Molecular mechanisms driving larval green color adaptation in swallowtail butterfly, Papilio memnon

Insects have evolved diverse anti-predator strategies, with coloration playing a central role through mechanisms such as mimicry and camouflage. Most swallowtail butterfly larvae switch from a black/white bird-dropping mimetic pattern to a green camouflage pattern at the final (5th) instar. In Papilio memnon, this switch from mimicry-like to camouflage also occurs at the final instar. But P. memnon initiates accumulating green coloration at the 3rd instar, earlier than its closely related species such as Papilio xuthus and Papilio polytes. This interspecific variation in the timing of green coloration provides a great model for understanding evolutionary and developmental mechanisms that drive the diversification of insect body coloration among closely related species. Through in vivo electroporation-mediated RNA interference (RNAi), we confirmed some bilin-binding proteins (BBPs), and juvenile hormone-binding proteins (JHBPs) as key effectors of green pigmentation in P. memnon. Further genomic analysis revealed that these BBP and JHBP genes are organized into conserved tandem clusters with the same gene order and transcriptional orientation across Papilio species. All BBPs and certain JHBPs within the corresponding clusters displayed markedly elevated expression in early-stage P. memnon larvae (green) relative to P. xuthus larvae (black). Together, our results suggest that heterochronic expression of conserved BBP and JHBP gene clusters contributes to the earlier onset of green coloration in P. memnon, potentially through evolutionary changes in upstream regulatory mechanisms shared by these genes.