Engineered action at a distance: Blood-meal-inducible paralysis in Aedes aegypti

BackgroundPopulation suppression through mass-release of Aedes aegypti males carrying dominant-lethal transgenes has been demonstrated in the field. Where population dynamics show negative density-dependence, suppression can be enhanced if lethality occurs after the density-dependent (i.e. larval) stage. Existing molecular tools have limited current examples of such Genetic Pest Management (GPM) systems to achieving this through engineering ‘cell-autonomous effectors’ i.e. where the expressed deleterious protein is restricted to the cells in which it is expressed–usually under the control of the regulatory elements (e.g. promoter regions) used to build the system. This limits the flexibility of these technologies as regulatory regions with useful spatial, temporal or sex-specific expression patterns may only be employed if the cells they direct expression in are simultaneously sensitive to existing effectors, and also precludes the targeting of extracellular regions such as cell-surface receptors. Expanding the toolset to ‘non-cell autonomous’ effectors would significantly reduce these limitations.Methodology/Principal findingsWe sought to engineer female-specific, late-acting lethality through employing the Ae. aegypti VitellogeninA1 promoter to drive blood-meal-inducible, fat-body specific expression of tTAV. Initial attempts using pro-apoptotic effectors gave no evident phenotype, potentially due to the lower sensitivity of terminally-differentiated fat-body cells to programmed-death signals. Subsequently, we dissociated the temporal and spatial expression of this system by engineering a novel synthetic effector (Scorpion neurotoxin–TetO-gp67.AaHIT) designed to be secreted out of the tissue in which it was expressed (fat-body) and then affect cells elsewhere (neuro-muscular junctions). This resulted in a striking, temporary-paralysis phenotype after blood-feeding.Conclusions/SignificanceThese results are significant in demonstrating for the first time an engineered ‘action at a distance’ phenotype in a non-model pest insect. The potential to dissociate temporal and spatial expression patterns of useful endogenous regulatory elements will extend to a variety of other pest insects and effectors.背景 通过大量释放携带显性致死转基因的埃及伊蚊（Aedes aegypti）雄蚊以实现种群抑制的策略已在田间得到验证。当种群动态呈现负密度依赖特性时，若致死效应发生在密度依赖阶段（即幼虫期）之后，种群抑制效果可得到增强。当前的分子工具仅能通过构建“细胞自主性效应因子（cell-autonomous effectors）”系统来实现此类遗传害虫管理（Genetic Pest Management, GPM），即表达的有害蛋白仅局限于其合成的细胞内——通常由构建系统所用的调控元件（如启动子区域（promoter regions））调控。这类技术的灵活性受到极大限制：具备特定空间、时间或性别特异性表达模式的调控区域，仅当其调控表达的细胞同时对现有效应因子敏感时才能被使用；同时该策略也无法靶向细胞表面受体等细胞外区域。将工具集拓展至“非细胞自主性效应因子（non-cell autonomous effectors）”将大幅缓解这些局限。 方法与主要发现 本研究尝试利用埃及伊蚊卵黄蛋白原A1（VitellogeninA1）启动子，驱动tTAV在吸血诱导下特异性在脂肪体中表达，以获得雌性特异性的晚期致死效应。最初使用促凋亡效应因子（pro-apoptotic effectors）的尝试未产生明显表型，这可能是因为终末分化的脂肪体细胞对程序性死亡信号的敏感性较低。随后，我们通过构建新型人工合成效应因子（蝎神经毒素–TetO-gp67.AaHIT），实现了该系统时空表达模式的解耦：该效应因子可从其合成的组织（脂肪体）中分泌出来，进而作用于其他部位的细胞（神经肌肉接头（neuro-muscular junctions））。最终在吸血后观察到了显著的暂时性麻痹表型。 结论与意义 本研究首次在非模式害虫昆虫（non-model pest insect）中实现了工程化的“远程作用”表型，这一成果具有重要意义。将有用的内源性调控元件的时空表达模式进行解耦的潜在应用价值，可拓展至多种其他害虫昆虫及效应因子的研究中。