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An Experimental Hut Evaluation of PBO-Based and Pyrethroid-Only Nets Against the Malaria Vector Anopheles funestus Reveals a Loss of Bed Nets Efficacy Associated With GSTe2 Metabolic Resistance

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An Experimental Hut Evaluation of PBO-Based and Pyrethroid-Only Nets Against the Malaria Vector Anopheles funestus Reveals a Loss of Bed Nets Efficacy Associated With GSTe2 Metabolic Resistance

Benjamin D Menze et al. Genes (Basel).

Abstract

Growing insecticide resistance in malaria vectors is threatening the effectiveness of insecticide-based interventions, including Long Lasting Insecticidal Nets (LLINs). However, the impact of metabolic resistance on the effectiveness of these tools remains poorly characterized. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we established that GST-mediated resistance is reducing the efficacy of LLINs against Anopheles funestus. Hut trials performed in Cameroon revealed that Piperonyl butoxide (PBO)-based nets induced a significantly higher mortality against pyrethroid resistant An. funestus than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control. Genotyping the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR = 11.7; p < 0.01). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR = 2.1; p < 0.01) and PermaNet 3.0 (side) (OR = 30.1; p < 0.001) but not for Olyset Plus. This study shows that the efficacy of PBO-based nets (e.g., blood feeding inhibition) against pyrethroid resistant malaria vectors could be impacted by other mechanisms including GST-mediated metabolic resistance not affected by the synergistic action of PBO. Mosaic LLINs incorporating a GST inhibitor (diethyl maleate) could help improve their efficacy in areas of GST-mediated resistance.

Keywords: Anopheles funestus; Long Lasting Insecticidal Nets; glutathione S-transferase; insecticide resistance; malaria; metabolic resistance; piperonyl butoxide.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Quality control before the experimental hut trial in Mibellon: Species composition in Mibellon: (A) Quality control of the efficacy of all the four nets checked against the susceptible laboratory strain of Anopheles gambiae Ngousso and LLINs efficacy testing using cone assays against the pyrethroid resistant An. funestus population from Mibellon, Cameroon; (B) Number of mosquitoes collected during the hut effect assessment; (C) Average of Anopheles funestus. ss collected by hut during the 18 days of the hut effect investigation.
Figure 2
Figure 2
Performance of the four LLINs in experimental hut trials against pyrethroid resistant An. funestus in Cameroon. (A) Mosquito species composition during the experimental hut study. (B) Proportion of mortality and blood feeding rate for the four LLINs against An. funestus.
Figure 3
Figure 3
Impact of the L119F-GSTe2 mediated metabolic resistance on bednet efficacy for blood feeding ability: (A) Genotype distribution of L119F-GSTe2 between blood fed and unfed mosquitoes after exposure to Olyset showing a significant increased ability to blood feed for resistant mosquitoes; (B) Genotype distribution of L119F-GSTe2 between blood fed and unfed mosquitoes after exposure to Olyset Plus showing a significant increased ability to blood feed for resistant; (C) Genotype distribution of L119F-GSTe2 between blood fed and unfed mosquitoes after exposure to PermaNet 2.0 showing an inverse marginal increased ability to blood feed for homozygote susceptible SS compared to homozygote resistant RR (p < 0.05); (D) Genotype distribution of L119F-GSTe2 between blood fed and unfed mosquitoes after exposure to PermaNet 3.0 showing an increased ability to blood feed of susceptible mosquitoes compared to resistant mosquitoes (R vs. S: OR = 0.29 p < 0.05).
Figure 4
Figure 4
Impact of the L119F-GSTe2 mediated metabolic resistance on bednet efficacy -exophily: (A) Genotype distribution of L119F-GSTe2 between indoor (Room) and outdoor (verandah) mosquitoes after exposure to Olyset showing no association; (B) Genotype distribution of L119F-GSTe2 between indoor (Room) and outdoor (verandah) mosquitoes after exposure to Olyset Plus showing a significant increased ability to exit the room when considering all mosquitoes; (C) A greater association is observed with exophily with Olyset Plus when only analyzing unfed mosquitoes; (D) Genotype distribution of L119F-GSTe2 between indoor (Room) and outdoor (verandah) mosquitoes after exposure to PermaNet 2.0 showing a significant association (RS vs. SS: OR = 1.35; p < 0.01); (E) Genotype distribution of L119F-GSTe2 between indoor (Room) and outdoor (verandah) mosquitoes after exposure to PermaNet 3.0 showing no association.
Figure 5
Figure 5
Association between L119F-GSTe2 mutation and ability to survive exposure to LLINs (cone assays) and WHO papers (bioassays): (A) Genotype distribution of L119F-GSTe2 between alive and dead mosquitoes after exposure to Olyset Plus showing no association; (B) Genotype distribution of L119F-GSTe2 between alive and dead mosquitoes after exposure to PermaNet 2.0 showing a significantly increased ability of resistant mosquitoes to survive (R vs. S: p < 0.01); (C) Genotype distribution of L119F-GSTe2 between alive and dead mosquitoes after exposure to PermaNet 3.0 showing a significantly greater ability of resistant mosquitoes to survive than that seen for PermaNet 2.0 (RR vs. SS: OR = 30.1; p < 0.001); (D) Genotype distribution of L119F-GSTe2 between alive (90 min) and dead (30 min) mosquitoes after exposure to deltamethrin showing a significantly greater ability of homozygote resistant mosquitoes to survive (RR vs. SS: OR = 4.6; p < 0.001); (E) Similarly for permethrin, homozygote resistant mosquitoes exhibit a significantly greater ability of to survive (RR vs. SS: OR = 4.8; p < 0.001); (F) For DDT, a much greater ability of mosquitoes with the resistance allele to survive exposure to DDT papers (RR vs. SS: OR = 66.7; p < 0.001).

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