School of Biological Sciences, Department of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, 92093, USA.
Infect Dis Poverty. 2024 Nov 11;13(1):83. doi: 10.1186/s40249-024-01256-7.
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.
针对蚊媒疾病的遗传生物控制干预措施需要专门释放雄性蚊子，因为只有雌性蚊子吸血并传播病原体。只释放雄性蚊子可以消除增加蚊子叮咬和传播病原体的风险，同时实现有效的种群控制。本研究的目的是为蚊子的早期幼虫阶段开发强大的性别分选方法，从而能够进行大规模的雄性仅释放，用于遗传生物控制干预。
为了解决亚洲疟蚊按蚊的性别分选问题，我们构建了 Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter（SEPARATOR）。这种基于显性荧光的方法以前在埃及伊蚊中被证明是有效的，它利用报告基因的性别特异性可变剪接来确保在发育早期雄性特异性表达。选择双性基因的性别特异性可变 RNA 剪接作为 SEPARATOR 的工程目标，因为它在昆虫中具有进化保守性。为了扩大 SEPARATOR 在遗传性别鉴定中的适用性，我们评估了 An. gambiae 双性基因（AngDsx）剪接模块在 An. stephensi 中的跨物种性别特异性 RNA 剪接活性。使用荧光立体显微镜验证了整个蚊子生命周期中雄性特异性增强型绿色荧光蛋白（EGFP）的表达。
我们的结果证实，SEPARATOR 调节 An. stephensi 中的雄性特异性 EGFP 表达，并能够从第一龄幼虫阶段可靠地进行阳性雄性选择。分子分析表明，雄性特异性 EGFP 表达依赖于双性基因的性别特异性剪接事件。此外，来自 An. gambiae 的剪接模块在 An. stephensi 中有效运作，表明这些物种之间的性别特异性剪接事件具有进化保守性。
SEPARATOR 不依赖于性染色体连锁，从而能够抵抗可能由减数分裂重组和染色体重排介导的断裂，使其非常适合大规模雄性释放。SEPARATOR 能够从第一龄幼虫阶段进行精确的雄性选择，这是一个重大进展，将有助于按蚊的遗传生物控制。
