A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study

Abstract

Background The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. Methods A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration–response curves for each insecticide–species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. Results Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species–insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. Conclusion Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide. Graphical abstract

Vegetable oil surfactants are synergists that can bias neonicotinoid susceptibility testing in adult mosquitoes

“…Our control experiments where female adults were exposed to linseed oil soap alone allowed us to rule out the possibility that this surfactant on its own had lethal effects on mosquitoes. Corbel et al [14] have also demonstrated Mero®, a formulation of rapeseed fatty acid esters had no insecticidal activity on Anopheles mosquitoes. Using estimates of 24-h LC50 and LC99, we then found that blends of clothianidin and linseed oil soap are highly potent leading to a drastic increase in shortterm lethal toxicity of the active ingredient.…”

“…The standard operating procedure for testing the susceptibility of adult mosquitoes recommends using Mero® as surfactant when exposing Anopheles and Aedes populations to neonicotinoids and butenolides [4,14]. A large-scale study conducted to test the efficacy of broflanilide, a new meta-diamide insecticide, on several Anopheles laboratory colonies from different African countries has noted a drastic increase in toxicity of the active ingredient due (which was not certified by peer review) is the author/funder.…”

“…Early efforts to implement a standard testing procedure were challenged by the low solubility of clothianidin observed when large quantities of active ingredient were being diluted in water, ethanol or acetone [10,11]. To circumvent this issue, it has become common to add a surfactant to insecticide solutions used for bottle bioassays [2,[12][13][14]. The WHO standard operating procedure for testing clothianidin susceptibility of adult mosquitoes recommends mixing the active ingredient with 1% (v/v) of a formulation of rapeseed fatty acid esters (Mero®, Bayer, Reading, UK) to improve solubility and uptake of the insecticide by mosquitoes [4].…”

What can be learned from the residual efficacy of three formulations of insecticides (pirimiphos-methyl, clothianidin and deltamethrin mixture, and clothianidin alone) in large-scale in community trial in North Benin, West Africa?

“…It might be appropriate to revisit the diagnostic dose as the longer the observation time, the more other factors could affect the mortality rate. Furthermore, an alternative WHO bottle bioassay recently described by Corbel et al [ 37 ] could also be considered for evaluating the susceptibility of Anopheles vectors to clothianidin.…”

Enhancing the efficacy of neonicotinoids against mosquitoes and overcoming resistance issues

“…coluzzii Ngousso) in order to determine LC50 and LC99 corresponding to the lowest concentrations required to kill respectively 50% and 99% of susceptible mosquitoes. After 1 h exposure to the insecticide, lethal concentrations were determined within 24 h and 72 h. By contrast to clothianidin whose toxicity has been tested with at least one susceptible strain 7,11,28,29 , there is no information on the lethal concentrations of acetamiprid, imidacloprid and thiamethoxam to African malaria mosquitoes. Therefore, we focused primarily on those three active ingredients.…”