2.5.3 Electroantennogram (EAG) recordings
Physiologically active antenna, cutting along the basal segment, was immediately placed between two glass electrodes that attached to an electrically conducting gel (Spectra 360 Electrode Gel) (Zhang et al., 2017). And 20 μL odor stimulus source was added on a filter paper strips (0.5 × 5 cm) that placed inside pasteur pipettes. The humidified air stream was supplied by a Syntech stimulus controller (CS-55 model, Syntech, Germany) at 50 cm/sec and maintained at a pulse time of 2 sec. The EAG responses of male antennae to odor compounds were recorded. After each sample was tested, hexane was used for testing as the control. Three biological replicates were performed for each odor compound combination in the experiment. Odor stimulus sources, sex pheromone mix components (Z8-12:Ac: E8-12:Ac = 9:1), were diluted with hexane to a final concentration of 1000 ppm. The proportion of mix components (Z8-12:Ac: E8-12:Ac = 9:1) was designed based on previous studies (Li et al., 2011).
EAG recordings were also used for evaluating functions of GmolCXE1 and GmolCXE5 in odor degradation. The purification of GmolCXE1 and GmolCXE5 was described in ‘method 2.6.1’. Odor stimulus sources included two ester sex pheromone components (Z8-12:Ac and E8-12:Ac), mix components (Z8-12:Ac: E8-12:Ac = 9:1) and their treatment samples. The ester sex pheromone components were diluted with hexane to a final concentration of 1000 ppm. Treatment samples showed that sex pheromone components were treated with 50 μg purified GmolCXE1 or GmolCXE5 for 1 h at 28 ℃ in 50 mM Tris-HCl (pH 7.4), then the reactions were extraction with 500 μl hexane. Three biological replicates were performed.