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

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.

Understanding the nature of target site resistance to pyrethroid insecticides is of paramount importance, given that this is the only class of insecticide approved for use in insecticide-treated bednets, which remain our most potent weapon against malaria transmission.

Huge collaborative efforts to sequence the genomes of wild caught mosquitoes from different malarial settings have highlighted a range of mutations, in the voltage-gated sodium channel that is the molecular target of pyrethroids, that may be associated with resistance. However, the strength of contribution of these mutations to insecticide resistance is unclear, as is how these mutations actually affect pyrethroid interaction. These are essential questions in order to: 1- improve diagnostic assays designed to track the most epidemiologically relevant mutations in a population; 2- open the possibility of rational design of improved insecticide formulations that are not subject to the same resistance.

The work proposed here takes a two-pronged approach: firstly, using CRISPR genome editing to introduce putative resistant mutations in vivo on a uniform genetic background to unambiguously measure their effect on resistance and any pleiotropic effects on mosquito fitness; secondly, by using an electrophysiology approach in parallel to measure, at fine scale, the nature of the alteration to the sodium channel conferred by these mutations.

Together, this information will allow a prioritisation of causative mutations according to effect size which can be used directly to improve the resolution of resistance monitoring in vector control programs, to better understand the dynamics of selection for resistance in the wild and, most importantly, allow more informed decisions when adapting control practice to combat resistance.