Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi.

Abstract

Genetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions. To address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR's applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope. Our results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species. SEPARATOR's independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.

Evidence profile

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laboratory

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published

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none

Conflict statement

Declarations Ethical approval and consent to participate All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as recommended by the National Institutes of Health and approved by the UCSD Institutional Animal Care and Use Committee (IACUC, Animal Use Protocol #S17187) and UCSD Biological Use Authorization (BUA #R2401). No human research was conducted so we did not need consent to participate in this study. Consent for publication Not applicable. Competing interests O.S.A. and S.W. have filed a patent on this technology. O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. All other authors declare no competing interests.

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