2.6 Electrophysiological assay
Electroantennograms (EAG) was used to determine the stimulus-dependent potential changes summed over the whole antenna. The ends of an isolated antenna cut from 2- to 3-day-old virgin male and female moth were connected by electroconductive gel (Signal gel, Parker laboratories inc, USA) to the two electrodes, respectively. Odorants for EAG analyses were selected based on a literature review as well as on compounds category. All components were dissolved in hexane to 100 ng/μL. A filter paper strip (2.5 cm × 1.0 cm) containing 10 µL of a test solution was inserted into a Pasteur pipette to deliver the stimuli, and the strip was left for 1 min to let the solvent evaporate before it was used for EAG measurement. The continuous airflow (30 mL/s) and the odor stimulating flow (0.2 s at 10 mL/s) were produced and controlled by a stimulus controller (CS-55, Syntech, Netherlands). At least 45 sec was allowed between two stimuli to provide time for recovery of antennal responsiveness. The antennal signal was amplified tenfold and converted to a digital signal by DC amplifier interface (IDAC, Syntech Inc., Netherlands). The signals were recorded with EAGPro software (version 2.0, Syntech Inc., Netherlands). A standard de canal was tested in the beginning and at the end of each recording to correct for the loss of sensitivity of the antennal preparation. Similarly, a control paraffin oil stimulation was done at the beginning and at the end of each recording to subtract the blank value from the antennal responses (Ren et al., 2017). For each compound, EAG responses of ten antennae from different adult beetles of each sex were recorded.
2.7 Tendency test of larvae to artificial diet
The responsiveness of larvae to the odor of artificial diet was investigated, twenty individuals of the 3th instar larvae which were allowed for starvation (24 h). After starvation, and the larvae were placed in a 20 cm diameter plastic Petri dish, which also contained diametrically opposite two points 11 cm hole, one with some artificial diet, the other set empty for control. After 10 min, larvae crawl to opposite sides of the hole (or in close proximity) were counted, as well as those which had not made any choice. Each experiment was repeated 5 times with unexperienced larvae, results were averaged, and standard deviations were calculated.