A sensory appendage protein protects malaria vectors from pyrethroids

Abstract

Pyrethroid-impregnated bed nets have driven considerable reductions in malaria-associated morbidity and mortality in Africa since the beginning of the century1. The intense selection pressure exerted by bed nets has precipitated widespread and escalating resistance to pyrethroids in African Anopheles populations, threatening to reverse the gains that been made by malaria control2. Here we show that expression of a sensory appendage protein (SAP2), which is enriched in the legs, confers pyrethroid resistance to Anopheles gambiae. Expression of SAP2 is increased in insecticide-resistant populations and is further induced after the mosquito comes into contact with pyrethroids. SAP2 silencing fully restores mortality of the mosquitoes, whereas SAP2 overexpression results in increased resistance, probably owing to high-affinity binding of SAP2 to pyrethroid insecticides. Mining of genome sequence data reveals a selective sweep near the SAP2 locus in the mosquito populations of three West African countries (Cameroon, Guinea and Burkina Faso) with the observed increase in haplotype-associated single-nucleotide polymorphisms mirroring the increasing resistance of mosquitoes to pyrethroids reported in Burkina Faso. Our study identifies a previously undescribed mechanism of insecticide resistance that is likely to be highly relevant to malaria control efforts.

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Fig. 1: CSP expression profiles.
Fig. 2: SAP2 mediates resistance to pyrethroid insecticides.
Fig. 3: SAP2 is upregulated and under selection in multiple countries across West Africa.

Data availability

All data analysed during this current study are described in the Article, Extended Data Fig. 19, Extended Data Table 1 and the Supplementary Information, or are available from the corresponding authors upon reasonable request. Source Data for Figs. 1, 2 are provided with the paper.

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Acknowledgements

We thank N. Grisales (Bakaridjan in 2013 and 2015), A. Sanou, M. Guelbeogo (Tiefora 2018, Tengrela 2011, 2012, 2014, 2016) and N. Lissenden (Tengrela 2018) for providing field collections; D. Neafsey and J. Tennessen for sharing Tengrela whole-genome sequence data; I. Iovinella for the provision of SAP1 and SAP3 plasmids; M. Bernardi for help with the generation of the map in Fig. 3a and the preparation of figures; F. Brown, S. Elg, P. Pignatelli and D. Au for providing technical support; H. Toé, B. Lambert and T. Churcher for supplying both the field data and figure in Extended Data Fig. 8; and D. Tsakireli and E. Morou for their help with the expression and characterization of SAP proteins. This study was funded by an MRC Skills Development Fellowship (MR/R024839/1) to V.A.I. and a Royal Society Challenge Grant (CH160059) to H.R. Mosquito collections in Burkina Faso were supported by EC FP7 Project grant no: 265660 ‘AvecNet’ and Wellcome Trust Collaborative Award (200222/Z/15/Z).

Author information

V.A.I. and H.R. conceived the experimental design. V.A.I. performed all transcriptomic expression experiments, RNAi and phenotyping experiments, data analysis, and PCR and associated sequencing experiments. A.A. and G.L. produced the transgenic lines and associated phenotypic characterisation. V.D. and J.V. performed the binding assays and associated protein expression experiments. N.J.H. analysed the data from the Anopheles gambiae 1000 Genomes Project and produced the haplotype SNP panel. M.M. provided all insectary support. V.A.I. and H.R. drafted the manuscript.

Correspondence to Victoria A. Ingham or Hilary Ranson.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Mara Lawniczak, James G Logan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Chemosensory protein cluster.

a, Schematic of the region surrounding the shared haplotype block found in the Anopheles gambiae 1000 Genomes Project data with all chemosensory proteins in the cluster highlighted in yellow. Genes displayed in order of appearance, from left to right, are as follows: AGAP008046, AGAP013713, AGAP008047, AGAP008048, AGAP008049, AGAP008050, AGAP008051 (SAP1), AGAP008052 (SAP2), AGAP008053, AGAP008054 (SAP3), AGAP008055 (CSP3), AGAP008056, AGAP029127 (CSP5, previously AGAP008058), AGAP008059 (CSP1), AGAP008060, AGAP008061 and AGAP008062 (CSP4). b, cDNA bootstrap consensus tree inferred from 1,000 replicates using the maximum likelihood method; the percentage of replicate trees with the associated clustering are shown next to the branches. Yellow indicates the sensory appendage proteins, orange the remaining chemosensory proteins in the 3R cluster and black dotted lines show CSP6, which is located on 2R. Alternative isoforms are represented with ‘-RX’, with ‘X’ proceeding alphabetically dependent on number of splice variants.

Extended Data Fig. 2 Overexpression of the CSP family in a multi-insecticide-resistant Anopheles population.

Left, mean relative fold change of each CSP in Tiassalé mosquitoes (blue) compared with the susceptible control N’Gousso mosquitoes (grey) as determined by qPCR. Right, mean relative fold change of each CSP in Tiassalé mosquitoes (blue) compared with the susceptible Kisumu population (grey). Points show three biological replicates. Data are mean ± s.d. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Statistical significance was calculated by an ANOVA followed by Dunnett’s post hoc test; P values are included in Supplementary Table 2.

Extended Data Fig. 3 Expression levels of non-induced chemosensory proteins after exposure to deltamethrin in Tiassalé mosquitoes.

a, Expression levels of the remaining four CSPs at various time points after exposure to deltamethrin in the multi-insecticide-resistant Tiassalé population. b, Tissue-specific induction of these four CSPs 4-h after exposure to deltamethrin. Data are mean ± s.d. of three biological replicates. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Statistical significance was calculated by an ANOVA followed by Dunnett’s post hoc test. If the data were non-normal, data were analysed using a Kruskall–Wallis test followed by a Dunn’s post hoc test; P values are included in Supplementary Table 2.

Extended Data Fig. 4 Efficacy of RNAi.

mRNA knockdown of whole female mosquitoes 72 h after injection compared with GFP-injected controls. Data are mean ± s.d. of three biological replicates.

Extended Data Fig. 5 Phenotype of other induced CSPs to a panel of insecticides.

a, Effect of attenuation of dsSAP3 on mortality after insecticide exposure in Tiassalé mosquitoes (right bars; deltamethrin, n = 4; permethrin, n = 5; α-cypermethrin, n = 3; DDT, n = 3; pirimiphos-methyl, n = 3; bendiocarb, n = 4) compared with dsGFP-injected controls (left bars, green patterned; deltamethrin, n = 5; permethrin, n = 5; α-cypermethrin, n = 5; DDT, n = 4; pirimiphos-methyl, n = 4; bendiocarb, n = 8). b, Effect of attenuation of dsCSP4 on mortality after insecticide exposure in Tiassalé mosquitoes (right bars; deltamethrin, n = 3; permethrin, n = 3; α-cypermethrin, n = 3; DDT, n = 3; pirimiphos-methyl, n = 3; bendiocarb, n = 3) compared with dsGFP-injected controls (left bars, green patterned; deltamethrin, n = 5; permethrin, n = 5; α-cypermethrin, n = 5; DDT, n = 4; pirimiphos-methyl, n = 4; bendiocarb, n = 8). c, Effect of attenuation of dsCSP6 on mortality after insecticide exposure in Tiassalé mosquitoes (right bars; deltamethrin, n = 6; permethrin, n = 4; α-cypermethrin, n = 4; DDT, n = 3; pirimiphos-methyl, n = 4; bendiocarb, n = 5) compared with dsGFP-injected controls (left bars, green patterned; deltamethrin, n = 5; permethrin, n = 5; α-cypermethrin, n = 5; DDT, n = 4; pirimiphos-methyl, n = 4; bendiocarb, n = 8). Analysis of mortality data was done using an ANOVA followed by a Tukey post hoc test; n.s indicates a non-significant change in mortality; *P ≤ 0.05. dsCSP6 μmortality = 11.7–31.6%, P = 0.0474. N indicates the number of individual mosquitoes used for phenotyping; points show the number of bioassay replicates per group. Data are mean ± s.d.

Extended Data Fig. 6 Characterization of SAP2 in the transgenic line.

a, Mean mRNA expression after SAP2 overexpression in the SAP2 × A10 transgenic line (n = 2) compared with SAP2 expression in the A10 × G3 control (n = 3). Data are mean ± s.d. and points show each biological replicate. b, mCherry under the PUBc A10 promoter demonstrating (i) ubiquitous expression; (ii) expression in the head; and (iii) expression in the legs as previously shown25; these results were tested across more than 100 independent mosquito screens. c, Intron splicing confirmed by PCR in A10 × SAP2 and negative control A10 mosquitoes compared with plasmid DNA of pUAS:SAP2. The size of the PCR product with and without the synthetic intron is 647 bp and 534 bp, respectively. MW, 100-bp DNA ladder. n = 2 A10 × SAP2 samples and n = 2 A10 control samples (each sample consists of a pool of 5 4-day-old, unfed females) were tested and experiments were repeated in 2 PCRs.

Extended Data Fig. 7 Effect of SAP2 RNAi injection on the fitness of Tiassalé mosquitoes.

a, Longevity of SAP2 RNAi (dsSAP2)-expressing mosquitoes (black) compared with dsGFP-expressing control mosquitoes (green). N indicates the number of individual mosquitoes used in each group; n.s represents P = 0.113 as calculated by a two-sided Mantel–Cox test. b, Life-history traits of dsSAP2-injected (black) and dsGFP-injected (green) females. (i) Number of eggs in each group 72-h after a blood meal (the median and interquartile range are shown). (ii) Proportion of females with eggs (dark shading indicates females with eggs, light shading without; P = 0.4382). (iii) Mortality after a blood meal (dark shading are females that are alive after a blood meal, light those that are dead; P = 0.0052). (iv) Blood feeding proportions (dark shading are blood-fed females, light non-blood-fed; P = 0.3257). Numbers show total numbers of individual females in each group. Significance in (i) was calculated by a two-tailed Mann–Whitney U-test (n.s represents P = 0.0657); significance in (ii), (iii) and (iv) was calculated using a χ2 test. **P ≤ 0.01.

Extended Data Fig. 8 Mortality of A. coluzzii field populations.

Temporal plot of mortality from 2011 to 2018 of A. coluzzii mosquitoes to 0.05% WHO tube deltamethrin exposure. Δ is the posterior median change in mortality from 2011 to 2018. N indicates the number of experiments included (minimum sample size for any given data point is 14). p indicates the posterior probability that resistance (the proportion of posterior samples for which the April 2018 mean exceeds the corresponding value in January 2011) has increased over the time period. The blue line indicates the posterior median of a logistic model fit to binomial test results; the two parameters of the logistic function were assigned using uninformative (Cauchy(0, 1)) priors. The model was fitted using Stan33 with 4 chains and 800 iterations per chain (400 of which were discarded as burn-in in each case); all parameters had Rhat < 1.1, indicating convergence. The shading indicates the 90% predictive interval on the mean. Data and figure were provided by H. Toé, B. Lambert and T. Churcher.

Extended Data Fig. 9 Sequencing of SAP2 primer binding sites.

Four N’Gousso and four Tiassale mosquitoes were sequenced across the primer binding sites. a, Complete conservation of the sequence was seen in the forward binding site. b, One N’Gousso mosquito was heterozygous at one base in the centre of the reverse primer binding site.

Extended Data Table 1 Expression of the CSP family in western Africa

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Ingham, V.A., Anthousi, A., Douris, V. et al. A sensory appendage protein protects malaria vectors from pyrethroids. Nature 577, 376–380 (2020) doi:10.1038/s41586-019-1864-1

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