Sex-ratio distorters enable pre-zygotic sexing for sex-specific transcriptomics in Anopheles gambiae embryos

The study of sex establishment at the onset of development is difficult due to the absence of early morphological differences between male and female embryos. This challenge is particularly relevant in non-model organisms such as the malaria mosquito Anopheles gambiae, where sex-determination mechanisms are not fully understood. We employed a pre-zygotic sexing (PZS) system based on synthetic sex-ratio distorters (X- and Y-shredders) to generate large-scale male- or female-biased embryo populations. Combining the PZS system with a hybrid long- and short-read RNA sequencing, we produced a comprehensive sex-specific transcriptomic dataset of early An. gambiae embryogenesis. Comparative analysis identified four sex-specific spliced genes interacting with the sex-determination network, providing candidates for future functional investigation. Together, the PZS system and sex-specific transcriptomic dataset provide a platform for dissecting early sex differences in An. gambiae, advancing both fundamental understanding of insect sex determination and informing novel vector control strategies.