Oviposition bioassay

The oviposition preference of gravid mosquitoes was tested in a dual-choice assay in a Bugdorm® insect rearing cage (polypropylene, 30 × 30 × 30 cm) as described previously with slight modification¹⁰. Briefly, two egg cups (PET, top opening = 7.0 cm, height = 6.0 cm, bottom = 5.0 cm) filled with 10 mL of dH2O as an oviposition substrate were placed in opposite corners of the assay cage. A pair of borosilicate glass vials (14.65 × 19 mm; Qorpak, Bridgeville, PA) with a screen top (10 × 10 mm) filled with 1 mL of test solution, labial active blend and solvent control were placed inside the egg cup (Fig. 5B). Experiments were started approximately 1 h prior to scotophase (11:00) by introducing 10 gravid females (48-hour pBF) into the assay cage through the ‘releasing chamber’ as described above, and the total number of eggs in the two ovipoistion cups were manually counted on the following day (09:00). Ethanol was used as a solvent control and to dissolve labial active blend. Ten bioassay cages were prepared each day with two replications for every treatment that includes control (solvent-solvent) and four concentrations (10⁻³, 10⁻⁴, 10⁻⁵ and 10⁻⁷ M) of labial active blend under the same climatic condition described above.