A major factor underlies biliverdin-mediated blue pigmentation in the Chinese oak silkworm Antheraea pernyi

Insect larval pigmentation is a key aspect of their ecology and development and often involves complex biochemical pathways. While carotenoids and flavonoids are well-studied contributors, the molecular mechanisms underlying biliverdin-associated pigmentation, responsible for striking blue-green hues in many insects, remain largely unknown. The Chinese oak silkworm, Antheraea pernyi, exhibits remarkable natural variation in larval epidermal coloration across strains, making it an excellent model for insect pigmentation. Here we report the discovery of six biliverdin-binding protein (BBP) genes (ApBBP1-6) in A. pernyi. Integrated transcriptomic and qRT-PCR analyses show that ApBBP1 gene activity is uniquely predominant in the larval epidermis, midgut, and fat body of the blue-skinned Lan strain but completely absent in the white-skinned Bai strain. Genomic comparisons revealed an ∼3.9 kb deletion in the Bai ApBBP1 locus that results in 214 bp truncation in the coding sequence. Cas9/sgRNA-mediated gene ablations of the Lan ApBBP1 locus resulted in significantly reduced ApBBP1 mRNA levels concomitant with a decreased biliverdin content in larval epidermis confirmed by metabolite profiling. The ablation converted the characteristic Lan strain blue larval skin to white. This work resolves a long-standing question regarding the molecular basis of biliverdin-derived coloration in insects and establishes A. pernyi as a powerful model for investigating the evolution and genetic regulation of complex pigmentary traits. Identification of ApBBP1 creates opportunities for exploring the conservation of this mechanism across Lepidoptera and the potential for engineering insect pigmentation.