2.9. Y-Tube Olfactometer Behavioral Bioassays

The Y-tube olfactometer mainly consisted of a Y-shaped glass tube (main arm, 30 cm; sidearm, 20 cm; inner diameter, 2.5 cm; side arm angle, 60 degrees), an air sampler, a drying tower, a gas scrubber, and a flow meter. Two gas-washing cylinders were successively connected with the flow meter, drying tower, air pump, and Y-shaped glass tube through rubber pipes. The testing compound (concentration 1 μg/μL in paraffin oil) and paraffin oil (as control) were taken and dropped respectively into a rubber septum (Pherobio Technology Co., Ltd., Beijing, China) and placed respectively into the side arms of the Y-tube. The flow rate of the atmospheric air sampler was adjusted to 500 mL/min.

A female adult beetle was introduced into the main arm at the downwind end of the Y-olfactometer. Each beetle was given 5 min to respond to the treatment, and the first choice that the beetle made for each of the lateral arms was recorded. The response was regarded as valid only if the beetle crawled more than 3 cm into a lateral arm and stayed for more than 1 min. The following measurements were recorded for all individual beetles: the number of beetles that crawled into a lateral arm of the Y-tube (responsive beetles), and the number of beetles that did not make any choice and stayed in the main arm for 5 min (non-responsive beetles).

For each compound, more than 90 beetles were tested individually until 90 responsive beetles were obtained. Each individual beetle was tested only once, and the Y-olfactometer was replaced after every 10 beetles were tested to eliminate the odor effect. The number of individual beetles that selected the compound-treated arm of the Y-olfactometer was statistically compared with the number of beetles that selected the solvent-treated arm using a Chi-square goodness of fit test.