2.3. Coupled Gas Chromatography-Electroantennographic Detection (GC-EAD)

The O. wightiana fruit volatile sample (2 μL) was injected in a Shimadzu GC-2010plus equipped with a DB-Wax column (30 m × 0.25 mm inner diameter × 0.25 μm film thickness; Agilent) and interfaced with the EAG apparatus. Helium (2.0 mL/min) was used as the carrier gas. The oven temperature was held at 40 °C for 2 min and programmed at 5 °C/min to 120 °C, then 15 °C/min to 240 °C and held for 4 min. The column effluent was split at a 1:1 ratio, with one part to a heated line into a humidified airstream (400 mL/min) which was directed at the antenna preparation, and the other to the Flame Ionization Detector (FID) of GC. Electroantennogram (EAG) recordings were made using Ag-AgCl glass microelectrodes filled with a Ephrussi-Beadle Ringer solution. The intact female fly was immobilized by cotton inside a pipette tip, while its head stretched out. The recording electrode was inserted into the distal region of the terminal antennal segment, while the reference electrode was positioned into the base of the antenna. The signals generated by the EAD and FID were passed through a Syntech IDAC-2 high-impedance amplifier and analyzed with Syntech GC-EAD software. FID peaks that elicited EAD responses for at least three runs were considered electrophysiologically-active and marked for identification by coupled gas chromatography-mass spectrometry (GC-MS).