Establishing a male-positive genetic sexing strain in the Asian malaria vector Anopheles stephensi

The notorious Anopheles mosquito species is known for transmitting malaria. Urbanization and climate change have increased urban transmission risks, posing new challenges for malaria control. Innovative biocontrol technologies are being investigated to address this issue. We introduce SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter), a robust sex-sorting method for Anopheles stephensi. This technique leverages the conserved sex-specific pre-mRNA splicing of the sex determination gene doublesex (dsx) to enable precise and efficient male selection from 1st instar larval stages through all subsequent developmental stages via positive EGFP selection. SEPARATOR represents a significant advancement in biocontrol strategies, enhancing the potential to control An. stephensi and related species.