CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes.
CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes.
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DOI：
10.1371/journal.pgen.1009556
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发表时间：
2021-07
期刊：
影响因子：
4.5
通讯作者：
Ranson H
中科院分区：
文献类型：
--
作者：
Grigoraki L;Cowlishaw R;Nolan T;Donnelly M;Lycett G;Ranson H
按蚊的抗药性是维持减少疟疾负担势头的主要障碍;减轻疟疾的战略需要更好地了解基本机制。杀虫剂的靶位点突变（最广泛使用的拟除虫菊酯类的电压门控钠通道）和解毒酶的过度表达是常见的，但它们对表型抗性的相对贡献仍然知之甚少。在这里，我们提出了一个基因组编辑管道，在An中引入单核苷酸多态性。我们已经用它来研究经典的kdr突变L1014 F（基于An.冈比亚编号），是分布最广的抗性等位基因之一。在完全易感的遗传背景中引入1014 F，增加了对所有测试的拟除虫菊酯和滴滴涕的抗性水平，从氯菊酯的9.9倍到滴滴涕的24倍。引入1014 F等位基因足以降低暴露于溴氰菊酯处理的蚊帐后蚊子的死亡率，即使是目前唯一的抗性机制。当1014 F与谷胱甘肽转移酶Gste 2的过表达相结合时，对二氯苯醚菊酯的抗性增加，进一步证明了体内靶位点抗性和解毒酶之间的关键综合效应。我们还表明，携带1014 F等位基因纯合性的蚊子表现出健身的缺点，包括在幼虫阶段的死亡率增加和繁殖力和成年寿命的减少，这可能会产生后果的选择，将适用于该等位基因在该领域的强度。按蚊对拟除虫菊酯抗药性的升级有可能降低我们在疟疾控制中最重要工具的有效性。研究杀虫剂抗药性的潜在机制对于设计缓解策略至关重要。在这里，使用基因组修饰的蚊子，我们在功能上表征了抗性蚊子中最普遍的突变，表明它对所有测试的拟除虫菊酯都具有相当高的抗性水平，并破坏了经拟除虫菊酯处理的蚊帐的性能。此外，我们表明，将这种突变与解毒酶水平升高相结合，进一步增加了耐药性。我们开发的管道提供了一种强大的方法来量化不同抗性机制组合对整体表型的贡献，提供了抗性监测和抗性影响预测之间缺失的环节。
Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field. Escalation of pyrethroid resistance in Anopheles mosquitoes threatens to reduce the effectiveness of our most important tools in malaria control. Studying the mechanisms underlying insecticide resistance is critical to design mitigation strategies. Here, using genome modified mosquitoes, we functionally characterize the most prevalent mutation in resistant mosquitoes, showing that it confers substantial levels of resistance to all tested pyrethroids and undermines the performance of pyrethroid-treated nets. Furthermore, we show that combining this mutation with elevated levels of a detoxification enzyme further increases resistance. The pipeline we have developed provides a robust approach to quantifying the contribution of different combinations of resistance mechanisms to the overall phenotype, providing the missing link between resistance monitoring and predictions of resistance impact.
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影响因子：
4.7
作者：
Hardstone, Melissa C.;Scott, Jeffrey G.
通讯作者：
Scott, Jeffrey G.
影响因子：
4.6
作者：
Lucas, Eric R.;Rockett, Kirk A.;Donnelly, Martin J.
通讯作者：
Donnelly, Martin J.
DOI：
10.1073/pnas.1711538114
发表时间：
2017-12-05
影响因子：
11.1
作者：
Li M;Bui M;Yang T;Bowman CS;White BJ;Akbari OS
通讯作者：
Akbari OS
影响因子：
11.8
作者：
Edi CV;Koudou BG;Jones CM;Weetman D;Ranson H
通讯作者：
Ranson H
影响因子：
5.3
作者：
Ffrench-Constant RH;Bass C
通讯作者：
Bass C