Zmlaccase2 is essential for cuticle pigmentation and development in the edible beetle Zophobas morio

Laccase2 is a multicopper oxidase that plays a central role in insect cuticle tanning and sclerotization, yet its molecular characteristics and biological functions remain poorly understood in the edible beetle superworm Zophobas morio. In this study, the laccase2 homolog, Zmlaccase2, was identified and functionally characterized in Z. morio. Sequence analysis revealed conserved multicopper oxidase domains and copper-binding motifs typical of insect laccase2 orthologs. Expression profiling showed that Zmlaccase2 is dynamically expressed from embryogenesis to adulthood, with expression peaks corresponding to periods of active cuticle remodeling, particularly during the prepupal and late pupal stages. RNAi-mediated silencing of Zmlaccase2 resulted in persistent cuticle hypopigmentation, with larval depigmentation persisting through pupal development and adult emergence, ultimately preventing normal adult cuticle melanization. Moreover, Zmlaccase2 knockdown significantly impaired larval growth and survival, accompanied by decreases in pupation and adult emergence rates. To further investigate its biological function, we established a CRISPR/Cas9-mediated genome-editing platform in Z. morio and generated Zmlaccase2 loss-of-function mutants. Consistently, Zmlaccase2 knockout individuals exhibited mosaic depigmentation and severe growth inhibition, and all tested mutants completely failed to pupate. Overall, our study demonstrates that Zmlaccase2 plays vital roles in cuticle tanning, growth, and development in Z. morio and provides both a valuable phenotypic marker gene and a functional genomic platform for this species.