Workshop 2 - Training course in data analysis for genomic surveillance of African malaria vectors
Module 2 - The genetic basis of metabolic resistance to insecticides in Anopheles mosquitoes#
Theme: Biology
This module provides a brief introduction to the biology of insecticide resistance caused by increased expression or modification of enzymes within Anopheles mosquitoes that can break down insecticide molecules before they have a chance to act.
Learning objectives#
After this module you will be able to:
Explain what metabolic resistance is
Describe which types of genes are involved in metabolic resistance
Explain how genetic changes such as copy number variation can cause metabolic resistance
Describe some recent research on metabolic resistance in An. gambiae s.l. and An. funestus
Lecture#
English#
%%html
<iframe width="560" height="315" src="https://www.youtube.com/embed/VCt5ZlBOa3I" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe>
Français#
%%html
<iframe width="560" height="315" src="https://www.youtube.com/embed/cXnuJe9asOw" title="YouTube video player" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe>
Please note that the code in the cells below might differ from that shown in the video. This can happen because Python packages and their dependencies change due to updates, necessitating tweaks to the code.
Types of insecticide resistance#
In workshop 1 we talked about target site resistance, where a single nucleotide polymorphism (or SNP) can alter the target site for an insecticide, resulting in resistance (we used the gene Vgsc as an example).
There is also behavioral resistance, where mosquitoes may change their behaviors to avoid insecticides, by biting outdoors or biting non-human hosts.
Cuticular resistance occurs where the thickening of different parts of the insect cuticle results in a reduction of an insecticide getting into an insect’s body.
Another type of resistance is metabolic resistance, which we will discuss in this module.
What is metabolic resistance to insecticides?#
Metabolic insecticide resistance involves the chemical transformation of an insecticide, preventing or reducing its ability to interact with its target site.
As an example, a metabolic resistance gene encodes a protein that directly interacts with an insecticide molecule, breaking it into pieces or otherwise altering it in a way that makes it non-toxic to the insect, so that it can be removed from the insect body.
What proteins are responsible for metabolic resistance?#
There are several families of enzymes that are known to be involved in metabolic insecticide resistance, including:
Cytochrome P450 monooxygenases (also sometimes called mixed-function oxidases or MFOs)
Glutathione S-transferases
Carboxylesterases
These proteins are highly diversified for many different functions, including normal detoxification pathways in insects and other organisms, including humans.
What genes are involved and where are they located in the genome?#
Genes associated with metabolic resistance are distributed across the Anopheles gambiae genome, they are not localized on any one chromosome or chromosome arm.
Here are some target-site resistant genes (like Ace1 and Vgsc) for reference, and some clusters of Cyp and Gst genes known to cause metabolic resistance, shown on the chromosomes of the An. gambiae genome.
Which genes cause metabolic resistance?#
Cytochrome P450 (Cyp) genes#
Cyp genes encode membrane-bound enzymes that break down compounds, primarily through oxidation. There are more than 100 Cyp genes in the An. gambiae genome. Cyp genes are associated with resistance to all known classes of insecticides. Insecticide resistance can occur when certain Cyp genes are expressed in greater quantities.
Cyp genes can be turned on (induced) by a chemical signal. For example, in the presence of insecticide or insecticidal activity, a molecule will initiate a signal that binds to an enhancer somewhere in the genome (not necessarily near the gene target). This enhancer will start or upregulate the transcription of one or more Cyp genes, which will then produce proteins or enzymes that will help to metabolize the insecticide.
Glutathione S-Transferase (Gst) genes#
Gst genes encode metabolic enzymes that can mediate resistance to organophosphates, organochlorines, and pyrethroid insecticides. These enzymes play a role in reducing oxidative stress. There are 31 known Gst genes in the Anopheles gambiae genome. Similar to Cyp genes, insecticide resistance can occur when certain Gst genes are expressed in greater quantities.
How can genetic changes cause metabolic resistance?#
Several different types of mutation that can cause a greater expression of genes causing metabolic resistance, including:
Copy number variation (CNV) - an increase the in the number of copies of a metabolic resistance gene
Cis-regulatory variation - a change in regulatory sequences near to or within a metabolic resistance gene
Trans-regulatory variation - a change in a regulatory gene or sequence in a different part of the genome
Genetic changes can also occur within metabolic resistance genes, causing them to have increase catalytic activity or specificity for certain insecticide molecules.
In this workshop we will focus primarily on CNVs, because these are a known to be an important cause of metabolic resistance in An. gambiae, but they are certainly not the whole story.
What are copy number variants (CNVs)?#
CNVs are a type of mutation that results in a change in the number of copies of a gene or sequence. This happens when a portion of a sequence (like in the box below) is copied and inserted somewhere else in the genome.
CNVs are particularly important for metabolic resistance because multiple copies of a gene can result in more proteins being produced.
So if a single gene produces some amount of protein, having multiple copies of a gene will produce more protein with the same input signals.
CNVs and metabolic resistance in An. gambiae#
Lucas et al. (2019) analysed CNVs in An. gambiae using whole-genome sequencing data from the Anopheles gambiae 1000 genomes project, and found that:
CNVs represent a major source of genetic variation in An. gambiae
CNVs can include multiple genes over hundreds of thousands of basepairs
Metabolic gene clusters were major CNV hotspots
There was evidence for strong positive selection for CNVs containing Cyp genes
Are other types of mutation associated with metabolic resistance?#
Yes. For example, SNPs associated with metabolic resistance have been found in An. gambiae and An. funestus. A SNP in Gste2, I114T, has been linked to increased resistance to DDT in An. gambiae (Mitchell et al. 2014). Another SNP in Gste2 found in a wild population of An. funestus, L119F, has been associated with better binding and increased clearance of DDT (Riveron et al. 2014).
Insertions affecting regulatory sequences can also be important. For example, Weedall et al. (2019) found a cis-regulatory polymorphism in An. funestus causing metabolic resistance to pyrethroids via increased expression of Cyp6p9a.
How do we identify genes involved in metabolic resistance?#
We can compare gene expression in insecticide resistant and susceptible lines or populations
We can look for changes before and after insecticide selection events
We can look for signals of evolutionary selection in populations with a known insecticide resistance phenotypes
Some examples of these types of studies:
Simma et al. (2018) used RNAseq to compare gene expression between insecticide-resistant and susceptible lines of An. arabiensis.
David et al. (2005) used a microarray to look at gene expression in permethrin and DDT-resistant strains of An. gambiae compared to a susceptible strain.
In both of these studies, Cyp and Gst genes were found to be upregulated in insecticide-resistant mosquitoes.
Functional validation of metabolic resistance genes#
Just because a gene is found to be expressed at higher levels in mosquitoes with insecticide resistance, doesn’t necessarily mean that the gene is a direct cause of metabolic resistance. Candidate resistance genes need to be functionally validated, which means performing lab experiments to demonstrate that they do directly cause resistance.
For example, Adolfi et al. (2019) showed that increased expression of three genes – Cyp6m2, Cyp6p3, and Gste2 – in an otherwise insecticide-susceptible line, conferred resistance to multiple classes of insecticides. This study confirmed that these genes are involved in metabolic resistance in An. gambiae, and that increasing the copy number of these genes causes resistance, and so CNVs could be used as markers for metabolic resistance.
How can we manage metabolic resistance in Anopheles mosquitoes?#
The addition of Pyperonyl Butoxide (PBO) to insecticide-impregnated nets rescues the mortality effect of pyrethroids by inhibiting the activity of cytochrome P450s. So, in places with highly pyrethroid-resistant populations of mosquitoes, where resitance is caused by Cyp-mediated metabolic resistance to insecticides, these new types of nets may help malaria control efforts.
Summary#
Emerging metabolic resistance is a challenge for malaria control and few molecular markers are currently available
Metabolic resistance genes include those in pathways already used for detoxification in mosquitoes, such as Cyp and Gst genes
Functional studies of genes are needed to reveal the mechanisms of metabolic resistance pathways
CNVs are a type of mutation that can lead to increased expression of multiple genes known to be involved in metabolic resistance and as such are potential molecular markers for metabolic insecticide resistance