Functional validation of a white pupae minimal gene construct in Ceratitis capitata (Diptera: Tephritidae)

Genetic sexing strains (GSS) are important tools for the sterile insect tech-nique (SIT), an environmentally friendly and species-specific insect pest control method. GSS feature sex-specific phenotypes, enabling sex sorting in mass-rearing facilities and male-only releases, which significantly improve the cost-effectiveness and efficiency of SIT programs. In classical GSS, sex linkage of marker gene(s), such as white pupae (wp), is achieved through an irradiation-induced translocation between the marker-carrying autosome and the Y chromosome. However, this approach may render GSS males semisterile. The recently proposed neo-classical GSS concept suggests using genome editing to achieve sex linkage by directly inserting the wild-type marker allele onto the Y chromosome, potentially yielding GSS males with higher fertility. In this study, we examined the Ceratitis capitata wp gene as a genetic marker for the neo-classical GSS concept and developed a minimal, intronless version of this gene, termed mini-wp. We demonstrate that a single copy of mini-wp is sufficient to restore the wild-type brown puparium phenotype and is functional when integrated at various positions within the C. capitata genome, including the X chromosome. Due to its smaller size (4689 bp, including 2000 bp of putative promoter region) relative to the full wild-type wp allele (20868 bp), mini-wp may facilitate its precise insertion into the Y chromosome, representing an important step toward realizing neo-classical GSS. Furthermore, the methodology developed for designing and testing mini-wp in medfly may be adapted to other Tephritid species with an identified wp gene.