Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays

Abstract

Abstract Background The World Health Organization (WHO) cone bioassay is routinely used for the evaluation of insecticide treated nets (ITNs) bioefficacy for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. This study investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. Methods Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and PBO) ITNs, using laboratory-reared pyrethroid resistant Anopheles funestus sensu stricto (FUMOZ) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO recommended test parameters with one variable changed in each, 1) environmental temperature during exposure: 22–23˚C, 26–27˚C, 29–30˚C, and 32–33˚C, 2) feeding: 6 hours sugar starved, blood-fed, or sugar-fed before exposure and 3) mosquito density per cone: 5, 10, 15, and 20. For each test, 15 net samples per treatment arm were tested with 4 cones per sample (N = 60). Mortality after 24, 48 and 72 hr post exposure to ITNs was recorded. Results There was a notable influence of temperature, feeding status and mosquito density on Anopheles funestus mortality for both types of ITNs. Mortality at 24 hours was significantly higher at 32–33˚C than 26–27˚C for MAGNet [19.33% vs 7%, OR = 3.96, 95% confidence interval (CI) (1.99–7.87), p < 0.001] and Veeralin [91% vs 47.33%, OR = 22.20 (11.45–43.05), p < 0.001]. Mosquito feeding status influenced the observed mortality. Relative to sugar fed mosquitoes, MAGNet induced higher mortality among blood-fed [7% vs 3%, OR: 2.23 (95% CI :0.94–5.27), p = 0.068] and significantly higher mortality among starved mosquitoes [8% vs 3%, OR: 2.88 (95% CI:1.25–6.63), p = 0.013] while Veeralin showed significantly lower mortality among blood-fed mosquitoes [43% vs 57%, OR:0.56 (95% CI:0.38–0.81), p = 0.002] and no difference for starved mosquitoes [58% vs 57%, OR:1.05 (95% CI:0.72–1.51),p = 0.816]. Mortality significantly increased with increasing mosquito density for MAGNet e.g., 5 vs 10 mosquitoes [7% vs 12%, OR: 1.81 (95% CI: 1.03–3.20), p = 0.040] and Veeralin [58% vs 71%, OR 2.06 (1.24–3.42) p = 0.005]. Conclusions The study highlights that the testing parameters, temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities.