Opening up Anopheles funestus to functional genetics and the study of insecticide resistance

A way to introduce precise genetic changes into an organism is essential for understanding how genes determine key biological characteristics. In the case of mosquito vectors such a technology can allow us to understand the genetic nature of traits that make it such a resilient transmitter of the malaria parasite. Half the world's population are at risk from malaria every year and, despite recent interventions that have had some success, close to half a million people die, most of these children. Anopheles funestus is a significant vector of malaria in many areas of sub-Saharan Africa, often being the dominant species responsible for the majority of transmission in some areas. Despite this, it is relatively poorly studied in the lab, in part because it can be difficult to rear in the laboratory, in part because a technology to genetically transform it - i.e. manipulate its genes to see what effect they have - has yet to be developed. This project will bring together two research teams with unique and complementary experience in the rearing of Anopheles funestus and in genetic transformation of mosquitoes, respectively.


We will establish new laboratory colonies of A. funestus, providing varied genetic populations as resource for the scientific community. We will use precise genome editing tools, such as CRISPR, which can function like a precise pair of molecular scissors to cut almost any mosquito DNA sequence of choice to introduce desired sequence changes there. Such a technology permits confirmation of the role of genetic sequences that might previously have been associated with important traits that determine the ability of this mosquito to transmit malaria. The focus of this research proposal will be to understand the genetic basis of insecticide resistance, which is currently jeopardising the gains achieved to date by using insecticide-treated nets and indoor spraying of insecticides. An understanding of the mechanism of resistance will enhance mosquito control through: 1) allowing a quantification of the different genetic contributions to resistance; 2) molecular screening to detect early the emergence of insecticide resistance and in a high throughput fashion; 3) adaptive rotation of insecticide classes in response to mechanism of resistance emerging, in order to prolong the utility of a given insecticide class 4) a comparison of the origins of insecticide resistance among Anopheles funestus and Anopheles gambiae

The study of the genetic basis of insecticide resistance in the important malaria vector Anopheles has lagged, due to a paucity of available expertise in rearing this mosquito and the resultant lack of genetic variation on which to select for traits of interest. Regions of the funestus genome associated with insecticide resistance have been identified yet the inability to easily rear and cross wild caught strains in the lab drastically limits the power and resolution to identify candidate genes that may be determining resistance, let alone identify the specific mutations that constitute a resistant allele. This not only precludes a full mechanistic understanding of the basis of resistance, it is an obstacle to developing molecular genotyping assays to detect resistance without the need to always do insecticide resistance assays in the field, which can be noisy, slow and logistically challenging.

The boon of CRISPR-mediated genome editing holds much promise for introducing alleles of choice into a given genetic background. Our groups have pioneered its use in the malaria vector Anopheles gambiae, and are starting to use it to decipher insecticide resistance in that species. However, there is a large and growing body of evidence that mechanisms of resistance have evolved very differently in these two species that are separated by ~30 million years of evolution, not only at the level of target gene families but also in the predominant mechanisms of rendering insecticides ineffective.

We include preliminary data that shows we have developed a CRISPR pipeline that can recapitulate insecticide resistance alleles, normally found in the field, on the standardised An funestus lab background. Now that we have developed and proven the technology we have planned the generation of a series of putative resistance alleles to understand their effect size, their mechanism of action and their interaction with other resistance mechanisms and their fitness effect.