4.9. Y-Tube Olfactometer Bioassay

The olfactory responses of female A. gifuensis to wheat plants and the plant volatile collections were detected in a Y-tube olfactometer (diameter, 1.5 cm; common tube length, 20.0 cm; length of each arm, 15.0 cm, and both arms were extended at 75°). Wheat plants were put into two custom-made glass chambers (1.0 L capacity), each contained 20 wheat plants. For the responses to volatile collections, an aliquot (10 μL) of each test solution was applied to a filter paper strip (2 × 1 cm) and the solvent was allowed to evaporate (1 min) before inserting the strip into an odor-source glass bottle connected to one arm of the olfactometer. Three treatments (24 h after treated with 1 mmol/L MeSA, 0.5 mmol/L MeSA and control) were compared pairwise. Each group was tested with 30 female adults and individuals with no responses were discarded.

According to GC-EAD and EAG assays, three compounds p-Cymene, Carveol, and m-diethyl-benzene were selected to identify their functional significance for A. gifuensis attraction. Chemicals were prepared in different concentrations using n-hexane as control (0.01, 0.1, 1, and 10 μg/μL). An aliquot (10 μL) of each test solution was applied to a filter paper strip (2 × 1 cm) and the solvent was allowed to evaporate (1 min) before inserting the strip into an odor-source glass bottle connected to one arm of the olfactometer. The control glass bottle connected to the other arm of the olfactometer contained a filter paper strip treated with 10 μL of hexane. Each test of the chemical required 20 female adults and individuals with no responses were discarded.

Airflow through the arms was 200 mL/min. To avoid interference from other light sources, the Y-tube olfactometer was held and placed inside a dark environment. Illumination was provided by a fluorescent bulb (ca. 100 lx). If an A. gifuensis adult crossed half the arm within 5 min, it was considered as having made a choice. Insects which did not make a choice within 5 min were considered as nonresponding [38]. After testing two insects, the two arms should be switched to avoid positional bias [42]. After testing five insects, the olfactometer was cleaned with anhydrous ethanol, rinsed with deionized water and dried in an oven.