2.3. Electrophysiological Recordings

Electroantennography was performed to determine the antennal responses of 18 insect species and one hybrid ant to EDP and EβF, and eight species to IPA. Most insects were not anesthetized prior to antenna excision, except for houseflies, M. domestica, yellow fever mosquitoes, A.aegypti and honeybee, A. mellifera. EAG were recorded using saline-filled capillary glass electrodes [9]. EAG responses of S. invicta to its own alarm pheromone (EDP) were determined for major workers and male and female alates. Only workers were used for the following social insects: S. richteri, S. invicta × S. richteri, A. picea, M. pharaonis, M. minimum, N. fulva, A. mellifera, and R. flavipes. Males and females were evaluated for the non-social insects including B. hilaris, C. novemnotata, M. domestica, A. aegypti, A. tumida, N. viridula, N. eichhorniae, and C. lectularius. Only females were evaluated for the two aphid species, A. merii and R. padi. Procedures for the EAG on a majority of the species tested were similar to those used for S. invicta with the exception of R. flavips, M. pharaonis, M. minimum, M. domestica, R. padi, and A. nerii because of their small size and/or morphology of the antennae.

For EAG, silver wires in two glass capillary (1.1 mm in diameter) electrodes filled with saline solutions served as the reference and recording electrodes. The antenna was excised using fine-tipped forceps. The base and tip of the antenna were connected to the reference electrode and the recording electrode based on procedures modified after Kaissling and Thorson (1980) [36]. Due to the small size of the antennae in R. flavipes, M. pharaonis, M. minimum, R. padi, and A. nerii, the reference electrode was connected to the isolated head, while the recording electrode was connected to the tip of the antenna. Since the housefly has aristate antenna, the reference electrode was inserted to the isolated head and the recording electrode was connected to the third antennal segment.

To test the EAG response, each pheromone was dissolved in pentane and a 10 μL aliquot of the solution was then applied to a Whatman filter paper strip (3 mm × 40 mm). The pentane was allowed to evaporate by gently shaking for 10 s under a fume hood. Then the strip was inserted into the glass Pasteur pipette (Fisher Scientific, Pittsburgh, PA, USA), and the tip and the end of the pipette were immediately sealed with parafilm. Filter paper strips treated only with pentane in a glass pipette served as the control. The tip of the pipette was fitted into a side port of an L-shaped glass tube (130 mm in length and × 12 mm in diameter) oriented 5 mm away from the antennal preparation. The antenna was exposed to a solvent control blank (10 μL pentane) at the start and the end of recordings for each antenna. The stimuli were provided as 0.5 s puffs of air into a continuous humidified air stream as generated by an air stimulus controller (CS-55, Syntech^®, Buchenbach, Baden-Württemberg, The Netherlands, Europe). EAG signals were recorded for 10 s, starting 1 s before the onset of the stimulus pulse. At least 1 min was allowed between each puff for the recovery of antennal receptors. The analog signal was detected through a probe (INR-II, Syntech^®), captured, and processed with a data acquisition controller (IDAC-4, Syntech^®), and later analyzed using EAGPro computer software (Syntech^® ).

In order to select an adequate concentration of EDP for measuring EAG response of other insects, a dose–response relationship between EDP and S. invicta was established. Four concentrations of EDP were used, including 0.1, 1, 10, 100 ug/μL dissolved in pentane. A linear relationship was found between 100 μg/μL and 0.1 μg/μL, and 100 μg/μL elicited the maximum EAG responses in S. invicta. Therefore, 100 μg/μL EDP was used for all EAG measurements for other insect species. For IPA and EβF, 100 μg/μL was also used for all the insect species.