CRISPR/Cas9-mediated knockout of serpin15 impacts reproduction and immunity in Plutella xylostella Linnaeus

Plutella xylostella represents a significant agricultural pest affecting cruciferous crops globally. The extensive use of synthetic insecticides has resulted in environmental contamination and resistance development, necessitating research into environmentally sustainable biopesticides. Serine protease inhibitors (serpins) serve essential functions in melanization during innate immunity, reproduction, and metamorphic development. Through proteomic analyses conducted across developmental stages of P. xylostella, serpin15 was identified as a crucial member of the typical inhibited serpin family, though its precise function remained undetermined. RT-qPCR analyses of gene expression patterns across tissues and developmental stages demonstrated that the serpin15 gene exhibits high expression in male adult gonads and reaches maximum levels in hemolymph. The serpin15 mRNA levels showed dynamic regulation in the midgut following Serratia marcescens (PS-1) infection, characterized by an initial decline followed by upregulation. CRISPR/Cas9-mediated knockout of serpin15 in homozygous lines led to decreased oviposition and embryonic hatching rates in offspring. Functional analyses confirmed that serpin15 inhibits phenoloxidase activity, while exogenous supplementation with recombinant serpin15 protein effectively suppressed hemolymph melanization, establishing its regulatory role in countering PS-1 through immune melanization. These findings demonstrate serpin15’s dual functionality in regulating both fecundity and immunity against PS-1 in P. xylostella. This research establishes a theoretical foundation for developing biocontrol strategies targeting insect immune and developmental systems.