A feeding female Anopheles gambiae mosquito. [CDC/James Gathany]
Researchers at the Liverpool School of Tropical Medicine have identified a previously unknown mechanism by which mosquitoes that carry the malaria parasite can become resistant to the insecticide used to impregnate bed nets. The scientists found that pyrethroid-resistant Anopheles gambiae and Anopheles coluzzii mosquitoes express high levels of a chemical binding protein known as sensory appendage protein (SAP2) in their legs, which comes in contact with the insecticide when the insects hit the Landing nets. Their studies linked high levels of SAP2 expression with resistance to pyrethroid insecticides in long-lived insecticidal nets (LLINs) that have so successfully reduced the incidence of malaria in many areas of Africa. Experiments showed that partial deletion of SAP2 made previously pyrethroid-resistant Anopheles mosquitoes susceptible to the insecticide, while overexpression of SAP2 made otherwise susceptible mosquitoes resistant to pyrethroids.
“We discovered an entirely new mechanism of insecticide resistance that we believe contributes to lower than expected effectiveness of bed nets,” said Victoria Ingham, PhD, postdoctoral fellow and lead author in the team’s article published in Nature. “The leg-based protein comes in direct contact with the insecticide when the insect lands on the net. This makes it an excellent potential target for future net additions to overcome this strong mechanism of resistance.” Ingham and colleagues reported their findings in an article entitled “A sensory limb protein protects malarial vectors from pyrethroids”.
The widespread use of pyrethroid-impregnated bed nets has helped lower malaria-related diseases and death rates in Africa since the beginning of the century, the authors say. “What is worrying, however, is that after many years of progress, progress in malaria control is now stalling. An estimated 219 million cases and 435,000 deaths from malaria across Africa in 2017 led to a reassessment of the effectiveness of primary prevention tools.” The widespread use of LLINs has forced selective pressures on the mosquitoes to develop mechanisms that confer resistance to pyrethroids. As a result, bed nets treated with insecticides have now been introduced, which contain the synergist piperonyl butoxide (PBO) in addition to pyrethroid insecticides. The synergist targets one of the most widespread and potent known mechanisms of resistance caused by cytochrome P450, restoring insects’ susceptibility to pyrethroids.
“By blocking this mechanism of resistance, PBO-pyrethroid nets restore susceptibility to insecticides, leading to a reduction in malaria incidence in areas where metabolic resistance prevails,” the authors say. However, not all pyrethroid-resistant mosquito populations can be attacked by the use of PBO to restore pyrethroid susceptibility, and Anopheles mosquitoes are developing new mechanisms of resistance. By screening gene expression data from mosquito populations in Burkina Faso and Ivory Coast, which are areas of “particularly high pyrethroid resistance and low PBO synergism,” the researchers found that a family of chemosensory proteins (CSPs) are known as sensory appendages Proteins, was overexpressed in resistant mosquito populations. CSPs are small, soluble proteins found only in arthropods that transport small hydrophobic molecules as part of a chemical communication system.
The team’s experiments initially showed that exposure to insecticides induced expression of the sensory appendage protein SAP2. They then showed that susceptibility to three different pyrethroid insecticides tested can be restored in highly pyrethroid resistant mosquitoes by using RNA interference (RNAi) to silence the SAP2 gene almost completely. Conversely, the team found: “… overexpression of SAP2 in an insecticide-prone population significantly increased pyrethroid resistance and linked the function of this protein directly to insecticide resistance.” Tests confirmed that SAP2 was directly linked to all three pyrethroid insecticides tested.
The team then analyzed the genomes of West African Anopheles populations over time using a combination of data from the Anopheles gambiae 1000 Genomes project and direct sequencing. The results showed that a “selective sweep” had taken place at the location where CSP genes were found over the period in which resistance to pyrethroid insecticides also increased. A selective sweep refers to when a beneficial mutation is fixed in a population and the frequency increases as a result of natural selection and also decreases the variation at linked sites.
“Our results show that SAP2, a chemosensory protein with no known function in insecticide resistance, plays a key role in conferring pyrethroid resistance in the A. gambiae species complex by binding insecticides at the first point of mosquito contact with bed nets. Commented the authors. “Due to its strong binding to pyrethroid insecticides, it is possible that SAP2 binds the insecticide directly, either preventing the insecticide from acting on the nervous system or making it easier to detoxify. In particular, the longitudinal sequencing of field samples and the available transcriptomic data from wild collections show that this mechanism is selected in several countries in West Africa, which underlines its relevance for the field settings. “
The researchers claim that the discovery of the new mechanism of resistance could help develop new synergists that could be used to restore pyrethroid susceptibility in some populations of resistant mosquitoes. “… The identification of this previously unspecified mechanism for sequestering insecticides offers the concrete possibility of restoring the effectiveness of pyrethroid insecticides in natural mosquito populations by identifying new targets for inhibitors that can be built into bed nets in a manner analogous to PBO in nets – this could prove crucial in eradicating malaria across Africa, ”the team concluded.
“Long-lasting bed nets treated with insecticides remain one of the most important malaria control measures,” said Dr. Hilary Ranson, senior author of the paper. “It is important that we understand and mitigate resistance within mosquito populations to ensure that the dramatic reductions in disease rates over the past few decades are not reversed. This newly discovered mechanism of resistance could provide us with an important target both for monitoring insecticide resistance and for developing new compounds that can block pyrethroid resistance and prevent the spread of malaria. “