Phenotypic resistance to pyrethroid associated to metabolic mechanism in Vgsc-L995F-resistant Anopheles gambiae malaria mosquitoes

Abstract

Background: The indiscriminate use of insecticides in agriculture and public health lead to a selection of resistance mechanisms in malaria vectors compromising vector control tools and strategies. This study investigated the metabolic response in the Vgsc-L995F Anopheles gambiae Tiassalé resistance strain after long-term exposure of larvae and adults to deltamethrin insecticide. Methods: Vgsc-L995F An. gambiae Tiassalé strain larvae were exposed over 20 generations to deltamethrin (LS) and adults to PermaNet 2.0 (AS) and combining exposure at larvae and adult stages (LAS) and compared to unexposed (NS) group. All four groups were subjected to the standard World Health Organization (WHO) susceptibility tube tests using deltamethrin (0.05%), bendiocarb (0.1%) and malathion (5%). Vgsc-L995F/S knockdown-resistance ( kdr) mutation frequency was screened using multiplex assays based on Taqman real-time polymerase chain reaction (PCR) method. Additionally, expression levels of detoxification enzymes associated to pyrethroid resistance, including CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and glutathione S-transferase GSTe2 were measured. Results: Our results indicated that deltamethrin resistance was a response to insecticide selection pressure in LS, AS and LAS groups, while susceptibility was observed in NS group. The vectors showed varied mortality rates with bendiocarb and full susceptibility to malathion throughout the selection with LS, AS and LAS groups. Vgsc-L995F mutation stayed at high allelic frequency level in all groups with a frequency between 87% and 100%. Among the overexpressed genes, CYP6P4 gene was the most overexpressed in LS, AS and LAS groups. Conclusion: Long-term exposure of larvae and adults of Vgsc-L995F resistant- An. gambiae Tiassalé strain to deltamethrin and PermaNet 2.0 net induced resistance to deltamethrin under a significant effect of cytochromes P450 detoxification enzymes. These outcomes highlight the necessity of investigating metabolic resistance mechanisms in the target population and not solely kdr resistance mechanisms prior the implementation of vector control strategies for a better impact.

Keywords: Anopheles gambiae; Côte d’Ivoire; Deltamethrin; P450 genes; PermaNet 2.0; Resistance selection; Tiassalé strain; Vgsc-L995F.

Figure 1.. Kaplan-Meier survival curves showing the cumulative survival over the 60 minutes exposure to diagnostic concentrations of deltamethrin (0.05%) in the WHO insecticide susceptibility assay.

The P-values represent the Log-rank test p-value. ( A) NS-LS; ( B); NS-AS; ( C) NS-LAS.

Figure 2.

Dynamics of mortality with bendiocarb (0.1%), deltamethrin (0.05%), malathion (5%) in An. gambiae Tiassalé divided into three groups ( A) LS, ( B) AS, ( C) LAS and ( D) NS. Error bars indicate 95% confidence intervals. The mortality threshold of 98% indicates full susceptibility according to WHO criteria .

Figure 3.. Dynamics of target site mutation Vgsc-L995F through resistance selection.

( A) LS group; ( B) AS group; ( C) LAS group and ( D) NS group. Percentages indicate the frequency of resistance alleles while bars indicate actual numbers. For each group, the Vgsc-L995F mutation was genotyped in individual mosquitoes.

Figure 4.. Differential expression levels for putative detoxifying genes measured in selected groups.

Fold changes give the change in expression level of mosquito populations at the 20th generation relative to the reference generation (parent G0). Fold changes were estimated using REST2009 analysis software for each gene. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.