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

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 comprises a 5883-bp open-reading frame that encodes a protein consisting of 1994 amino acids, which contain 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 wild-type (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 were 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 suggesteds that PxOsp plays a vital role in female reproduction, particularly in ovary and egg development. Disrupting PxOsp results in 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. © 2024 Society of Chemical Industry.

The Potential Role of the Piwi Gene in the Development and Reproduction of Plutella xylostella.

Piwi proteins play a significant role in germ cell development and the silencing of transposons in animals by associating with small non-coding RNAs known as Piwi-interacting RNAs (piRNAs). While the Piwi gene has been well characterized in various insect species, the role of the Piwi (PxPiwi) gene in the diamondback moth (Plutella xylostella), a globally distributed pest of cruciferous crops, remains unclear. Expression analysis demonstrated the upregulation of PxPiwi in pupae and testes. Furthermore, we generated a PxPiwi-knockout mutant using CRISPR/Cas9 technology, which resulted in a significantly prolonged pupal stage and the failure of pupae to develop into adults. Additionally, the knockdown of PxPiwi, through RNA interference (RNAi), led to a substantial decrease in the oviposition and hatchability of P. xylostella. These findings indicate that PxPiwi is specifically expressed and essential for the development and reproduction of P. xylostella. This is the first report indicating the involvement of the Piwi gene in the development of lepidopteran insects, except for reproduction and germ cell development, which provides a foundation for future investigations into the functions of PxPiwi.