A horizontally acquired gene mediates insect cocoon pigmentation in the eri silkmoth, Samia ricini

Holometablous insects make cocoons during larval-pupal metamorphosis to protect the pupal phase. The materials used for cocoon construction vary widely, in which Lepidopteran insects typically secrete silk to form cocoons, which display diverse colors. The eri silkworm, Samia cynthia ricini, is an economically important domesticated species that mostly produces white cocoons, with some varieties producing red cocoons. The enzyme kynureninase (KYNU), acquired from bacteria by horizontal gene transfer (HGT), has previously been implicated in insect coloration, while the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) has been identified as a red pigment. However, exactly how KYNU is involved in cocoon pigmentation remains unclear. Here we report that a horizontally transferred bacterial gene encoding KYNU regulates red cocoons formation. Metabolomic analysis revealed a high accumulation of 3-HAA in red cocoons, confirming its role as the primary pigment and associating the coloration with the tryptophan metabolism. q-PCR analysis indicated that SrKYNU is highly expressed in the silk glands, and significantly downregulated in the red cocoon strain compared to the white cocoon strain. Genomic sequencing identified a 141-bp deletion in the upstream regulatory region of KYNU in the red cocoon strain compared to the white cocoon strain. Dual-luciferase assays confirmed that this deletion significantly reduced promoter activity. CRISPR/Cas9 knockout of SrKYNU in the white-cocoon strain resulted in mutants producing red cocoons with elevated 3-HAA content. These findings reveal that the horizontally transferred gene SrKYNU exhibits tissue-specific expression and regulates cocoon coloration in S. ricini, illustrating that horizontal gene transfer can play an important role in regulating an insect physiological process.