Testing Anopheles larvae and adults using standard bioassays reveals susceptibility to chlorfenapyr (pyrrole) while highlighting variability between species

A standard test is available for assessing the susceptibility of adult Anopheles mosquitoes to chlorfenapyr, a new active ingredient in insecticide-treated nets. However, for a new insecticide with a unique mode of action, testing both larvae and adults using different routes of exposure is crucial to a comprehensive evaluation of susceptibility and to identifying potential selection pressures that may drive resistance. We followed WHO guidelines to assess the lethal toxicity of chlorfenapyr and monitor Anopheles susceptibility. Based on the median lethal concentration (LC50), larvae of the pyrethroid-susceptible colonized strain An. coluzzii Ngousso were 16-fold more susceptible to chlorfenapyr than immature stages of another susceptible colony: An. gambiae Kisumu. Larval bioassays indicated 99.63 +- 0.2% mortality after 24 h at a discriminating concentration of 100 ng/ml in Anopheles gambiae and An. coluzzii larvae collected from seven locations in urban and rural areas of Yaounde, Cameroon. By contrast, exposing emerging female adults from these populations to the recommended discriminating concentration (100 ug Active Ingredient (AI)/bottle) in bottle bioassays revealed variable mortality after 72 h, with values below the threshold of susceptibility (98%) in several tests. Anopheles coluzzii larvae and adults were fully susceptible, but mortality rates were slightly lower in An. gambiae adults compared to larvae (94 +- 1.5% vs 100%, Fisher exact test, p < 0.001). Piperonyl butoxide antagonized the activity of chlorphenapyr in An. gambiae adults. 100 ng/ml provides sufficient discriminative power for assessing the susceptibility of An. gambiae and An. coluzzii larvae to chlorfenapyr. Testing An. gambiae adults with 100 ug AI/bottle is likely to reveal inconsistent mortality values making it difficult to detect any emergence of resistance. Exploring different tests and accounting for variability between species are key to a reliable monitoring of Anopheles susceptibility to chlorfenapyr.