2.11. Electroantennography (EAG)

The olfactory sensitivity of male and female S. granarius antennae to ascending concentrations of the three hop extracts was assessed by EAG using the technique reported in previous studies [29,30]. Briefly, the head of a 2- to 3-week-old insect was excised from the prothorax using a scalpel and mounted between two glass capillary electrodes (Microglass, Naples, Italy) filled with Kaissling saline solution [31]. The recording electrode (diameter~100 μm) was put in contact with the dorsal surface of the antennal club while the neutral electrode was inserted into the base of the head. The electrical continuity between the antennal preparation and an AC/DC UN-6 amplifier in DC mode was maintained using AgCl-coated silver wires. The amplifier was connected to a PC equipped with the EAG 2.0 program (Syntech Laboratories, Hilversum, The Netherlands).

Five two-fold dilution of n-hexane, methanol, and acetone hop cone extracts (100, 50, 25, 12.5, 6.25 µg/µL) in the corresponding solvents (Sigma–Aldrich, Milan, Italy) were prepared. The test stimulus (10 µL of an extract solution) was adsorbed onto a filter paper (Whatman No. 1) strip (2 cm²) inserted in a Pasteur pipette (15 cm long) after solvent evaporation. The vapor stimuli (3 cm³) were puffed, using a disposable syringe, for 1 s into a charcoal-filtered and humidified air stream (500 mL/min) flowing over the antenna through a stainless-steel delivery tube (Ø 1.0 cm) with the outlet positioned~1 cm from the antenna. Control (10 μL of a solvent) and standard (10 μL of a 10 μg/μL (Z)-3-hexenol solution) stimuli were also applied at the beginning of the experiment and after each group of 5 test stimuli. The intervals between stimuli were 1 min. Each dose of the three hop extracts was tested on three antennae from different males and females.

EAG responses were measured by the maximum amplitude of negative polarity deflection (-mV) elicited by a stimulus [32]. To compensate for solvent and/or mechanosensory artefacts, the amplitude (mV) of the EAG responses to each test stimulus was subtracted by the mean EAG response of the two nearest solvent controls [33]. To compensate for the decrease in the antennal responsiveness during the experiment, the resulting EAG amplitude was corrected according to the reduction of the EAG response to the standard stimulus [34]. Dose–response curves were calculated based on the corrected EAG values. To verify antennal activation, the corrected mean male and female EAG response to the last dilution of each hop extract was compared to “0” value using one-sample Student’s t-test and regarded as “activated” if significant at p = 0.05. Saturation level was taken as the lowest dilution at which the mean response was equal to or less than the previous one [35]. Mean male and female EAG responses to each stimulus were compared using a Student’s t-test for independent samples at p = 0.05. Since no significant differences were found between the male and female EAG responses to each test stimulus, individual male and female EAG responses were pooled and analyzed together. For each dose tested, the mean EAG responses of adult insects to the three hop extracts were submitted to ANOVA followed by Tukey HSD test (p = 0.05). Levene’s test was used to assess homogeneity of variances.