Insecticide Resistance Profiling of Anopheles coluzzii and Anopheles gambiae Populations in the Southern Senegal: Role of Target Sites and Metabolic Resistance Mechanisms

Abstract

The emergence and spread of insecticide resistance among the main malaria vectors is threatening the effectiveness of vector control interventions in Senegal. The main drivers of this resistance in the Anopheles gambiae complex (e.g., An. gambiae and Anopheles coluzzii) remains poorly characterized in Senegal. Here we characterized the main target site and metabolic resistances mechanisms among the An. gambiae and An. coluzzii populations from their sympatric and allopatric or predominance area in Senegal. Larvae and pupae of An. gambiae s.l. were collected, reared to adulthood, and then used for insecticides susceptibility and synergist assays using the WHO (World Health Organisation) test kits for adult mosquitoes. The TaqMan method was used for the molecular characterization of the main target site insecticide resistance mechanisms (Vgsc-1014F, Vgsc-1014S, N1575Y and G119S). A RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chaine Reaction) was performed to estimate the level of genes expression belonging to the CYP450 (Cytochrome P450) family. Plasmodium infection rate was investigated using TaqMan method. High levels of resistance to pyrethroids and DDT and full susceptibility to organophosphates and carbamates where observed in all three sites, excepted a probable resistance to bendiocarb in Kedougou. The L1014F, L1014S, and N1575Y mutations were found in both species. Pre-exposure to the PBO (Piperonyl butoxide) synergist induced a partial recovery of susceptibility to permethrin and full recovery to deltamethrin. Subsequent analysis of the level of genes expression, revealed that the CYP6Z1 and CYP6Z2 genes were over-expressed in wild-resistant mosquitoes compared to the reference susceptible strain (Kisumu), suggesting that both the metabolic resistance and target site mutation involving kdr mutations are likely implicated in this pyrethroid resistance. The presence of both target-site and metabolic resistance mechanisms in highly pyrethroid-resistant populations of An. gambiae s.l. from Senegal threatens the effectiveness and the sustainability of the pyrethroid-based tools and interventions currently deployed in the country. The Kdr-west mutation is widely widespread in An. coluzzii sympatric population. PBO or Duo nets and IRS (Indoor Residual Spraying) with organophosphates could be used as an alternative measure to sustain malaria control in the study area.

Keywords: An. coluzzii; An. gambiae; N1575Y; Senegal; kdr; malaria; metabolic resistance; pyrethroid.

Figure 1

Susceptibility profile of Anopheles gambiae s.l. to insecticides. Recorded mortalities following 60-min exposure of Anopheles gambiae s.l. from Fatick, Tambacounda and Kedougou to different insecticides. Data are shown as mean ± standard error of the mean (SEM).

Figure 2

Results of resistance intensity and synergist tests. Resistance intensity in Tambacounda (A) and Kedougou (B); activities of PBO combined to permethrin, and deltamethrin on An. gambiae s.l. from Kedougou (C). Data are shown as mean ± standard error of the mean.

Figure 3

Genotyping of resistance markers in An. coluzzii, An. gambiae s.s. and their hybrids. Distribution of Kdr(s) genotypes (A) and alleles (B) and N1575Y genotypes (C) in the different species. Kdr − LL = Kdrw susceptible homozygous; Kdr − LF = Kdrw heterozygous; Kdr − FF = Kdrw resistant homozygous; Kdr − LS = Kdre resistant heterozygous; Kdr − SS = Kdre resistant heterozygous; Kdr − FS = Kdrw and Kdre resistant; 1575 − YY = Resistant homozygous; 1575−NY = Resistant heterozygous; 1575 − NN = Susceptible homozygous.

Figure 4

Genetic diversity parameters of Vgsc in An. coluzzii, An. gambiae and their hybrids from Senegal in relation to the species. (A) Haplotype network in relation to the species composition; (B) phylogenetic trees (using a maximum likelihood method) and the nucleotide diversity of the L1014F mutation in Senegal (C).

Figure 5

Differential expression by quantitative reverse-transcription polymerase chain reaction of the major insecticide resistance genes in An. gambiae in Senegal compared with the susceptible Kisumu. Error bars represent standard error of the mean. *—statistically significant p ≤ 0.05.