MOLECULAR MECHANISMS OF MULTIPLE INSECTICIDE RESISTANCE IN Anopheles funestus sensu stricto AT AKAKA-REMO, SOUTHWESTERN NIGERIA

Abstract

The use of insecticides against malaria vectors has been a good approach to control malaria, but its efficacy is threatened by mosquito resistance to the lethal doses. In Nigeria, Anopheles funestus is gradually becoming an important malaria vector, especially in rural areas. However, despite recurring cases of Insecticide Resistance (IR) in some Anopheles species, there is a dearth of information on the molecular mechanisms of IR in An. funestus. Therefore, this study was designed to investigate the molecular mechanisms of IR in An. funestus at Akaka-Remo, Southwestern Nigeria. Early morning collections of adult mosquitoes were conducted from October 2014 to April 2015 in 30 rooms (25 houses) at Akaka-Remo (population density = 5,585). The mosquitoes were identified with standard keys, but only An. funestus (F0) was identified to sub-species level by Polymerase Chain Reaction (PCR). The F0 was analysed for Plasmodium Infection Rate (PIR) by TaqMan real-time PCR, and screened for Knockdown resistance (Kdr) mutations using next-generation sequencing. The gene sequences generated were analysed with bioinformatics tools. The F0 was further subjected to forced-egg laying technique to generate the F1. The mortality rate of 2-5 day old F1 exposed to permethrin- (0.75%), deltamethrin- (0.05%), DDT- (4%), dieldrin- (4%) and bendiocarb (0.1%)-treated papers was determined using WHO standard procedures. The contribution of detoxifying enzymes to IR was assessed by first exposing the F1 to three standard synergists: Piperonyl Butoxide (PB:4%), S,S,S-Tributyl Phosphorotrithioate (STP:0.25%) and Diethyl Maleate (DM:8%); and to permethrin and DDT, respectively. Microarray and real-time PCR were used to identify differentially expressed genes in permethrin and DDT-resistant populations. The frequency of resistant and susceptible alleles in DDT (L119F-GSTe2) and dieldrin (A296S-RDL) resistance markers was determined in the F0 and F1 genotype using TaqMan real-time PCR. Data were analysed using descriptive statistics and Student’s t-test at 0.05. A total of 375 mosquitoes were collected and identified as An. funestus (83.8%), An. gambiae (6.9%), Culex species (5.6%), Aedes species (1.3%) and Mansonia species (2.4%). All Anopheles funestus were further identified as An. funestus sensu stricto, with a PIR of 8.0%; and no Kdr mutations. Permethrin, deltamethrin, DDT, dieldrin, and bendiocarb induced 68.0, 87.0, 10.0, 8.0 and 84.0 % mortalities, respectively in the F1, indicating resistance. Exposure of the F1 to the three synergists with permethrin induced 100% mortality each; while PB, STP and DM with DDT, respectively induced 30.0, 81.8 and 71.4% mortalities. These suggest that detoxifying enzymes contributed to IR in the mosquitoes. Overexpressed genes in the resistant populations were GSTe2, GSTd3, GSTd1-5, GSTU2, CYP6P2, CYP6P9a, CYP6P4a and CYP9K1. The frequency of resistant alleles in L119F-GSTe2 and A296S-RDL was significantly high: F0=77.0%, F1=80.0% and F0=76.0%, F1=90.0%, respectively compared to the susceptible alleles: F0=23.0%, F1=20.0% and F0=24.0%, F1=10.0%, respectively. Overexpression of detoxifying genes and the high frequency of resistant-associated mutations were responsible for multiple insecticide resistance in the Anopheles funestus at Akaka-Remo.