Masculinization of XX female embryos by a Cctraep-specific maternal RNA interference in the agricultural pest Ceratitis capitata

The agricultural pest Ceratitis capitata, also known as the Mediterranean fruitfly or Medfly, is a fruitcrop pest of economic relevance in different continents. The Medfly has been the first non-drosophilid genetically transformed fly, paving the way for new biotechnology-based pest control strategies. Furthermore, it is an experimentally tractable emerging model, in which transient and transgene-mediated RNAi have also been successfully used. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The sexing method is based on female-specific conditional lethality. We have previously discovered that female sex determination is promoted in XX individuals of Ceratitis by an autoregulating master gene, Cctransformer (Cctraep), encoding a female-specific splicing factor and acting as an epigenetic switch device, ON in females and OFF in males. In XX individuals, Cctraep controls doublesex (Ccdsx) and fruitless (Ccfru) female-specific splicing and epigenetically maintains its own activity by a positive feedback loop. In XY individuals, a Y-linked M factor, still to be identified, represses the activity of Cctraep leading to male-specific dsx and fru expression. We have used transgene-mediated RNA interference (RNAi), an effective method for inhibiting gene expression, to switch OFF Cctraep in XX transgenic flies by Cctra-specific dsRNA molecules. Serendipitously, we have obtained a Ceratitis transgenic line, expressing Cctra-specific dsRNA molecules maternally and masculinizing very effectively almost all XX progeny. XX males compete efficiently with XY males in matings and, although these males lack the Y chromosome, they are fertile. XX males are able to effectively transfer GFP-marked sperms in female sperm storage structures during mating. We propose that this strain is a prototype for a novel sexing system based on masculinization, rather than female-specific lethality. This sexing system could be a valuable alternative for SIT, because it could avoid the loss of 50% of the reared progeny (females), doubling the production of male-only flies in mass rearing facilities.