CRISPR/Cas9 mediated knockout of laccase2 impairs cuticle tanning and pigmentation in the tobacco cutworm, Spodoptera litura

Abstract

Cuticle tanning is a vital physiological process in insects, mediated by the phenol oxidase Laccase2 (Lac2). To elucidate the functional role underlying cuticle formation in the economically important pest Spodoptera litura, SlitLac2 was functionally characterized in this study. The identified SlitLac2 encodes a protein containing conserved Cu-oxidase domains and a conserved cysteine-rich region, exhibiting high sequence simility to other lepidopteran Lac2 orthologs. Quantitative expression analysis revealed peak expression of SlitLac2 in 3rd instar larvae, with 251.45-, 25.06-, 34.43-, 341.04-, 31.67-, 158.04-, 5.68-, 228.43- and 668.31-fold higher expression levels than those in eggs, 1st, 2nd, 4th, 5th, 6th larvae, pupae, male adults and female adults, respectively. While tissue-specific profiling showed predominant expression in the epidermis, displaying 678.28-, 6.18-, 51.35-, 28.21- and 176.59-fold expression levels, compared to those in the hemolymph, head, malpighian tubule, fat body and midgut, respectively. Knockout of SlitLac2 severely impaired normal cuticle tanning of the 3rd, 4th, 5th, 6th instar larvae and pupae, ultimately leading to failure of adult eclosion. This study provides the first functional characterization of SlitLac2 in S. litura, establishing its critical role in cuticle formation and offering new insights into the molecular basis of insect development. Our findings identify SlitLac2 as a potential target for developing novel pest control strategies against this agriculturally significant species.