Dual-choice behavioral assay

To perform the behavioral assay, a dual-choice olfactometer was utilized in this study ( Figure 1A ). The entire apparatus was made of transparent glass and consisted of two flat-bottom spherical chambers (height: 40 cm, width: 40 cm). Each chamber was equipped with an air inlet on the side and a lid on the top, allowing for the delivery of clean air to each chamber and preventing any potential air leakage, respectively. The chambers were connected by an arm (length: 40 cm, internal diameter: 10 cm) equipped with an insect release chamber (height: 15 cm, width: 15 cm) at the midpoint. The aphid-infested or uninfested cotton plants were placed in the side chambers, and a constant charcoal-filtered humid air flow was delivered to the side chambers at the rate of 0.3 L/min and exhausted from the release chamber. The H. variegata individuals (fifty males, females, or larvae) were introduced simultaneously into the insect release chamber and allowed to make choices. The number of insects entering each chamber was counted every 30 minutes for a period of 6 hours, and the cumulative count was subjected to subsequent data analysis. Insects remaining in the release chamber or lateral arms were considered to have no response. The experiments were carried out with five replications under conditions similar to colony maintenance.

Behavioral responses of Hippodamia variegata to healthy plants and plants damaged by cotton aphid. (A) Schematic diagram of the dual-choice behavioral assay setup. (B) Bar charts demonstrate behavioral tendencies of third instar larvae, adult males and adult females towards healthy and damaged plants (proportion of insects that made choices). The data are shown as mean ± SEM. *** demonstrates significant differences (P < 0.001) analyzed by a chi-squared test. Pie charts illustrate the overall proportion of all tested insects, including those that made no choices and remained unresponsive.