Whole genome analysis of toxic Papilionidae butterflies utilizing aristolochic acid, <i>Pachliopta aristolochiae</i> , and <i>Byasa alcinous</i>

Several species of toxic butterflies are known, including those from the Troidini tribe of the Papilionidae, which accumulate aristolochic acid from their host plants in Aristolochiae. However, the molecular mechanisms involved in utilizing aristolochic acid remain unknown. Toxic butterflies often exhibit warning colouration to signal their toxicity to predators, a complex adaptive trait with toxin utilization. In this study, we sequenced, assembled, and annotated the genomes of 2 toxic Troidini butterflies, Pachliopta aristolochiae (312.1 Mb, 13,497 genes) and Byasa alcinous (257.6 Mb, 14,669 genes), and conducted comparative genomics to identify genes involved in toxin utilization and warning colouration. Comparative analysis across 11 species revealed 31 gene families significantly expanded and 417 genes under positive selection. Additionally, 442 genes were highly expressed in the red spots on the hindwings of P. aristolochiae. The genes shared within these lists may be involved in the formation of the complex adaptive traits of toxin utilization and warning colouration. Functional analysis using RNAi confirmed the involvement of ebony, laccase2, and tyrosine hydroxylase (TH) in warning colouration. This research marks a significant starting point in understanding the genetic basis of aristolochic acid utilization and the formation of warning colouration, providing the first list of candidate genes.