equilibration buffer for 15 column volumes. Bound protein was eluted
with 5 column volumes of 20 mM Tris, 300 mM NaCl, 500 mM imida-
zole, pH 8.0. Fractions with an absorbance at 59 nm (Bradford assay)
greater than 0.5 were pooled, concentrated and buffer-exchanged
into 50mM Tris-HCl pH 8.0, 25mM NaCl, 10% glycerol with dialysis
membrane (6-8kDa MWCO) overnight at 4 °C.
1-NPN binding competition assays
The affinity of various insecticides for SAP1, SAP2 and SAP3 was meas-
ured indirectly by competitive binding assays, which determined the
displacement of the ligands from the SAPs by the fluorescent probe
1-NPN. Purified recombinant SAP proteins were mixed with different
concentrations of insecticides (in methanol) and 1-NPN (in methanol)
in a final volume of 200 μl of 20 mM Tris-HCl, pH 8.0, 100 mM NaCl
(containing 1.5% methanol). The final concentration of recombinant
SAP protein and 1-NPN in the assays was 10 μM and 5 μM, respectively.
Insecticide concentration ranged from 0.0625 to 20 μM. The probe
was excited at 337 nm and emission spectra were recorded between
380 nm and 460 nm (peak emission in the presence of recombinant
SAP is at 386–388 nm). Emission spectra were recorded on an Infinite
M-200 fluorimeter (Tecan Trading) using black 96-well plates (Greiner
Bio-One). Graphs were produced using GraphPad Prism 8.0.2. A non-
linear fit using logged concentrations were used to fit best-fit lines and
calculate the IC 50 values. The calculated log(IC 50 ) values and associated
standard errors were used to fit an ANOVA to compare the binding
specificity of the three proteins.
Microarray data
Microarray data were retrieved from the IR-TEx application^10 (https://
http://www.lstmed.ac.uk/projects/ir-tex)..)
Field collections
Field collections of A. gambiae s.l. were performed by colleagues at
the Liverpool School of Tropical Medicine in collaboration with the
Centre National de Recherche sur le Paludsime (CNRFP) in Banfora
district, Burkina Faso. The mosquitoes used in this study originated
from Tengrela, (10° 38′ 7.53′′ N; 4° 48′ 48.35′′ W) in 2011, 2012, 2014,
2016 and 2018, Bakaridjan (10° 24′ 26.34′′ N; 4° 33′ 44.78′′ W) in 2013
and 2015 and from Tiefora (10° 37′ 54.02′′ N; 4° 33′ 22.85′′ W) in 2018. All
gDNA extractions were subject to SINE PCR^30 to determine the species
of each mosquito before sequencing.
Scan for natural selection
We computed iHS^20 and XP-EHH^21 statistics on phased sequences for
regions 1.5 Mb upstream and downstream of the chemosensory locus
for all available A. gambiae West African mosquito populations found
in phase 2 of the Anopheles gambiae 1000 Genomes Project using
scikit-allel v.1.1.10 (https://doi.org/10.5281/zenodo.822784). A peak
was apparent in iHS in the Guinean A. gambiae population (highest
value at 3R: 4,845,138) and a smaller peak was present in Burkina Faso,
and a XPEHH peak was present in Cameroon.
Clustering analysis and SNP panel
We carried out haplotype clustering analysis around the chemosensory
locus and identified 13 haplotypes that differed by fewer than 20 SNPs
across a window 25 kb upstream and downstream of the limits of SAP2.
This is a firm proxy for identity by descent^19 in an organism with π ≈ 0.01,
indicative of selection acting at this region. These 13 shared haplotypes
were observed in Guinea, Burkina Faso and Cameroon.
These 13 haplotypes were used to define 55 SNPs with high FST
between this cluster and the wild-type A. gambiae populations. A SNP
panel screen was designed to encompass a range of these SNPs showing
the highest difference between wild-type and cluster SNPs, primers for
the two panels were as follows: forward, CAAGCATTGCGCCATCGT;
reverse, GAGAAGATGATACTGAGCGG. PCR was performed on gDNA
from individual A. gambiae from Bakaridjan in 2013 (42 individuals)
and 2015 (50 individuals) and Tiefora in 2018 (40 individuals). SNP
genotypes from genomic sequence data from individual A. coluzzii
samples from Tengrela from 2011 (21 individuals), 2012, 2014 and 2016
(72 individuals for each year) were provided by the Broad Institute;
the PCR panel was carried out on samples from the same site in 2018
(20 samples). The panel was analysed using Phusion High-Fidelity
DNA Polymerase (Thermo Fisher) following the manufacturer’s instruc-
tions and the following cycles were used: 98 °C for 30 s, 35 cycles
of 98 °C for 7 s, 65 °C for 10 s, 72 °C for 15 s and a final hold at 72 °C
for 15 min. The resultant product was purified using a QiaQuick Gel
Extraction Kit (Qiagen) following the manufacturer’s instructions and
sent for Sanger sequencing at Eurofins Genomics using the reverse
primer. SNPs were identified using Benchling (Biology Software;
https://benchling.com), the resultant change in minor allele frequency
was calculated and a trend line was fitted to each SNP using GraphPad
Prism 8.0.2.Phylogeny
cDNA sequences for each of the CSPs were retrieved from VectorBase^31
and aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/
clustalo/). These data were loaded into Mega 7.0^32 and a maximum
likelihood tree with 1,000 bootstraps was performed.Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this paper.Data availability
All data analysed during this current study are described in the Article,
Extended Data Fig. 1–9, Extended Data Table 1 and the Supplementary
Information, or are available from the corresponding authors upon rea-
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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 contributions V.A.I. and H.R. conceived the experimental design. V.A.I. performed all
transcriptomic expression experiments, RNAi and phenotyping experiments, data analysis,