Transfluthrin Susceptibility Tests for Mosquitoes

Physiological susceptibility tests for transfluthrin were conducted for each mosquito strain before the start of semi-field experiments. The tests were performed using tube test bioassays following World Health Organization (WHO) guidelines [25 ]. As there is no recommended discriminating dose of transfluthrin for testing the susceptibility status of these mosquitoes, transfluthrin-impregnated papers at the doses proposed by Sukkanon et al. [26 (link)] were used. Five serial dilutions of emulsifiable concentrate (EC) were prepared by mixing with acetone and silicone oil in individual Falcon tubes. The concentrations of EC transfluthrin were 0.00125%, 0.0025%, 0.005%, 0.01%, 0.02%, 0.04%, 0.08% and 0.1% for Anopheles, and 0.003125%, 0.00625%, 0.125%, 0.025%, 0.05% and 0.1% for Ae. aegypti. Whatman grade 1 filter papers (12 × 15 cm; Whatman International, Banbury, UK) were prepared by impregnation with the concentrations of EC transfluthrin. For each filter paper, 2 ml of diluted EC transfluthrin was used. The impregnated papers were air-dried in the shade at ambient temperature, then wrapped in aluminium foil and refrigerated at 4 °C before use in the tests that were carried out on the same day. The papers were destroyed after the experiment.
One hundred and fifty non-blood-fed, 3–5-day-old mosquitoes were exposed to the transfluthrin-treated paper or to the control for 1 h. The mosquitoes were then provided with 10% sucrose solution and maintained at approximately 27 °C and 80% RH for the determination of 24 h mortality. Each dilution was tested four times.
The discriminating concentration (DC) for Anopheles (Table 4) was used to test the susceptibility status of An. gambiae (Kisumu strain; KDR) and An. funestus (FUMOZ strain). The same procedure was used as in the susceptibility test, and the same numbers of mosquitoes were exposed to the transfluthrin-treated paper as per the obtained DC.