Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Abstract

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a locality situated 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F ₁ adults. Bioassays were performed to assess resistance profile to the four insecticides classes. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was the most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (<40% mortality). An. gambiae s.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the significant Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Keywords: Anopheles funestus; Anopheles gambiae; Cameroon; Insecticide resistance; Malaria; Plasmodium infection; Vector control.

Figure 1.. Map of Elende study area; showing its close proximity to the Nsimalen- International Airport of Yaoundé, capital city of Cameroon.

Figure 2.. Results of WHO insecticides susceptibility test.

( a) Susceptibility profile of female An. funestus s.s. (F1 progeny; N=638) from Elende following exposure to various public health insecticides. ( b) Susceptibility profile of Elende female An. gambaie s.s. (F1 progeny; N=621). ( c) Resistance intensity of An. gambiae s.s. (F1; N=358) on exposure to 5x and 10x permethrin and deltamethrin each; N is the total number of mosquitoes tested. Error bars represent standard error of the mean.

Figure 3.. Susceptibility profile of An. funestus s.s. to synergist and cone assays from Elende.

( a) Activities of PBO synergist assay on An. funestus s.s. (F1 population; N=489). ( b) Recorded mortalities following 3-min exposure by cone assay of An. funestus s.s. (F1 generation; N=230) from Elende to Olyset, Olyset Plus, PermaNet 2.0, PermaNet 3.0 (side) and PermaNet 3.0 (roof); N is the total number of mosquitoes tested. Data are shown as mean±SEM.

Figure 4.. Temporal distribution of resistance markers in Elende.

( a) An. funestus s.s: Frequency of the L119F-GSTe2 conferring DDT resistance; A296S-RDL mutation conferring dieldrin resistance; Cyp6P9a related with pyrethroid resistance. ( b) An. gambiae s.s: Frequency of the L1014F conferring pyrethroids and DDT kdrW resistance; L1014S related with pyrethroids and DDT kdrE resistance; G119S ace-1 conferring carbamate and organophosphate resistance. RR, homozygote for resistant allele; RS, heterozygote; SS, homozygote for susceptible allele.

Figure 5.. Differential expression by qRT-PCR of the major Cytochrome P450 genes ( CYP325A, CYP6P5, CYP6P9a and CYP6P9b) in An. funestus s.s. in Elende compared with the susceptible A. funestus s.s. strain FANG.

Error bars represent standard errors of the mean.