Development of gene-edited Zeugodacus tau and Bactrocera zonata white pupae lines in support of sterile insect technique applications

Zeugodacus tau and Bactrocera zonata, native to South and Southeast Asia, are invasive quarantine pests causing significant economic damage to over 50 crops. The sterile insect technique (SIT), a proven method for controlling tephritid pests such as the Mediterranean fruit fly (Ceratitis capitata), could offer a sustainable control solution for these species. However, the absence of genetic sexing strains (GSS), essential for efficient and cost-effective SIT programs, hampers their operational use. The white pupae (wp) gene is a promising selectable marker for GSS development, but its applicability in these species has not been demonstrated. In this study, we identified and disrupted the orthologous wp gene in both species using CRISPR/Cas9 gene editing, targeting exon 3 as a first step in a proposed “neoclassical approach” to GSS development. Embryo microinjection resulted in high survival rates, and several homozygous mutant lines were established and validated through sequencing and protein structure modeling. Four wp mutant lines were generated in each species. Zeugodacus tau mutants exhibited reduced apical wing spots, while B. zonata mutants displayed partial loss of pigmentation in the thorax and abdominal tergites. These results confirm the wp gene as a functional and transferable GSS marker and demonstrate the potential of CRISPR/Cas9 for rapid GSS development in non-model tephritids. This work lays the foundation for future SIT applications against Z. tau and B. zonata, illustrating the transformative potential of genome editing in insect pest management.