The role of epigenetics in polyphenisms and transgenerational immune priming in Lepidoptera

Abstract

Examples of phenotypic plasticity, referring to the capacity to express multiple phenotypes from a single genotype in response to environmental conditions, are abundant in lepidopterans. However, the exact extent and mechanistic foundations of the phenomenon are unresolved. For example, the bivoltine European map butterfly (Araschnia levana, L. 1758, Nymphalidae) displays a seasonal polyphenism characterized by the formation of two remarkably distinct dorsal wing phenotypes in the spring (pupal diapause) and summer (direct development) generations respectively, which depends on larval photoperiod and temperature. We investigated, if the polyphenism extends to the larval stage and found that, following the injection of a bacterial entomopathogen (Pseudomonas entomophila), larvae of the spring generation succumbed to infection later, displayed higher antibacterial activity in the haemolymph, and expressed higher levels of antimicrobial peptides than larvae of the summer generation. These findings likely point towards a bolstered immune phenotype in preparation of overwintering. Furthermore, from physiological experiments by Koch and Bückmann (1987), it was known, that the expression of the seasonal polyphenism depends on the photoperiodically controlled timing of the release of 20-hydroxyecdysone in pupae. Encouraged by profoundly different transcriptomic profiles in spring- and summer-primed prepupae, we hypothesised that post-transcriptional epigenetic regulators such as microRNAs (miRNAs) might be involved in controlling the seasonal polyphenism in this species. Using microarrays containing over 2,000 conserved insect miRNAs, we detected several hundred miRNAs differentially expressed between larvae (and pupae) primed for either developmental trajectory. Also, by employing in silico target prediction, we identified numerous targets of miRNAs that were differentially regulated in the two generations including one miRNA (miR‐2856‐3p) that potentially controls the expression of a diapause bioclock protein, which is vital to determine the moment when diapause should be broken in the Silkworm (Bombyx mori, L. 1758, Lepidoptera: Bombycidae). These results suggest a significant involvement of epigenetic regulatory systems in the expression of seasonal phenotypes in A. levana. Moreover, for many insect orders, including the Lepidoptera, it was known that parents invest in their offspring by relaying the experience of their own microbial environment to the offspring generation, thus conferring a (plastic) degree of protection. This phenomenon is known as trans-generational immune priming (TGIP) and had, inter alia, been observed in the Tobacco hornworm, Manduca sexta (L. 1763, Sphingidae). However, it was unknown if, in line with Bateman’s principle, the transgenerational immune phenotype was sex-specifically expressed. Moreover, the mechanistic underpinnings of the previously observed effects were unclear. We used this model species to investigate these questions by feeding larvae of the parental generation with fluorescently labelled bacterial fragments or non-pathogenic Escherichia coli or the entomopathogen Serratia entomophila. We found that maternal TGIP depended on the translocation of bacterial structures from the gut lumen to the eggs. Also, we observed sexand/or microbe-specific differences in the expression profiles of immunity-related genes as well as genes encoding enzymes involved in the regulation of histone acetylation and DNA methylation in larvae of the offspring generation. These larvae further displayed shifts in both DNA methylation and histone acetylation pointing towards the existence of multiple routes for TGIP in M. sexta. Moreover, the entomopathogen S. entomophila appeared to be capable of interfering with TGIP in the host. In summary, we contributed to the body of knowledge of lepidopteran phenotypic plasticity by widening the extent of known intra- and intergenerational phenotypes and demonstrating the involvement of the epigenetics in the regulation of polyphenisms and TGIP in A. levana and M. sexta respectively.