Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification

Abstract

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Keywords: Anopheles; Azole fungicides; Deltamethrin; FUMOZ-R; Malaria; P450; Pyrethroid resistance; Sodium channel; Synergists.

Graphical abstract

Fig. 1

Chemical structures of transfluthrin and its derivatives used in this study.

Fig. 2

Concentration response curves for common pyrethroids (A) and transfluthrin derivatives (B) measured on functionally expressed house fly voltage-gated sodium channels (VGSC) using a fluorescence-based membrane potential cation influx assay. Data are mean values ± standard deviation (SD) (n = 12). C, D Pearsonʼs correlation analysis between in vitro VGSC EC₅₀-values and in vivo LC₅₀-values obtained from glazed tile bioassays against female adults of Anopheles funestus strains FANG (C) and FUMOZ-R (D). Data points circled with a dashed line represent deltamethrin, cypermethrin and permethrin.

Fig. 3

Tarsal uptake of different synergists by adult females of Anopheles funestus strain FUMOZ-R by contact exposure for 30 min on glazed tiles. A Three different synergists were tested in vivo: piperonyl butoxide (PBO; 100 mg/m²), triflumizole (100 mg/m²) and 1-aminobenzotriazole (1-ABT; 250 mg/m²). B Amount of synergist internalized and detected in mosquito legs and bodies analyzed by UPLC/MS. Different letters denote a significant difference (One-way ANOVA, Tukeyʼs post-hoc comparisons, P < 0.05). Data are mean values ± standard deviation (SD) (n = 20).

Fig. 4

Heat maps displaying the synergistic ratio obtained in glazed tile bioassays for different pyrethroid insecticides in Anopheles funestus strain FANG (susceptible) and FUMOZ-R (resistant) upon contact exposure to piperonyl butoxide (PBO) (A), 1-amino-benzotriazole (1-ABT) (B) and triflumizole (C), directly prior to insecticide application. Synergistic ratios (SR) were calculated by dividing the LC₅₀ of the insecticide by the LC₅₀ of the insecticide + synergist (see Supplementary Table S3 for details).

Fig. 5

Metabolism of different cytochrome P450 coumarin and resorufin model substrates by microsomal preparations of Anopheles funestus, strain FUMOZ-R (A) and strain FANG (B) in comparison to FUMOZ-R with those substrates displaying highest cytochrome P450 activity. Data are mean values ± standard deviation (SD) (n = 4). Significant differences are denoted by ∗∗∗∗P ≤ 0.0001. Substrate abbreviations: EC, 7-ethoxycoumarin; PC, 7-pentoxycoumarin; MFC, 7-methoxy-4-trifluoromethylcoumarin; EFC, 7-ethoxy-4-trifluoromethylcoumarin; BFC, 7-benzyloxy-4-trifluoromethylcoumarin; BOMFC, 7-benzyloxymethoxy-4-trifluoromethylcoumarin; MR, 7-methoxyresorufin; ER, 7-ethoxyresorufin; PR, 7-n-pentoxyresorufin; BR, 7-benzyloxyresorufin ether; BOMR, 7-benzyloxymethoxyresorufin.

Fig. 6

Expression level (log-scale) of CYP6P9a and CYP6P9b in Anopheles funestus strains FUMOZ-R and FANG measured by qPCR. The expression level was normalized to RPS7 and Act (5c) reference genes. Data are mean values ± 95% CI (n = 4 FUMOZ-R and n = 3 for FANG).