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Background: Increasing metabolic resistance in malaria vector mosquitoes resulted in the development of insecticide-treated nets (ITNs) with active ingredients (AI) that target them. Bioassays that accurately measure the mortality induced by these AIs on ITNs are needed. Mosquito metabolic enzyme expression follows a circadian rhythm. Thus, this study assessed (i) influence of the time of day of mosquito exposure and (ii) timing of assessment of mortality post exposure (24 and 72 h) to ITNs against vectors that are susceptible to pyrethroids and those with metabolic and knockdown resistance mechanisms., Methods: Two cone bioassay experiments were conducted following World Health Organization (WHO) guidelines. Firstly, on ITNs incorporated with 2 g AI/kg of deltamethrin (DM) alone, or combined with 8 g AI/kg piperonyl butoxide (PBO) synergist, during the day (9:00-14:00 h) and repeated in the evening (18:00-20:00 h). This was followed by a confirmatory experiment during the afternoon (12:00-14:00 h) and repeated in the night (22:00-24:00 h) using mosquitoes unexposed or pre-exposed to PBO for 1 h before exposure to DM ITNs. Each net piece was tested with a minimum of eight cones per time (N = 24). The outcome was mortality after 24 h (M24) or 72 h (M72) of holding., Results: The cone bioassays performed using metabolic resistant mosquitoes during the evening showed significantly lower M24 than those performed in the day for DM: odds ratio (OR) 0.14 [95% confidence interval (CI) 0.06-0.30, p < 0.0001] and DM PBO [OR 0.29 (95% CI 0.18-0.49, p < 0.0001). M72 was higher than M24 for metabolic resistant mosquitoes exposed to DM [OR 1.44 (95% CI 1.09-1.88), p = 0.009] and DM PBO [OR 1.82 (95% CI 1.42-2.34), p < 0.0001]. An influence of hour of experiment and time of assessment was not observed for mosquitoes that had knockdown resistance or that were pyrethroid-susceptible., Conclusions: Time of day of experiment and hour of assessment of delayed mortality after exposure of mosquitoes are important considerations in evaluating insecticides that interact with mosquito metabolism to counter metabolic resistant mosquitoes. This is important when evaluating field-aged ITNs that may have lower concentrations of AI., (© 2024. The Author(s).)

Background: Insecticide-treated net (ITN) durability is evaluated using longitudinal bioefficacy and fabric integrity sampling post-distribution. Interceptor ® G2 was developed for resistance management and contains two adulticides: alpha-cypermethrin and chlorfenapyr; it is a pro-insecticide that is metabolized into its active form by mosquito-detoxifying enzymes and may be enhanced when the mosquito is physiologically active. To elucidate the impact of bioassay modality, mosquito exposures of the alphacypermethrin ITN Interceptor ® and dual adulticide Interceptor ® G2 were investigated., Methods: This study evaluated the performance of Interceptor ® G2 compared to Interceptor ® against local strains of mosquitoes in Tanzania. Unwashed and 20× times washed nets were tested. Efficacy of ITNs was measured by four bioassay types: (1) World Health Organisation (WHO) cone test (cone), (2) WHO tunnel test (tunnel), (3) Ifakara ambient chamber test (I-ACT) and (4) the WHO gold standard experimental hut test (hut). Hut tests were conducted against free-flying wild pyrethroid metabolically resistant Anopheles arabiensis and Culex quinquefasciatus. Cone, tunnel and I-ACT bioassays used laboratory-reared metabolically resistant An. arabiensis and Cx. quinquefasciatus and pyrethroid susceptible Anopheles gambiae sensu stricto and Aedes aegypti., Results: Against resistant strains, superiority of Interceptor ® G2 over Interceptor ® was observed in all "free-flying bioassays". In cone tests (which restrict mosquito flight), superiority of Interceptor ® over Interceptor ® G2 was recorded. Mortality of unwashed Interceptor ® G2 among An. arabiensis was lowest in hut tests at 42.9% (95% CI: 37.3-48.5), although this increased to 66.7% (95% CI: 47.1-86.3) by blocking hut exit traps so mosquitoes presumably increased frequencies of contact with ITNs. Higher odds of mortality were consistently observed in Interceptor ® G2 compared to Interceptor ® in "free-flying" bioassays using An. arabiensis: tunnel (OR = 1.42 [95% CI:1.19-1.70], p < 0.001), I-ACT (OR = 1.61 [95% CI: 1.05-2.49], p = 0.031) and hut (OR = 2.53 [95% CI: 1.96-3.26], p < 0.001). Interceptor ® and Interceptor ® G2 showed high blood-feeding inhibition against all strains., Conclusion: Both free-flying laboratory bioassays (WHO Tunnel and I-ACT) consistently measured similarly, and both predicted the results of the experimental hut test. For bioefficacy monitoring and upstream product evaluation of ITNs in situ, the I-ACT may provide an alternative bioassay modality with improved statistical power. Interceptor G2 ® outperformed Interceptor ® against pyrethroid-resistant strains, demonstrating the usefulness of chlorfenapyr in mitigation of malaria., (© 2022. The Author(s).)