In the arms race of humans verses malaria-spreading mosquitos, the insects have developed a number of genetic tricks to thwart pyrethroids, a class of insecticides used to keep their populations at bay. For instance, in mosquitos with a particular mutation, the insecticides can’t bind to their target. Metabolic resistance, which allows mosquitos to digest the chemicals and detoxify them, has been harder to pin down genetically, leaving scientists without markers to easily track the spread of various metabolic tactics.
Now, researchers have found a marker, a single mutation, linked to pyrethroid detoxification in populations of southern Africa’s Anopheles funestus, a major malaria vector, and can use it to monitor the spread of resistance, the scientists reported March 20 in Science Translational Medicine.
“It’s a magnificent piece of work. It’s bridging a large gap,” says Jo Lines, a malaria entomologist at the London School of Hygiene and Tropical Medicine who was not part of the project.
Malaria prevention relies on controlling its mosquito vector and pyrethroids are an important tool towards that end. A recent study estimated that insecticide use prevented upwards of 663 million cases of the disease between 2000 and 2015, with pyrethroid-treated bed nets accounting for roughly 70 percent of those. “All this is threatened by the rise of insecticide resistance in mosquito populations,” says Gareth Weedall, a coauthor of the new paper and a geneticist at Liverpool John Moores University in the United Kingdom.
Most of that genetic diversity has been swept away and only the resistant allele is left, pretty much.—Gareth Weedall, Liverpool John Moores University
Because resistance can arise through multiple mechanisms and multiple genes, metabolic evasion is tricky to study. This resistance, in particular, involves genes encoding cytochrome P450, a family of enzymes found previously to break down pyrethroids. While the gene identities were known, the locus that promotes their overexpression in resistant mosquitos wasn’t. So to study the resistance, researchers relied on RNA sequences to capture the genes’ expression. But RNA can be difficult to work with, especially in the field, because it isn’t very stable.
To sniff out possible expression enhancers, Weedall and his collaborators started with analyzing the gene expression of several mosquito populations from different regions in Africa. This led them to a cluster of genes that seemed unique to mosquitos from southern Africa, which they investigated further using whole genome sequences for mosquitos resistant to permethrin, one type of pyrethroid.
Their detective work pointed to a culprit stretch of DNA that appears to have racked up genetic mutations and accumulated a large insertion of genetic material that drives the genes encoding cytrochrome P450. “It’s not just a mutation. It’s a geneticist’s dream of complex genetic changes,” says Lines. These elaborate mechanisms are evidence for the strong selective pressures the mosquitoes experienced, he says. The investigators also identified a single mutation in a promoter region that correlated with, but is not necessarily responsible for, the overexpression of these genes in resistant mosquitos.
This single mutation handed the researchers a diagnostic to screen for pyrethroid resistance. Using PCR, they could now look for the allele in a mosquito’s DNA, which is much easier to work with than RNA, and will let scientists track the spread of this resistance. “We can track that beautifully—and do it using specimens that were dried, that were pickled in alcohol, doesn’t matter, we can do it—because DNA is so stable,” says Lines.
They applied their diagnostic to samples from 2014 collected from around the African continent to get a snapshot of the allele’s distribution and found the resistant allele highly prevalent across southern Africa. It had even become the dominant variation in some populations, for instance, from Malawi and Mozambique. A previous sampling of these two locations in 2002, before the widespread use of pyrethroid-treated bed nets, had found much greater variation in this region of the genome, says Weedall. “Most of that genetic diversity has been swept away and only the resistant allele is left, pretty much,” he says.
In experimental huts in the field with populations of lab-reared mosquitos, the researchers compared how effective pyrethroid-treated bed nets were in killing resistant and susceptible insects and took a census of alleles present in dead and live mosquitos after exposure. For one type of treated bed net, researchers found that more than 90 percent of the surviving mosquitos had one or two copies of the resistance allele. And while another type of bed net was more effective at killing insects with the resistance allele, humans sheltered beneath them still got bitten, meaning that they could still get infected with malaria even if the insect eventually died.
The hut experiments “are particularly impressive and really useful and important,” says Chris Bass, an entomologist at the University of Exeter in the United Kingdom who was not part of the work.
Altogether, the work paints a picture of pyrethroid resistance’s spread. Under selection by pyrethroid use, the allele seems to have permeated mosquito populations of southern Africa, though it hasn’t yet become the dominant variant in further north and its yet unclear if it will become so. Now that the researchers have a genetic marker, they can monitor the allele’s future spread and the increase of this resistance in mosquito populations.
This marker only applies to one type of resistance while other mechanisms may be at work in populations of mosquitos elsewhere in Africa. To understand the genetic basis of those methods of evasion and develop a traceable marker, “we have to replicate this work for potentially all of those candidate genes,” says Weedall. And this is just one species of malaria spreading mosquitos—similar work could help track metabolic resistance in the other species of Anopheles, including A. gambiae.
It’s important to watch the spread of resistance, as “there’s a large part of the world that's just getting doused in pyrethroid insecticides and it’s a major selection pressure on lots of different insects,” says Weedall.
“The reliance on a single or small number of insecticides is just asking for trouble in the sense that resistance will very rapidly evolve,” says Bass. “This is a nice illustration of that.”
G.D. Weedall et al., “A cytochrome P450 allele confers pyrethroid resistance on a major African malaria vector, reducing insecticide-treated bednet efficacy,” Science Translational Medicine, doi:10.1126/scitranslmed.aat7386, 2019.