Electroantennographic recording (EAG)

The EAG responses of P. spumarius male and female antennae to increasing concentrations (0.01–100 µg/µL) of the three EOs were measured by the EAG technique similar to that used in previous studies^33,53. In order to prevent the rapid evaporation of test compounds, EOs were dissolved in mineral oil (Sigma-Aldrich, Milan, Italy) and stored at −20 °C until needed.

For the test, a single adult was dissected between the head and the thorax and a glass micropipette (0.2–0.3 mm i.d.) filled with 0.1 M KCl solution, acting as the indifferent electrode, was inserted into the head. The last antennal segment was put in contact with the end of a similar pipette which provided the recording electrode. AgCl coated silver wires were used to maintain the electrical continuity between the antennal preparation and an AC/DC UN-6 amplifier in DC mode connected to a PC equipped with the EAG 2.0 program (Syntech). A stream of charcoal-filtered humidified air (500 ml/min) was directed constantly onto the antenna through a stainless steel delivery tube (1 cm i.d.) with the outlet positioned at approximately 1 cm from the antenna. Twenty five microliters of each stimulus were absorbed onto a filter paper (Whatman No. 1) strip (1 cm × 2 cm) inserted in a Pasteur pipette (15 cm long) and used as an odour cartridge. Over 1 s, 2.5 cm³ of vapour from an odour cartridge were blown by a disposable syringe into the air stream flowing over the antennal preparation. Intervals between stimuli were 1 min. Standard (25 μl of (Z)-3 Hexen 1 ol) and control (25 μl mineral oil) stimulus were applied at the beginning and at the end of the experiment. In addition, the standard stimulus was applied after each group of two test odours, to evaluate the gradual decrease in the antennal sensitivity over time.

For each population (Campobasso, Benevento), the EAG responses were recorded from at least 13 antennae of different males and females.

The amplitude (mV) of the EAG response to each test stimulus was adjusted to compensate for solvent and/or mechanosensory artefacts according to Raguso & Light⁵⁴. To compensate for the decrease of the antennal responsiveness during the experiment, the resulting EAG amplitude was corrected according to the reduction of the EAG response to the standard stimulus⁵⁵. The Student’s t-test for independent samples were used to compare the mean EAG responses of specimens of each sex collected in the two different areas (Campobasso, Benevento) and those of males and females of the same area.

In dose–response curves, the activation threshold was considered to be the first dose at which the mean response was higher than “0” value using Shapiro-Wilk test for normality followed by one-sample Student’s t-test (P = 0.05)³³. Saturation level was taken as the lowest dose at which the mean response was equal to or less than the previous one⁵⁶. The non-parametric Kruskal-Wallis test for multiple independent comparisons followed by pairwise Mann–Whitney U-test comparisons (P < 0.05) were used to compare the mean EAG responses of each sex to different doses of each EO and to the same dose of different EOs.

Data were processed by Statistical Package for Social Sciences (SPSS), version 25.0 per Windows software (SPSS Inc., Chicago, IL).