DsiRNA‐mediated silencing of Ceratitis capitata transformer or transformer‐2 leads to masculinization of XX embryos and systemic gene silencing in ovaries

Ceratitis capitata (medfly), a major agricultural pest, is predominantly controlled using chemical insecticides, which pose environmental risks. Ecosustainable alternatives, such as the Sterile Insect Technique (SIT), rely on the mass release of sterile male‐only progeny. However, sexing of male offspring requires the elimination of females during development. To overcome the loss of 50% of the progeny, the sex reversal of females into XX fertile males at embryonic stages by dsRNA injections was effective but not scalable. This study demonstrates the efficacy of Dicer‐substrate small interfering RNAs (DsiRNAs) as an alternative to long double‐stranded RNAs (dsRNAs) for targeting the sex determination genes Cctransformer and Cctransformer‐2 in the medfly, which causes full masculinization of XX individuals. Injection of DsiRNAs into XX embryos induced the expression of male‐specific Cctra isoforms within a few hours, resulting in the development of adult masculinized XX flies. Additionally, thoracic injection of DsiRNAs in adult females achieved systemic gene silencing, reducing Cctra/Cctra‐2 transcript levels in the ovaries by 75%–80% within 48 h. The reduced size of DsiRNAs compared to dsRNAs enhances their potential for alternative delivery methods, including embryo permeabilization, electroporation, and feeding in larvae or adult females. These findings provide a potential foundation for future scalable conditional masculinization of XX individuals rather than relying on female lethality, doubling male‐only productivity. Developing novel sexing methods as an alternative to transgenic approaches will expand the applicability of SIT.