Abstract
Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control.Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre.The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools.
Highlights
The cytochrome c reduction assay produced an activity of 92.04 cytochrome c reduced/min/mg protein ± 10.70 (n 1⁄4 3) of the cytochrome P450 reductase, higher than obtained from co-expression of CYP6P3 from An. gambiae, but lower than obtained from co-expression with CYP6M2 (Muller et al, 2008; Stevenson et al, 2011)
Highest activity was obtained with permethrin which exhibited catalytic activity half the value established for An. gambiae CYP6M2 (Stevenson et al, 2011) but with comparable catalytic efficiencies due to higher affinity exhibited by CYP6P4
These catalytic efficiencies of An. arabiensis CYP6P4 with permethrin were similar to the values we have established for the An. funestus CYP6P9a and CYP6M7 resistance genes (Riveron et al, 2014)
Summary
Within the last ten years the burden of malaria has been greatly reduced in sub-Saharan Africa thanks to the scale-up of pyrethroidimpregnated long-lasting insecticidal treated nets (LLINs) (WHO, 2014) and indoor residual spraying (IRS) (WHO, 2006). By 2013, these interventions, in addition to chemotherapy, have helped reduce malaria-related mortality in the WHO African region by 54% (WHO, 2014). One of the challenges threatening these malaria intervention tools is the widespread resistance to the major insecticides used in LLINs and IRS, notably from the Anopheles gambiae complex and Anopheles funestus group (Coetzee and Koekemoer, 2013; Corbel and N'Guessan, 2013). Ibrahim et al / Insect Biochemistry and Molecular Biology 68 (2016) 23e32 some times over relatively small distances (Ranson et al, 2009), implementation of any resistance management demands sound knowledge of dominant vector species distribution, behaviours, insecticide susceptibility/resistance status, and most importantly the molecular mechanisms of the resistance (Coetzee et al, 2000; Corbel and N'Guessan, 2013; Gatton et al, 2013)
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