1：Experimental Design and Method Selection:

Used chemical (odorants) and optogenetic (CsChrimson activation with red LED light) methods to activate ORNs. Employed a larval tracking assay to measure behavioral responses, including navigational parameters like run length, run speed, number of runs per trajectory, and curve rating. Principal component analysis and hierarchical cluster analysis were used for data interpretation.

2：Sample Selection and Data Sources:

Drosophila melanogaster larvae (Canton-S wild-type and UAS-IVS-CsChrimson crossed with OrX-Gal4 lines) were used. Third-instar larvae (96-120 hours after egg laying) were selected for experiments. Data were collected from video recordings of larval movements.

3：List of Experimental Equipment and Materials:

Equipment includes CCD camera (Basler Ace series), IR LEDs (850 nm), red LED light strips (630 nm), Raspberry Pi microprocessor, Jaz spectrometer (Ocean Optics), power supply (Rigol DP832), and software (MATLAB, R, Statistica). Materials include odorants (Sigma-Aldrich), paraffin oil, agarose, all-trans-retinal (ATR), sucrose, dimethyl sulfoxide (DMSO), and filter papers (GE-Whatman).

4：Experimental Procedures and Operational Workflow:

Larvae were pre-exposed to odorants or optogenetically stimulated. For optogenetics, larvae expressing CsChrimson in specific ORNs were exposed to red light at different frequencies (0.04 Hz, 1 Hz, constant). Larval movements were tracked in a 22-cm x 22-cm arena, and data were processed using custom MATLAB routines.

5：04 Hz, 1 Hz, constant). Larval movements were tracked in a 22-cm x 22-cm arena, and data were processed using custom MATLAB routines. Data Analysis Methods:

5. Data Analysis Methods: Statistical analyses included Kolmogorov-Smirnoff test, Kruskal-Wallis multiple comparison test, and Student's t-test. Data were normalized and analyzed using PCA and hierarchical clustering in R.