untergasser et al., ), and available primer pairs were selected based on the lowest number of potential self‐annealing structures and primer loops. Gene‐specific primers were designed based on nucleotide sequences derived from previously published RNA‐Seq data (Vilcinskas & Vogel, ) (Table ). The ribosomal protein L10 gene ( RPL10 ) was used for normalization and PBS controls served as calibrator.…”

Seasonal phenotype‐specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana

“…A. levana caterpillars were collected in the vicinity of Albach in Hesse either in June (long‐day condition) or August (short‐day condition), fed with stinging nettle cultivars and reared in captivity for RNA isolation and microarray analysis as previously described (Freitak, Knorr, Vogel, & Vilcinskas, 2012; Mukherjee & Vilcinskas, 2014; Vilcinskas & Vogel, 2016). Total RNA was isolated from eight specimens exposed to long‐day conditions (18‐hr daylight, collected in June) and eight specimens conditioned under short‐day conditions (8‐hr daylight, collected in August).…”

“…It remains enigmatic how such environmental stimuli are converted via the same genome into morphologically distinct phenotypes. However, we recently found that day length during larval development leads to phenotype‐specific transcriptional reprogramming in the A. levana prepupae (Vilcinskas & Vogel, 2016). We identified numerous genes that are differentially expressed during metamorphosis, reflecting adaptations favoring either accelerated metamorphosis and egg production or diapause and overwintering.…”

“…These microarrays have previously been designed to study the differential expression of miRNAs in another lepidopteran species, the greater wax moth Galleria mellonella , during development as well as in response to infection (Mukherjee & Vilcinskas, 2014, Mukherjee et al, 2017). We analyzed RNA samples from last‐instar larvae and pupae originating from A. levana caterpillars exposed to either short‐day (short‐day larvae, short‐day pupae) or long‐day (long‐day larvae, long‐day pupae) conditions to identify differentially expressed miRNAs that may regulate seasonal polyphenism (Vilcinskas & Vogel, 2016). We predicted multiple targets of miRNAs including the above‐mentioned diapause bioclock protein gene using previously established techniques on base‐pair complementarity and minimum free energy hybridization.…”