Fitness cost of target-site and metabolic resistance to pyrethroids drives restoration of susceptibility in a highly resistant Anopheles gambiae population from Uganda

Abstract

Background: Insecticide resistance threatens the effectiveness of malaria vector control, calling for an urgent need to design suitable resistance management strategies. Here, we established the resistance profiling of an Ugandan Anopheles gambiae population to insecticides using WHO procedures and assessed the potential restoration of susceptibility in the hybrid line Mayuge/KISUMU in an insecticide-free environment for eighteen (18) generations.

Results: This An gambiae population exhibited a very high intensity of resistance to permethrin, deltamethrin, and alphacypermethrin with a consistent loss of efficacy of all long-lasting insecticidal nets (LLINs) tested including PBO-based and new generation nets Interceptor G2 (IG2) and Royal guard. Molecular analysis revealed a fixation of the L1014S-kdr mutation together with the overexpression of some P450 metabolic genes (CYP6Z1, CYP9K1, CYP6P1, 3 & 4) besides the cuticular resistance-related genes (CYP4G16) and sensorial appendage proteins (SAP1, SAP2, and SAP3) but no GSTe2 overexpression. In the absence of selection pressure, the mortality rate after exposure to insecticides increased significantly over generations, and restoration of susceptibility was observed for most of the insecticides in less than 10 generations. Accordingly, a significant reduction in the frequency of KdrE was observed after 13 generations coupled with reduced expression of most metabolic resistance genes.

Conclusions: The results of this study show that the high intensity of pyrethroid resistance observed in An gambiae from Uganda associated with the loss of efficacy of LLINs could compromise vector control efforts. The study also highlights that an early rotation of insecticides could help manage resistance to insecticides by restoring the susceptibility. However, the persistence of Kdr mutation together with overexpression of some metabolic genes after many generations in the absence of selection pressure indicates the potential implication of modifiers alleviating the cost of resistance which needs to be further investigated.

Fig 1. Susceptibility profile of An. gambiae s.l population from Mayuge.

A) susceptibility profile of females An. gambiae; B) resistance intensity with 5× and 10× the diagnostic concentrations of permethrin and deltamethrin and Alphacypermethrin C) effect of pre-exposure to synergist PBO against pyrethroids. D) Susceptibility profile and intensity of females An. arabiensis and E) bio-efficacy of different commercial LLINs against An. gambiae. Results are average of percentage mortalities ± SEM; Results are average of percentage mortalities from four replicates each ± SEM.

Fig 2. Influence of of the L1014S-KdrE on key life traits of An. gambiae.

(A) and (B) Schematic representation of the impact of L1014S genotypes on egg-laying success with odd ratio (OR); (C) Distribution of the L1014S genotypes at different developmental stages of the hybrid MYG/KIS; D) the proportion of pupae obtained in D7, D9 and D11 of development; E) influence of L1014S on the adult longevity of An. gambiae.

Fig 3. Susceptibility profile of the hybrid MYG/KIS to permethrin over 13 generation in the absence of selection.

Results of WHO tube bioassays with permethrin 1x, 5x and 10x and PBO+ permethrin 1x. Results are average of percentage mortalities ± SEM of four replicates.

Fig 4. Evaluation of the reversal to susceptibility in the hybrid colony MYG/KIS.

Changes in the L1014S genotypes (A) and allele (B) over13 generations in the insecticides free-environment; F represents each generation.

Fig 5. Differential expression by quantitative reverse-transcription polymerase chain reaction of key metabolic genes in the hybrid MYG/KIS over generation compared to field F1 unexposed.

Histograms represent the fold-change of the genes in the hybrid MYG/KIS mosquitoes across generations relative to the pyrethroid-susceptible KISUMU laboratory strain.

Fig 6. Evaluation of the restoration of susceptibility in the hybrid colony MYG/KIS.

Determination of susceptibility to all the four classes of insecticides, resistance intensity with 5× and 10× the diagnostic concentrations of pyrethroids and effect of pre-exposure to synergist PBO against pyrethroids type I and type II in comparison with field F0. Results are average of percentage mortalities from four replicates each ± SEM.