378 | Nature | Vol 577 | 16 January 2020Article
(CSP3 (AGAP008055) and CSP5 (AGAP008058)) and one in the abdo-
men carcass (CSP1 (AGAP008059)).
We performed a time course to determine whether any of the eight
CSPs were induced by exposure to pyrethroids in the resistant strain
(Fig. 1b and Extended Data Fig. 3a) and found that four of the eight CSPs
were significantly induced by deltamethrin, the pyrethroid that is most
widely used in LLINs, including the constitutively overexpressed SAP2
and CSP6, in addition to SAP3 and CSP4 (AGAP008062) (Fig. 1b). Examin-
ing the tissue specificity of the induction 4 h after exposure showed that
SAP2, SAP1 (AGAP008051) and CSP3 were induced in multiple tissues,
including the legs, whereas CSP4, CSP6 and CSP1 showed induction in
a single tissue (Fig. 1c and Extended Data Fig. 3b).CSPs confer resistance
Having established that CSPs are highly overexpressed in the append-
ages of pyrethroid-resistant mosquitoes, and expression of a subset of
this family is further induced by insecticide exposure, we next silenced
these CSPs using RNA interference (RNAi) (Extended Data Fig. 4) in
females from the highly pyrethroid-resistant Tiassalé colony before
exposing the mosquitoes to a panel of insecticide classes (pyrethroids,
carbamates and organophosphates) that are widely used in public
health initiatives. Notably, silencing of SAP2 almost completely restored
susceptibility to the pyrethroid deltamethrin, and also significantly
increased the susceptibility to the other two pyrethroids (permethrin
and α-cypermethrin) that were used in these analyses (Fig. 2a). No
change in mortality was observed after exposure to the other insecti-
cide classes, indicating that this mechanism is specific to pyrethroid
insecticides. We further explored this notable phenotype in a secondmulti-insecticide-resistant population from Burkina Faso (Banfora) and
again found that mortality of the mosquitoes was significantly restored
after exposure to deltamethrin (Fig. 2a). Knockdown of the other three
CSPs (SAP3, CSP4 and CSP6) had no effect on mortality to any insecticide
class, except in the case of CSP6 knockdown, which also significantly
increased susceptibility to deltamethrin in Tiassalé mosquitoes—
although not to the same extent as SAP2 knockdown (Extended Data
Fig. 5). Conversely, overexpression of SAP2 in an insecticide-susceptible
population (Extended Data Fig. 6) significantly increased pyrethroid
resistance, directly linking the function of this protein to insecticide
resistance (Fig. 2b). To check for adverse changes to the life-history
traits of mosquitoes owing to the disruption of SAP2 expression, we
recorded the survival, blood-feeding ability and egg production in SAP2
RNAi-injected females and found no significant changes compared to
a GFP-injected control, indicating that the mortality that is observed is
due to a direct effect of SAP2 on the insecticide and not reduced overall
fitness (Extended Data Fig. 7).
To determine a putative mode of action through which a chemosen-
sory protein could confer resistance, we next investigated whether
these proteins act as pyrethroid-binding proteins. We heterologously
expressed SAP2 and two closely related CSPs (SAP1 and SAP3) in Escheri-
chia coli and carried out competitive binding assays with the fluorescent
marker N-phenyl-1-naphthylamine and a panel of insecticides (Fig. 2c).
SAP2 bound to all three pyrethroid insecticides tested: deltamethrin
(half-maximum inhibitory concentration (IC 50 ) = 3.99 μM); permethrin
(IC 50 = 4.77 μM); and α-cypermethrin (IC 50 = 3.74 μM), but did not bind
to pirimiphos-methyl (an organophosphate) or bendiocarb (a carba-
mate). The IC 50 values were similar to those found for the most impor-
tant cytochrome p450s that are responsible for metabolic clearance*****NSNS
**Mortality (%)0255075100n =9 10051391081399794809673218975781Mortality (%)020406080100n = 279 369***abcTiassalé, Deltamethrin
Permethrin
α-CypermethrinDDT
Pirimiphos-methyl (10 min exposure)
BendiocarbBanfora, Deltamethrin1-NPN uorescence (%)020406080100Deltamethrin (μM)0510 15 20 25020406080100Permethrin (μM)0510 15 20 25020406080100α-Cypermethrin (μM)0510 15 20 25SAP1
SAP2
SAP3NSFig. 2 | SAP2 mediates resistance to pyrethroid insecticides. a, Effect of SAP2
knockdown on mortality in multi-insecticide-resistant Anopheles populations
in response to a panel of insecticides (right bar) compared with GFP-injected
controls (left bar). Tiassalé, deltamethrin (blue; GFP, n = 5; SAP2, n = 5);
permethrin (brown; GFP, n = 6; SAP2, n = 7); α-cypermethrin (dark grey; GFP,
n = 5; SAP2, n = 5); DDT (yellow; GFP, n = 4; SAP2, n = 3), primiphos-methyl (light
grey; GFP, n = 4; SAP2, n = 4), bendiocarb (purple0; GFP, n = 9; SAP2, n = 4) and
Banfora, deltamethrin (grey blue; GFP, n = 4; SAP2, n = 5). Indicated n numbers
are the total number of females used across all replicates. b, Transgenic
overexpression of SAP2 in susceptible G3 mosquitoes reduces mortality after
permethrin exposure. Bars represent control (grey; n = 15) and SAP2
overexpression (white; n = 17). Indicated n numbers are the total number of
females used across all replicates. c, Competitive binding assays of the three
SAP proteins to three pyrethroid insecticides. Only instances with binding
shown; no binding was found for SAP3 and SAP1 with permethrin; SAP3 with
α-cypermethrin; any SAP with bendiocarb or pirimiphos-methyl. 1-NPN,
N-phenyl-1-naphthylamine. The data are mean ± s.d. *P ≤ 0.05; **P ≤ 0.01;
***P ≤ 0.001; NS, not significant (P > 0.05). Statistical significance in a and b was
calculated using an ANOVA test followed by a Tukey post hoc test; P values are
included in Supplementary Table 2.