Electroantennography (EAG)

The response of M. domestica adult female antennae (3 days old) to sweet basil EO, methyl chavicol, linalool and neem oil was recorded using an electroantennographic system (Syntech). The dual electrode probe was used for mounting the antennal prep. The head of the adult female fly was decapitated and mounted on one electrode and the proximal tip funiculus to another electrode using a conductive gel (Spectra 360 Parker, Orange, New Jersey). The clean air (activated charcoal filtered) was continuously flushed over the antennae. The EO and its constituent’s methyl chavicol, linalool and neem oil were diluted in HPLC grade dicholoromethane at concentration of 1 μg μL^-1. Dichloromethane alone was used as a control. One μl of the aliquot amounting to 1 μg of the test compound placed on Whatman filter paper strips (Advantec 5C (110 mm) Japan of 2 cm length and 4 mm diameter) was dried for 5 min in fume hood, and then it was inserted into the Pasteur pipettes. This setup was connected to stimulus controller (CS 05 Syntech) by Tygon silicone tube. The first puff was blown off after 30 seconds of loading filter paper. After sixty seconds, the antennae were exposed to vapour phase of the stimulus through pipette placed 15 mm upstream from the antennae that had continuous air stream (pulse time 0.5 seconds, continuous flow 25 ml/s, pulse flow 21 ml/s) as suggested by Venugopal and Subaharan (2019). Between the stimulus puffs, a time delay of 20 s was maintained. The antennal responses were recorded through a high impedance probe that was in turn connected to amplifier (IDAC-4, Syntech), and the signals were recorded with EAG software (Syntech). Responses were expressed as a summated response of neurons, sorted according to shape and amplitude, emitted during 1 s after the onset of the stimulation. The control stimulus was at the beginning, middle and end of each session. EO and its constituents and neem oil were tested on six fly antennae with four replications of per stimuli per antennae in randomized manner.