Y-tube behavioral assay

We conducted Y-tube olfactometer trials to determine the behavioral tendencies of adult males, adult females, and third instar larvae of H. variegata towards the compounds, identified from infested cotton plants. The olfactometer tubes (diameter: 2.5 cm) were made of glass and consisted of a 20 cm main stem, two 20 cm lateral arms, and a 45° angle between arms. The Y-tube was placed inside a steel chamber (1m × 0.8 m × 0.8 m) which was equipped with a camera and 40-W fluorescent lamps providing uniform lighting (~2000 lux). A video monitor was used to observe the behavioral responses of insects during the assays. A 500 ml glass conical flask served as the odor source container. The lid of each flask contained an air inlet tube connected to a clean air source, and an outlet tube connected to the olfactometer arm. Each compound was individually formulated in light mineral oil to desired concentrations (0.1, 1, 10, and 100 mg/ml), applied as a 20 µl sample on a filter paper strip (50 × 5 mm) and promptly placed inside the treatment flask. Mineral oil alone was used as a control. The stimuli were delivered to the olfactometer arms at a constant air flow rate of 0.3 l/min. Insects were introduced individually at the base of the central arm and given 5 minutes to make a choice. A choice was defined when an insect reached to the midpoint of the lateral arm and remained there for at least 10 seconds. If an insect did not make a choice during this period, it was recorded as no choice and excluded from data analysis. Each insect was used only once. After testing a total of four insects, we switched the position of the treatment and control arms to avoid any positional bias. The Y-tube olfactometer was replaced with a clean one after testing eight individuals. For each odorant, 50 adult males, adult females, or larvae were tested. All olfactometer assays were conducted between 9:00 am to 5:00 pm, under conditions similar to colony maintenance. The entire experiment (testing 50 individuals for each odorant) was repeated 5 times at different days and data are shown as mean ± SEM.