A Functional Analysis of Resistance to Pyrethroid Insecticides in the malaria vector Anopheles gambiae

疟疾每年感染2亿人，是许多国家，特别是撒哈拉以南非洲国家的巨大健康和经济负担。控制这种疾病的最好方法是减少传播这种寄生虫的蚊子(“媒介”)的数量，并减少它们与人类的互动。事实上，自千年之交以来，每年死于疟疾的人数减少了一半以上，这在很大程度上是由于大规模使用经杀虫剂处理的蚊帐。经杀虫剂处理的蚊帐提供保护，既充当阻止蚊子到达人类叮咬的物理屏障，又通过接触杀虫剂杀死蚊子。只有一类杀虫剂适合涂在蚊帐上：拟除虫菊酯。这是因为它们的活性持续时间很长，而且对人类的毒性非常低。然而，对这类杀虫剂的抗药性迅速上升，对减少这种疾病取得的成果构成了威胁。因此，为了避免操作上的失败，我们需要找到管理这种耐药性的方法：及早发现耐药性；相应地更换媒介控制工具；开发不受相同耐药性机制影响的新的或改良的杀虫剂。实现这一目标的关键是了解杀虫剂在与蚊子分子靶标相互作用时是如何发挥作用的。DNA测序和“基因组监测”--野外采集蚊子基因组--的巨大进步表明，电压门控钠通道存在DNA突变，而钠通道是拟除虫菊酯在昆虫神经膜上靶标的通道蛋白。然而，除了这一“确凿证据”，我们几乎不知道突变(有时单独发生，有时成群结队地发生)对抗性强度或对所使用的特定类型的拟除虫菊酯(有几种可用)的单独贡献。这一点很重要，因为为了有一个有效的早期预警系统来检测耐药性，至关重要的是要知道每个突变或其组合的“严重程度”。用早期天气预警系统来类比，一个可以区分迎面而来的强风和飓风的检测系统，在允许人们做好准备和做出相应反应方面具有价值。钠通道突变的影响，以及它们如何设法阻止拟除虫菊酯分子干扰它，对于准确理解这种迄今为止非常成功的杀虫剂是如何工作的，是一条至关重要的信息。它应该为设计不受现有突变影响的现有拟除虫菊酯的变种打开大门，从而延长这些杀虫剂的保质期。此外，如果出现与我们所描述的功能相似的新突变，我们预测抗药性出现的能力将会增加。这里提出的工作旨在通过使用最先进的基因组编辑技术将相同的突变引入具有标准化遗传背景的蚊子，来评估在野外捕获的蚊子中发现的不同突变的影响。这将允许直接比较不同的突变并排和相互结合，不仅在赋予杀虫剂抗性的程度方面，而且在蚊子是否有其他相关的适应成本方面。我们还将使用电生理学来确定每个突变在改变钠通道的不同属性方面的影响。这将使我们能够确定哪些突变对抗药性最重要，并帮助我们理解为什么钠通道的变化为这些突变蚊子提供了生存优势。然后，我们的方法将为我们提供一组前所未有的关于拟除虫菊酯抗药性性质的数据，这些数据可以用来更好地了解它是如何出现的，以及如何规划策略来减轻其影响。

Malaria infects 200 million people every year and is a huge health and economic burden on many countries, particularly those in sub-Saharan Africa. The best way to control the disease is by reducing the number of mosquitoes ('vectors') that transmit the parasite responsible, and to reduce their interactions with humans. Indeed, since the turn of the millennium, the number of annual deaths from malaria has more than halved and this is largely due to the large-scale use of insecticide-treated bednets. Insecticide-treated nets provide protection by acting as both a physical barrier that stops the mosquito reaching the human to bite and by killing the mosquito through contact with insecticide. There is only one class of insecticide suitable for coating the net: the pyrethroids. This is due to their long duration of activity and their very low toxicity to humans. However, the rapid rise of resistance to this class of insecticide is a threat to the gains made in reducing this disease. Therefore, to avoid operational failure we need to find ways to manage this resistance by: detecting resistance early; changing vector control tools accordingly; developing new or modified insecticides that are not compromised by the same resistance mechanism. Essential to this goal is an understanding of how the insecticide works when interacting with its molecular target in the mosquito.Huge advances in DNA sequencing and 'genomic surveillance' - sampling mosquito genomes in the field - have pointed to the presence of DNA mutations in the voltage gated sodium channel, which is the channel protein that pyrethroids target in the membrane of insect nerves. However, other than this 'smoking gun' we have little idea of the individual contribution of the mutations (which sometimes occur alone and sometimes occur together in numbers) to the strength of the resistance or to the particular type of pyrethroid used (several are available). This is important because in order to have an effective early warning system to detect resistance it is vital to know the 'severity' of each mutation, or combination thereof. To use an analogy with an early weather warning system, a detection system that can distinguish an oncoming brisk wind from a hurricane has value in allowing one to prepare and react accordingly.The impact of mutations in the sodium channel, and how they manage to stop the pyrethroid molecule from interfering with it, is a vital piece of information for understanding exactly how this class of insecticides, so successful to date, works. It should open the door to designing variants of the currently available pyrethroids that are not compromised by the existing mutations, thereby extending the shelf life of these insecticides. Furthermore, our power to predict the emergence of resistance will be increased if new mutations should arise that are functionally similar to those we have characterised.The work proposed here looks to assess the effect of different mutations identified in field-caught mosquitoes by using state of the art genome-editing techniques to introduce the same mutations into a mosquito with a standardised genetic background. This will allow the direct comparison of the different mutations side by side and in combination with each other, in terms not only of the magnitude of insecticide resistance conferred but also if there are other associated fitness costs in the mosquito. We will also use electrophysiology to determine the effect of each mutation in changing different properties of the sodium channel. This will allow us to identify which mutations are most important for resistance and help us understand why changes in their sodium channels provide those mutant mosquitoes with a survival advantage..Together then, our approaches will provide us with an unprecedented set of data on the nature of pyrethroid resistance, which can be used to better understand how it emerges and how to plan strategies to mitigate its effect.