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.