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

疟疾每年感染2亿人，对许多国家，特别是撒哈拉以南非洲国家造成巨大的健康和经济负担。控制这种疾病的最佳方法是减少传播寄生虫的蚊子（“媒介”）的数量，并减少它们与人类的相互作用。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，这主要是由于大规模使用经杀虫剂处理的蚊帐。经杀虫剂处理过的蚊帐提供保护，它既是一种物理屏障，阻止蚊子到达人体叮咬，又通过与杀虫剂接触杀死蚊子。只有一种杀虫剂适合用于蚊帐涂层：拟除虫菊酯。这是因为它们的活性持续时间长，对人类的毒性很低。然而，对这类杀虫剂的抗药性迅速上升，威胁到在减少这一疾病方面取得的进展。因此，为了避免操作失败，我们需要找到管理这种抗药性的方法：及早发现抗药性;相应地改变病媒控制工具;开发新的或改良的杀虫剂，这些杀虫剂不会受到相同抗药性机制的影响。要实现这一目标，关键是要了解杀虫剂在与蚊子体内的分子靶点相互作用时是如何起作用的。DNA测序和“基因组监测”（实地对蚊子基因组进行采样）的巨大进展表明，电压门控钠通道中存在DNA突变，这是拟除虫菊酯在昆虫神经细胞膜中靶向的通道蛋白。然而，除了这个“确凿的证据”之外，我们对突变（有时单独发生，有时一起发生）对抗性强度或所用拟除虫菊酯的特定类型（有几种可用）的单独贡献几乎一无所知。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，了解每种突变的“严重性”或其组合至关重要。以天气预警系统为例，一个能够区分强风和飓风的探测系统，可以帮助人们做出相应的准备和反应。钠通道突变的影响，以及它们如何阻止拟除虫菊酯分子干扰钠通道，对于了解这类迄今为止非常成功的杀虫剂的确切作用机制，是一个至关重要的信息。它应该为设计目前可用的拟除虫菊酯的变体打开大门，这些变体不会受到现有突变的影响，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，我们预测耐药性出现的能力将增加。这里提出的工作旨在评估野外捕获的蚊子中鉴定的不同突变的影响，方法是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中。这将允许直接比较不同的突变并排和彼此组合，不仅在所赋予的杀虫剂抗性的大小方面，而且在蚊子中是否存在其他相关的适应性成本方面。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特性中的作用。这将使我们能够确定哪些突变对耐药性最重要，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耐药性性质的数据，这些数据可用于更好地了解它是如何出现的，以及如何规划减轻其影响的策略。

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.