CRISPR/Cas9‐based <i>white pupae</i> mutant lines in <i>Bactrocera</i> spp. for sterile insect technique applications

The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae. The sterile insect technique (SIT) is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates, reducing reproduction and eventually pest numbers. The effectiveness of the SIT against insect pests can be enhanced through male-only releases, achieved via genetic sexing strains (GSS) that enable early-stage sex separation. To overcome limitations faced when developing a new GSS through the classical genetic approach, a novel "neoclassical approach" has been proposed, focusing on the identification of genetic markers, the induction of desired phenotypes through genome editing, and the linkage of selectable markers to male sex. In this study, we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species. The white pupae orthologous genes have been identified, and, through CRISPR/Cas9 mutagenesis, the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae species. These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species, supporting the development of genetic sexing systems for SIT-based pest management.