Standardising bioassays for passive spatial emanators against mosquito vectors

The increasing use of passive spatial emanators (SEs) for vector control has highlighted the need for standardised laboratory methods for bioefficacy testing. Peet–Grady (PG) chamber assays are widely used for volatile insecticides; however, key methodological parameters remain poorly characterised. Here, we investigated how ventilation (airflow and baffle configuration), mosquito holding cage design, and cage mesh structure influence mosquito responses to a transfluthrin (TF) passive emanator. Susceptible and pyrethroid-resistant Anopheles gambiae mosquitoes were exposed to TF, and responses were assessed using flight inhibition (FI) and 24-hour mortality. The assay was highly reproducible, with 83% agreement among replicate comparisons in FI dynamics. Ventilation had the greatest effect, with airflow differences producing 6- to 19-fold changes in the odds of FI. Time to FI thresholds was up to 4-fold shorter under medium airflow without a baffle than under low airflow with a baffle. Cage mesh structure influenced FI, with larger mesh apertures producing 10- to 20-fold higher odds of FI, whereas cage geometry had little effect, supporting higher-throughput dual-cage configurations. These findings demonstrate that ventilation, mosquito holding cage geometry, and cage mesh structure strongly influence mosquito exposure in PG-chamber assays and should be standardised for passive spatial emanator bioassays.