3.5. Electroantennography (EAG) Analysis

Peripheral olfactory responses were recorded from antennae of male C. capitata using a Syntech EAG system (Syntech Original Research Instruments, Hilversum, Netherlands) and methods developed by Kendra et al. [37,38,42]. Test substrates consisted of the 29 compounds, each 20 mg neat material. The standard reference sample (positive control) was tea tree oil, 20 mg (Essential Oil India-SAT Group, Kannauj, India), shown previously to elicit strong EAG responses in male medflies [15]. Each substrate was placed into a separate 250 mL hermetic glass bottle equipped with a lid containing a short thru-hull port (Swagelok, Solon, OH, USA) and silicone septum (Alltech, Deerfield, IL, USA). Sample bottles were sealed and equilibrated overnight at 24°C to allow for headspace saturation with volatiles.

Freshly dissected antennal preparations (whole head mounts) were secured between electrodes using salt-free conductive gel (Spectra 360, Parker Laboratories, Fairfield, NJ, USA) and placed under a stream of humidified air, purified with activated charcoal granules, at a flow rate of 400 mL/min. Using gas-tight syringes (SGE Analytical Science, Victoria, Australia), samples of saturated vapor were withdrawn from the test bottles, injected into the airstream, and presented to the antennae. In each recording session, samples (fixed 1 mL doses) were delivered in the following order: the tea tree standard, test chemicals in random order, a clean air injection (negative control), and a final standard injection. There was a 2-min interval (clean air flush) between injections to prevent antennal adaptation (diminished EAG response resulting from repeated exposure to chemical stimuli). Due to the large number of test chemicals, EAG analyses were conducted using four groupings; each group compared olfactory responses to seven or eight chemicals, randomly chosen, and responses were measured from ten replicate females.

EAG responses to test substrates were measured initially in millivolts (peak height of depolarization) and then normalized to percentages relative to the EAG response obtained with the reference sample. Normalization corrects for time-dependent variability (gradual decline) in antennal performance and allows for comparison of relative EAG responses obtained with different substrates [37,43,44,45] and with different cohorts of insects [37,38]. Finally, any response recorded with the negative control was subtracted from the normalized test responses to correct for “pressure shock” caused by injection volume. All statistical analyses were performed using the corrected normalized EAG values.