Molecular Drivers of Multiple and Elevated Resistance to Insecticides in a Population of the Malaria Vector Anopheles gambiae in Agriculture Hotspot of West Cameroon

Abstract

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F0) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.

Keywords: Anopheles gambiae; Cameroon; cytochrome P450s; malaria; pyrethroids; resistance escalation.

Figure 1

Susceptibility profile of An. gambiae s.s. to insecticides and effect of pre-exposure to synergist PBO against permethrin, alpha-cypermethrin and deltamethrin. Recorded mortalities following 60-minute exposure of An. gambiae s.s. from Mangoum to different insecticides. Data are shown as mean ± standard error of the mean (SEM). Blue line corresponds to 80% mortality and red line to 98% mortality.

Figure 2

(A) Temporal variation in susceptibility profile of An. gambiae s.s. (F₀ & F₁) to different doses of permethrin. (B) Susceptibility profile of F₀ and F₁ An. gambiae s.s. to different doses of permethrin per collection time point (May & November). Recorded mortalities following 60-minute exposure of An. gambiae s.s. from Mangoum. Data are shown as mean ± standard error of the mean (SEM). Student’s test was used for comparisons. p value code: ns: p > 0.05. *: p ≤ 0.05, **: p ≤ 0.01.

Figure 2

(A) Temporal variation in susceptibility profile of An. gambiae s.s. (F₀ & F₁) to different doses of permethrin. (B) Susceptibility profile of F₀ and F₁ An. gambiae s.s. to different doses of permethrin per collection time point (May & November). Recorded mortalities following 60-minute exposure of An. gambiae s.s. from Mangoum. Data are shown as mean ± standard error of the mean (SEM). Student’s test was used for comparisons. p value code: ns: p > 0.05. *: p ≤ 0.05, **: p ≤ 0.01.

Figure 3

Bio-efficacy of different commercial LLINs against An. gambiae s.s. in Mangoum. Results of cone bioassays with Olyset^®Net, Olyset^®Plus, PermaNet^®2.0, PermaNet^®3.0 (side and roof), Duranet^®, Royal guard^®, Inteceptor^® and Interceptor G2^® (% Mortality 72 h). Results are average of percentage mortalities ± SEM of five replicates. Mortality < 50% (blue line): No efficient, 50% < Mortality ≤ 80%: minimal efficacy, Mortality ≥ 80% (green line): optimal efficacy.

Figure 4

Genotyping of resistance markers in F₀ An. gambiae s.s. from Mangoum.

Figure 5

Analysis of the polymorphism of a portion of the voltage-gated sodium channel (VGSC) gene spanning the L1014F/S mutation. (A) Maximum likelihood phylogenetic tree of VGSC fragment with previously recorded 1014F/S haplotypes across Africa. (B) Templeton–Crandall–Singh network for the VGSC haplotypes in F₀ mosquitoes from Mangoum.

Figure 6

Differential expression by quantitative reverse-transcription polymerase chain reaction of the major insecticide resistance genes in An. gambiae in Mangoum compared with the susceptible Kisumu. (A) Permethrin, (B) deltamethrin. Error bars represent standard error of the mean at 95% confidence interval, with significance * p ≤ 0.05, ** p ≤ 0.01 as calculated by Kruskal–Wallis test for between exposed groups comparisons (in red) and Dunnett’s test for comparing several each exposed groups with a unexposed (in purple). The red line represent the two-fold change threshold. n.s: non significant.

Figure 6

Differential expression by quantitative reverse-transcription polymerase chain reaction of the major insecticide resistance genes in An. gambiae in Mangoum compared with the susceptible Kisumu. (A) Permethrin, (B) deltamethrin. Error bars represent standard error of the mean at 95% confidence interval, with significance * p ≤ 0.05, ** p ≤ 0.01 as calculated by Kruskal–Wallis test for between exposed groups comparisons (in red) and Dunnett’s test for comparing several each exposed groups with a unexposed (in purple). The red line represent the two-fold change threshold. n.s: non significant.