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 that is used to impregnate bed nets. The scientists found that pyrethroid-resistant Anopheles gambiae and Anopheles coluzzii mosquitoes expressed high levels of a chemical binding protein known as sensory appendage protein (SAP2), in their legs, which comes into contact with the insecticide when the insects land on the nets. Their studies linked high expression of SAP2 with resistance to pyrethroid insecticides in long-lasting insecticidal nets (LLINs) that have been so successful in reducing the incidence of malaria in many areas of Africa. Experiments showed that partial deletion of SAP2 rendered previously pyrethroid-resistant Anopheles mosquitoes susceptible to the insecticide, whereas overexpressing SAP2 rendered otherwise susceptible mosquitoes resistant to pyrethroids.

“We have found a completely new insecticide resistance mechanism that we think is contributing to the lower than expected efficacy of bed nets,” said Victoria Ingham, PhD, postdoctoral research associate and first author on the team’s published paper in Nature. “The protein, which is based in the legs, comes into direct contact with the insecticide as the insect lands on the net, making it an excellent potential target for future additives to nets to overcome this potent resistance mechanism.” Ingham and colleagues reported their findings in a paper titled, “A sensory appendage protein protects malaria vectors from pyrethroids.”

The widespread use of pyrethroid-impregnated bed nets has been instrumental in reducing malaria-associated disease and death rates in Africa since the beginning of the century, the authors said. “However, worryingly, after many years of progress, gains in malaria control are now stalling, with an estimated 219 million cases and 435,000 malaria-associated deaths across Africa in 2017 prompting a re-examination of the effectiveness of the primary prevention tools.” The widespread use of LLINs has forced selection pressures on the mosquitoes to develop mechanisms that confer resistance to pyrethroids. As a result, insecticide-treated bed nets containing the synergist piperonyl butoxide (PBO) in addition to pyrethroid insecticides have now been introduced. The synergist targets one of the most widespread and most potent known resistance mechanisms caused by cytochrome P450s, and so re-establishes insect susceptibility to pyrethroids.

“By blocking this resistance mechanism, PBO–pyrethroid nets restore insecticide susceptibility, leading to a reduction in malaria cases in areas in which metabolic resistance prevails,” the authors stated. However, not all pyrethroid-resistant mosquito populations can be targeted by the use of PBO to restore pyrethroid susceptibility, and Anopheles mosquitoes are developing new resistance mechanisms. By screening gene expression data from mosquito populations in Burkina Faso and the Côte d’Ivoire, which are areas with “particularly high pyrethroid resistance and low PBO synergism,” the investigators found that a family of chemosensory proteins (CSPs), known as sensory appendage proteins, was overexpressed in resistant mosquito populations. CSPs are small, soluble proteins that are only found in arthropods, and they transport small hydrophobic molecules as part of a chemical communication system.

The team’s experiments first showed that insecticide exposure induced expression of the sensory appendage protein, SAP2. They then demonstrated that susceptibility to three different pyrethroid insecticides tested could be restored in highly pyrethroid-resistant mosquitoes, by using RNA interference (RNAi) to almost completely silence the SAP2 gene. Conversely, the team noted, “ … overexpression of SAP2 in an insecticide-susceptible population significantly increased pyrethroid resistance, directly linking the function of this protein to insecticide resistance.” Tests confirmed that SAP2 bound directly 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 from direct sequencing. The results indicated that a “selective sweep” had occurred at the locus where CSP genes are found, over the time period during which resistance to pyrethroid insecticides also increased. A selective sweep refers to when a beneficial mutation becomes fixed in a population and increases in frequency as a result of natural selection, and also reduces variation in linked sites.

“Our results show that SAP2, a chemosensory protein with no previous known function in insecticide resistance, has a key role in conferring pyrethroid resistance in the A. gambiae species complex through the binding of insecticides at the first point of mosquito contact with bed nets,” the authors commented. “Given its strong binding to pyrethroid insecticides, it is possible that SAP2 acts by sequestering the insecticide directly, thus either preventing the function of the insecticide on the nervous system or facilitating its detoxification. Notably, longitudinal sequencing of field samples and available transcriptomic data from wild collections show that this mechanism is being selected for in multiple countries in West Africa, highlighting its relevance in field settings.”

The researchers claim that discovery of the new resistance mechanism may help in the development of new synergists that could be used to restore pyrethroid susceptibility in some populations of resistant mosquitoes. “… the identification of this previously undescribed insecticide sequestration mechanism offers the concrete possibility to restore the effectiveness of pyrethroid insecticides in natural mosquito populations through the identification of new targets for inhibitors that can be incorporated into bed nets, in an analogous manner to the incorporation of PBO into nets—this may prove to be critical for the elimination of malaria across Africa,” the team concluded.

“Long-lasting insecticide-treated bed nets remain one of the key interventions in malaria control,” stated Hilary Ranson, PhD, senior author on the paper. “It is vital that we understand and mitigate for resistance within mosquito populations in order to ensure that the dramatic reductions in disease rates in previous decades are not reversed. This newly discovered resistance mechanism could provide us with an important target for both the monitoring of insecticide resistance and the development of novel compounds able to block pyrethroid resistance and prevent the spread of malaria.”

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