Abstract: 【Aim】 The study aims to establish a suitable olfactory behavior assay system for small insects such as Drosophila melanogaster based on trajectory tracking in four-quadrant olfactometer by analyzing the olfactory behavior of D. melanogaster under different experimental conditions. 【Methods】 The movement trajectory of D. melanogaster in the four-quadrant olfactometer was tracked, and the residence time and moving speed within each field arena was analyzed using Ethovision XT software. Meanwhile, the effects of airflow velocity and ordor placement on the attraction of apple vinegar and the repellence of Eucalyptus globulus leaf oil to D. melanogaster were investigated. 【Results】 The airflow velocity suitable for olfactory behavior analysis of D. melanogaster was from 300 to 1 200 mL/min, and 300 mL/min airflow was recommended as the optimal airflow velocity at which the moving speed of D. melanogaster individuals was more uniform. Ordor placement had no effect on the olfactory behavior of D. melanogaster, but had some impact on the attraction index of ordor. Single ordor showed better efficiency. Meanwhile, the efficiency of single individual test was better than that of multiple individual test. 【Conclusion】 The olfactory behavior assay system based on trajectory tracking developed in this study can be used for screening and identifying effective components in semiochemical compounds of insects. Better efficiency can be obtained by testing each insect individually with a suitable airflow velocity and single ordor. Multiple individual test could be used in the primary screening of compounds.

Key words: Drosophila melanogaster, olfactory behavior, airflow velocity, ordor placement, residence time, attraction index, trajectory tracking, fourquadrant olfactometer