Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa

Abstract

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance.

Figure 1

Assessment of diagnostic dose of chlorfenapyr using the susceptible lab strain Kisumu. Percentage mortality (24 h, 48 h and 72 h) of the susceptible lab strain Kisumu after exposure to chlorfenapyr (with acetone as solvent). LC50 represents the concentration able to kill 50% of mosquitoes and LC99 the concentration able to kill 99%.

Figure 2

Comparative susceptibility pattern of field An. gambiae and An. funestus using the LC₅₀ obtained with kisumu. Percentage mortality (24 h, 48 h and 72 h) after exposure to chlorfenapyr 10 µg/ml (with acetone as solvent); * indicate significant difference compared to Kisumu.

Figure 3

Susceptibility profile of An. funestus and An. gambiae s.l to chlorfenapyr 100 µg/ml across Africa. Mortality rate of An. funestus (A) and An. gambiae (B) from different sites 72 h after exposure to chlorfenapyr 100 µg/ml.

Figure 4

Susceptibility profile of An. funestus s.s (Mibellon) and An. gambiae (Mibellon and Nkolondom) populations to chlorfenapyr 100 µg/ml at different time points. Error bars represent standard error of the mean. Min minutes.

Figure 5

Results of tests with PBO, DEM and DEF. Effect of pre-exposure to inhibitors PBO, DEM and DEF against chlorfenpayr 10 μg/ml for An. funestus (from Mibellon and Elende) and An. gambiae (from Mibellon and Nkolondom) and 100 μg/ml for An. gambiae from Nkolondom. Results are average of percentage mortalities from four-five replicates each. The bars represent the standard error on the mean (SEM), * indicate the level of significance and NS represents no significant difference.

Figure 6

Association between pyrethroids resistance markers and resistance to chlorfenapyr in An. gambiae. Distribution of the L1014F-Kdr_w genotypes (A) and alleles (B) among the dead and alive mosquitoes after exposure to chlorfenapyr and (C) and (D) for I114T-GSTe2. R represents the resistant allele while S represent the susceptible allele.

Figure 7

Association between the L119F-GSTe2 mutation and resistance to chlorfenapyr in An funestus. Distribution of genotypes and alleles among the dead and alive mosquitoes after exposure to chlorfenapyr. R represents the resistant allele while S represents the susceptible allele.