2.2. Electroantennography (EAG)

Dose-response experiments using (E)-2-octenyl acetate were carried out. Electroantennograms (EAG) were conducted using aliquots of 2 μL of test solution of (E)-2-octenyl acetate (Bedoukian Research, Danbury, CT, USA) dissolved in acetone, pipetted onto a piece of filter paper (Whatman, grade 1). Tested doses of (E)-2-octenyl acetate were 0.02, 0.2, 2, 20, and 200 µg; acetone was used as a control. The loaded paper was exposed to the air for 40 s to allow the solvent to evaporate and was then placed inside a glass Pasteur pipette. Puff stimuli were blown into an airstream that passed over the antennal preparation using a flow controller (model CS-05; Syntech, Hilversum, the Netherlands) to generate a 1.5-s stimulus at 1-min intervals, with a flow rate of 1.5 L/min. The signals generated by the antennae were passed through a high-impedance amplifier (model IDAC-4, Syntech) and were recorded with custom software (Syntech, Hilversum, the Netherlands). For the EAG preparations, adult and nymphs were anesthetized by refrigeration at −4°C for 40 s; the head was excised and mounted on a glass capillary tube reference electrode (1.5 mm diameter) filled with 0.1 M KCl solution and connected with silver wire to the amplifier. The recording electrode was a similar glass capillary in contact with the tip of the antenna. The capillary tubes were drawn to a fine point using a microelectrode puller (PC-10, Narishige, Tokio, Japan) to achieve a diameter enabling insertion into the antennal tip.