Knockout of ovary serine protease Leads to Ovary Deformation and Female Sterility in the Asian Corn Borer, Ostrinia furnacalis

Abstract

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOspF mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular.

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

“…Moreover, the amino acid sequence of PxOsp shares almost 50% similarity with B. mori, S. liturs, and O. furnacalis, indicating that Osp homologous are highly conserved in lepidopteran insects and present in almost all insect species. 23,24,[48][49][50] The stage-specific expressions of the PxOsp gene showed an increasing trend from the pupal stage and peaked at the adult stage. The relative expression levels of PxOsp exhibited at least a 10-fold increase in female adults compared to other developmental stages (from egg to pupae).…”

“…This expression pattern of PxOsp is almost synchronized with the Osp expression patterns of other lepidopteran insects such as B. mori, S. litura, and O. furnacalis. 23,24 The tissue-specific expression profile revealed that PxOsp exhibited nearly 270-fold higher expression in the ovaries of adult females compared to other tissues, indicating its specificity for ovary development. Similar expression profiles are also observed in other insect species, such as Nilaparvata lugens, Pteromalus puparum, O. furnacalis, B. mori, and S. litura.…”

“…Similar expression profiles are also observed in other insect species, such as Nilaparvata lugens, Pteromalus puparum, O. furnacalis, B. mori, and S. litura. 23,24,48,50 CRISPR/Cas9-mediated mutagenesis is a powerful genome editing technology to explore the functions of different genes in nonmodel species such as diamondback moth. 46,47 Here, CRISPR/Cas9 technology was used to target the exon 3 of the PxOsp gene and obtain three types of homozygous mutations (−2 bp, (+1 and −23) bp and −50 bp).…”

“…18,37 Previously, CRISPR/ Cas9-mediated knockout of the Osp gene of in S. litura, B. mori, and O. furnacalis caused the female sterility, and the mutant female produced a significantly lower number of eggs with a limited or zero hatching rate. 23,24 Similarly, in D. melanogaster, the mutation in the SpNudel gene also caused the female sterility by disturbing the oogenesis. 18,19,21 We observed that disruption of PxOsp somehow affected the ovary development of P. xylostella.…”

“…19,22 In lepidopteran insects such as B. mori, S. litura, and Ostrinia furnacalis the mutation of the Osp gene causes female sterility, emphasizing its conserved role in female fertility. 23,24 These studies emphasize the significance of the Osp gene in the female fertility of various insect species, which sets the stage for us to explore the function of the Osp gene in the diamondback moth.…”