Wound-induced eyespots on butterfly wings at the intersection of immune response and pigmentation development

Butterfly eyespots are striking examples of evolutionary novelty arising through the repurposing of ancestral genetic pathways, including pathways involved in wound healing. Given the activation of the immune system during wound healing, and the known links between immunity and pigmentation, we hypothesized that the immune response triggered by wounding contributes to the formation of wound-induced ectopic eyespots on butterfly wings. We tested this hypothesis by wounding the wings of Bicyclus anynana pupae and modulating immune challenge levels with application of varying quantities of heat-killed bacteria. These led to up-regulation of defense-related genes in the wounded wing as well as in the contralateral non-wounded wing. Immune activation did not affect the likelihood of ectopic eyespot formation, but it did significantly influence the extent of pigmentation changes around injury sites: stronger immune activation produced larger ectopic eyespots. This effect was localized to the treated wing and did not affect contralateral, control wound-induced eyespots. Additionally, stronger immune challenges led to smaller overall wing size and to relatively smaller native eyespots. Our findings reveal that immune activation contributes to the development of wound-induced eyespots and impacts both pigmentation pattern formation and wing size regulation. These results underscore the complex interplay between immune function and developmental processes, and provide new insights into the origins of lineage-specific morphological innovations.