Multi-insecticide resistant malaria vectors in the field remain susceptible to malathion, despite the presence of Ace1 point mutations

Abstract

Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d'Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases - including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 - overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.

Fig 1. Map of the three larval sampling sites in southern Côte d’Ivoire.

In Tiassalé and Agboville we collected Anopheles larvae from irrigated rice fields, whereas in Dabou they were collected from standing water in vegetable fields. The map was created using ArcGIS v10.6.1 (ESRI Inc., Redlands, CA, USA) with the base map Sentinel-2 cloudless - https://s2maps.eu by EOX IT Services GmbH (contains modified Copernicus Sentinel data 2020).

Fig 2. Phenotypic insecticide resistance in Anopheles coluzzii from southern Côte d’Ivoire assessed with WHO discriminating concentrations and dose-response bioassays.

(A) Outcome of the standard WHO insecticide susceptibility tests using discriminating concentrations of four insecticides. The solid symbols represent the average mortalities and the vertical solid lines with whiskers represent their 95% confidence intervals (CIs) as estimated with GLMs. Note that for the Tiassalé population we observed 100% mortality 24 h post malathion exposure, but in order to estimate a 95% CI, we artificially added one survivor into the model. The total number (n) of tested mosquitoes per field site and insecticide is indicated below the corresponding average mortality. Horizontal dashed lines represent thresholds to interpret the WHO susceptibility tests [6]: i) A mosquito population is susceptible to the tested insecticide if the observed mortality is between 98% and 100% (above upper line); ii) if mortality is between 90% and 97% resistance is possible (between lines); and iii) if mortality is below 90%, resistance is confirmed (below lower line). Dose-response curves for (B) deltamethrin and (C) malathion for the three An. coluzzii field populations from southern Côte d’Ivoire and the insecticide susceptible Ngousso laboratory colony. The curves show the dose-response relationship between the mortality rate on the y-axis and the percentage of insecticide on the filter paper on the log₂-transformed x-axis. Symbols represent the actually observed mortality rates per dose, while the curves are the estimates of the average as estimated with GLMs. The shaded areas indicate the 95% CIs. Vertical lines indicate the discriminating concentration for insecticide susceptibility as recommended by the WHO.

Fig 3. Results of differential gene expression analysis comparing each multi-resistant field population (Agb_C, Dab_C and Tia_C) against two susceptible laboratory colonies combined as a group (Lab2_C: Mali-NIH and Ngousso).

Volcano plots showing the expression profile for comparisons (A) Agb_C vs. Lab2_C; (B) Dab_C vs. Lab2_C; and (C) Tia_C_ vs. Lab2_C. The genes highlighted in colour belong to protein families known to be involved in insecticide resistance and those that we detected significantly enriched with the GSEA for at least one site. Genes above the horizontal dotted line passed our threshold for significance (FDR ≤ 0.01). Genes on the right of the vertical line (log₂FC > 0) were higher expressed in the field than in the lab populations, whereas genes on the left of the vertical line (log₂FC < 0) were lower expressed in the field than in the lab populations. (D) Venn diagram showing the number of significantly (FDR ≤ 0.01) over- (log₂FC > 0, red) and under- (log₂FC < 0, blue) expressed genes.

Fig 4. Heat maps visualising differential expression of the three major insecticide detoxifying gene families between multi-resistant Ivorian (Agb_C, Dab_C, Tia_C) and susceptible laboratory colonies (Lab2_C).

(A) Ace1 and 12 out of 41 carboxylesterases (COEs); (B) 8 out of 33 glutathione S-transferases (GSTs); and (C) 41 out of 110 cytochrome P450-dependent monooxygenases (P450s) for which **FDR ≤ 0.01 in at least one comparison. The number displayed on the coloured tiles shows the log₂ fold change (FC) with tiles in red depicting overexpression (log₂FC > 0) and blue underexpression (log₂FC < 0). Levels of significance *FDR ≤ 0.05; **FDR ≤ 0.01; and ***FDR ≤ 0.001. Tiles were left empty when FDR > 0.05.

Fig 5. Linear relationship in fold changes measured in P450s between RNA-seq and RT-qPCR.

The RT-qPCR log₂ fold change (log₂FC) is predictive of the RNA-seq log₂FC (R² = 0.923, p < 0.001). Comparison groups were insecticide-unexposed mosquitoes from the field (n = 5 for each site: Agb_C, Dab_C and Tia_C) against all unexposed mosquitoes from the two laboratory colonies Mali-NIH and Ngousso as a group (n = 10, Lab2_C).

Fig 6. Bar plots showing qPCR results on leg DNA for two target-site mutations.

(A) Vgsc-L995F mutation and (B) Ace1-G280S mutation frequency (% below bar) in 40 insecticide-unexposed individuals per field population. Panel (C) depicts in which combination the resistance-associated alleles occurred in the 55 RNA-sequenced individuals. See S4 Fig for the combined Vgsc-L995F and Ace1-G280S genotype of the 40 individuals per field population.