Insect body pigmentation and coloration are critical to adaption to the environment.To explore the mechanisms that drive pigmentation,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura.Ebony is crucial to melanin synthesis in insects.By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S.litura embryos,we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage.Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony.Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S.litura as well as other lepidopteran pests.