Exploring the role of ovary‐serine protease gene in female fertility of diamondback moth using CRISPR/Cas9

Abstract BACKGROUND Oogenesis is a complex pathway necessary for proper female reproduction in insects. Ovary‐serine protease ( Osp ) is a homologous gene of serine protease Nudel ( SpNudel ) and plays an essential role in the oogenesis and ovary development of Drosophila melanogaster . However, the function of Osp is not determined in Plutella xylostella, a highly destructive pest of cruciferous crops. RESULTS The PxOsp gene comprised a 5883‐bp open‐reading frame that encoded a protein consisting of 1994 amino acids, which contained four conserved domains. PxOsp exhibited a high relative expression in adult females with a specific expression in the ovary. Through the utilization of CRISPR/Cas9 technology, homozygous mutants of PxOsp were generated. These homozygous mutant females produced fewer eggs (average of 56 eggs/female) than wildtype (WT) females (average of 97 eggs/female) when crossed with WT males, and these eggs failed to hatch. Conversely, mutant males produced normal progeny when crossed with WT females. The ovarioles in homozygous mutant females were significantly shorter (5.02 mm in length) and contained fewer eggs (average of 3 eggs/ovariole) than WT ovarioles (8.09 mm in length with an average of 8 eggs/ovariole). Moreover, eggs laid by homozygous mutant females were fragile, with irregular shapes and unable to maintain structural integrity due to eggshell ruptures. However, no significant differences were observed between WT and mutant individuals regarding developmental duration, pupal weight, and mating behavior. CONCLUSION Our study suggested that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. Disrupting PxOsp results in a recessive female sterility while leaving the male reproductive capability unaffected. This report represents the first study of a haplosufficient gene responsible for female fertility in lepidopteran insects. Additionally, these findings emphasize PxOsp as a potential target for genetically‐based pest management of P. xylostella . This article is protected by copyright. All rights reserved.