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

Background The standard operating procedure for testing the susceptibility of adult mosquitoes to clothianidin, a neonicotinoid, recommends using a vegetable oil ester as surfactant. However, it has not yet been determined if the surfactant is an inert ingredient or if it can act as a synergist and bias the test. Methodology/Principal Findings Using standard bioassays, we tested the synergistic effects of a vegetable oil surfactant on a spectrum of active ingredients including four neonicotinoids (acetamiprid, clothianidin, imidacloprid and thiamethoxam) and two pyrethroids (permethrin and deltamethrin). Three different formulations of linseed oil soap used as surfactant were far more effective than the standard insecticide synergist piperonyl butoxide in enhancing neonicotinoid activity in Anopheles mosquitoes. At the concentration used in the standard operating procedure (1% v/v), vegetable oil surfactants lead to more than 10-fold reduction in lethal concentrations, LC 50 and LC 99 , of clothianidin in a multi-resistant field population and in a susceptible strain of Anopheles gambiae . At 1% or 0.5% (v/v), the surfactant restored susceptibility to clothianidin, thiamethoxam and imidacloprid and increased mortality to acetamiprid from 43 ± 5.63% to 89 ± 3.25% (P<0.05) in resistant mosquitoes. By contrast, linseed oil soap had no effect on the level of resistance to permethrin and deltamethrin suggesting that the synergism of vegetable oil surfactants may be specific to neoniconoids. Conclusions/Significance Our findings indicate that vegetable oil surfactants are not inert ingredients in neonicotinoid formulations, and their synergistic effects undermine the ability of standard testing procedures to detect early stages of resistance.