In order to evaluate whether the CRMs be more effective at higher concentrations of each AI, we used cheesecloth (100 % cotton, Loins Services, Inc. Charlotte, NC) as a common CRM surrogate material sprayed with technical grade AIs dissolved with acetone. We prepared solutions (containing 1% of each AI above) with each technical grade insecticide in acetone (Table 1) and sprayed 2 ml of each insecticide solution uniformly on glass petri dishes (5 cm diameter) containing a 4.8 cm diameter piece cheesecloth by using an artist’s airbrush sprayer (Badger 100 series, Badger Corporation, Franklin Park, IL, US) which is commonly used in stored product insect research (e.g. Arthur and Morrison 2020; Morrison et al. 2021). A 1% concentration of AI was used to approximate the far higher concentration of AI in commercially available incorporated long-lasting materials compared to the concentration typically used in direct spray applications. The insecticide-treated cheesecloth was allowed to dry at room temperature overnight (~18 h) inside a fume hood to avoid affecting the behavior of insects.
Cohorts of 20 mixed-sex (~1:1 M: F sex ratio) adult beetles were exposed continuously up to 96 h or 1 weeks in the laboratory on cheesecloth (e.g., as an absorptive CRM surrogate material) at constant conditions (27.5° ± 0.1 C, 65% RH, 14:10 L:D) in an environmental chamber. Exposure times were added iteratively at the same 1% concentration to yield a sufficient number of points to calculate median lethal time (LT50) up to 1 week (Supplemental Table 1). At each time point, the condition of insects was checked, and rated as alive, affected, or dead condition according to the definitions in (Morrison et al. 2018) for each of the different treatments. Dead insects were completely immobile, even after prodding, and were removed from the tested arenas, but retained in analyses. There were 4 replicate cohorts for each treatment combination of insecticide, exposure, phosphine susceptibility, and species.