Efficient sex separation by exploiting differential alternative splicing of a dominant marker in Aedes aegypti.

Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions. Author summary: Female mosquitoes, exclusive blood-feeders and carriers of dangerous human diseases, necessitate elimination before implementing genetic biocontrol methods. Enter SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter), a robust sex-sorting approach with key attributes: (i) harnessing male-specific expression through sex-specific alternative splicing (SSAS) of a safe, bright fluorescent marker; (ii) early larval sex-sorting and beyond; (iii) adaptable high-throughput sorting with COPAS; (iv) independence from sex-chromosome linkage; (v) genetic stability, resistant to meiotic recombination or chromosomal rearrangements; (vi) allowing positive selection of males for added reliability; (vii) transferable to various species through ubiquitous sex-specific alternative splicing, cross-species compatible promoters, and markers; and (viii) enabling early-stage mosquito separation for efficient male mosquito production. SEPARATOR reliably separates sexes during Aedes aegypti larval and pupal stages, and facilitates transcriptome sequencing of early larval males and females, revealing sex-specific mosquito genes. This method streamlines male production for release programs, offering cross-species adaptability to support genetic biocontrol initiatives. [ABSTRACT FROM AUTHOR]