2.5.2 Y-tube assays
Behavioral assays are performed with a glass Y-tube olfactometer (10 cm body length; 8 cm arm length; 1.5 cm inner diameter; 60 degree for the angle of two arms). Air aspirated with a vacuum pump (Qianxi Air Company, Beijing, China) was filtered through an activated-charcoal filter and a sorbent cartridge (Porapak Q, 50 mg, 80/100 mesh, Supelco, Bellefonte, PA, USA) with a 0.3 L/min flow rate. Experimental samples (fresh ripe fuji apples or crown pears / 30 virgin female adults for 3 days after eclosion / 10-3 M ethyl butanoate and 10-3 M ethyl hexanoate from crown pear volatiles) and control samples (DEPC water/ 10-3 M HPLC purity grade hexane) were placed into the two arm sides respectively. Each individual larva or male adult was placed at the entrance of the olfactometer. The number of larvae or male adults in each arm exceeded 2 cm and stayed for 30s were recorded within 5 min. If larva or male adult made no choice within 5 min, it was not counted. For each experiment, 30 male adults or 30~60 larvae were tested in the Y-tube olfactometer. All assays were performed in the behavior room at 25 ± 0.5℃ and 70% relative humidity. Behavioral assays of male adults were operated under red light (0.61 lux). The olfactometer was washed with detergent and water before each experiment. Each experiment was repeated for three times.