Disruption of Spodoptera exigua serine protease 2 (Ser2) results in male sterility by CRISPR/Cas9 technology

Abstract BACKGROUND Sperm development and behavior present promising targets for environmentally safer, target‐specific biorational control strategies. Serine protease in seminal fluid proteins plays a crucial role in the post‐mating reproductive processes of lepidopteran pest insects. The serine protease 2 has been identified as the initiatorin of the seminal fluid protein in Lepidoptera, and its loss of function leads to male sterility. Nevertheless, the genetic pattern of this gene mutation and the impacts of various mutant genotypes on the hatchability of the eggs of pests remain unclear. RESULTS This study focused on the cloning of Spodoptera exigua serine protease 2 ( SeSer2 ), which is specifically expressed in male moths. The open reading frame of SeSer2 consists of 843 nucleotides, encoding 280 amino acids with structural characteristics typical of serine proteases in the S1 family. To validate the functional role of SeSer2 in the fertility of S. exigua , a targeted ~3574‐bp deletion of SeSer2 was introduced using the CRISPR/Cas9 genome editing system, leading to premature truncation of the SeSer2 protein. The SeSer2 mutation had no significant impact on the growth and development of individuals of either sex. However, disruption of SeSer2 resulted in heritable male sterility. Although females mated with SeSer2 −/− ( SeSer2 knockout homozygote) males laid eggs normally, these eggs failed to hatch. SeSer2 +/− ( SeSer2 knockout heterozygote) male moths crossed with female moths produced viable offspring, indicating the gene's recessive role in egg hatching. CONCLUSION These findings strongly support the conclusion that the Ser2 gene is essential for male reproductive success in diverse lepidopterans. Targeting the Ser2 gene holds promise as a foundational element of a novel pest control strategy. © 2024 Society of Chemical Industry.