CRISPR/Cas9-mediated knockout of ovary-related serine protease gene induces a recessive female sterility in the diamondback moth

Abstract

Abstract Oogenesis is a complex pathway necessary for the proper female reproduction in insects. Ovary-serine protease (Osp) is a homologous gene of serine protease nudel (SpNudel) and plays an essential role in oogenesis in Drosophila melanogaster. However, the role of Osp remains unclear in Plutella xylostella. PxOsp consisted of a 5883-bp open reading frame encoding a protein of 1994 amino acids with four conserved domains. PxOsp exhibited a high relative expression in adult female with a specifical expression in the ovary. Through the utilization of CRISPR/Cas9 technology, homozygous mutants of PxOsp were generated. These homozygous mutant females produced fewer eggs than wildtype (WT) females 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 mutant females were significantly shorter in length and contained fewer eggs compared to WT ovarioles. Moreover, eggs laid by mutant females were fragile, with irregular shapes and unable to maintain the structural integrity due to eggshell ruptures. However, no significant differences were observed between WT and mutant individuals in terms of developmental duration, pupal weight, and mating behavior. Our study suggests that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. The disruption of PxOsp leads to a recessive female sterility without affecting male reproductive capability. 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.