Usage of DrosoMating and CAD Parameters of the Chamber

(A) Typing input speed and input board numbers before selecting analyzing area (upper). Schematic representation of the region selection process (lower). Columns should be selected in a clockwise direction starting from the top left corner.. (B) Selection of reference flies for tracking accuracy. Any three reference flies were chosen for the analysis. (C) Use the X-axis, Y-axis, and threshold regulator to adjust the selection area to fit each well. Click “Enter” to run the program. Output file will be in the video folder: [Video Name]_output_[board numbers].csv (D) CAD exploded 3D view of the chamber instruction. (E) CAD parametric data of the chamber (dimensions in mm). (F) 3D diagram of the completed chamber

Comparison of the Accuracy of DrosoMating with Manual Scoring

(A) Copulation latency (CL) of Canton-S males was analyzed using DrosoMating and compared with manual recordings. DBMs represent the ‘difference between means’ for the evaluation of estimation statistics. Asterisks represent significant differences, as revealed by the Student’s t test (* p<0.05, ** p<0.01, *** p<0.001). See the Background for a detailed description of the CL assays used in this study. (B) Courtship index (CI) of Canton-S was analyzed by DrosoMating and manual recording. CI was measured by [courtship time/ (mating time-courtship time) *100%]. For detailed methods, see the BACKGROUND for a detailed description of the CI assays used in this study. (C) Mating Duration (MD) of Canton-S was analyzed by DrosoMating and manual recording. See the BACKGROUND for a detailed description of the MD assays used in this study.

Behavioral assays of Drosophila males under different rearing conditions.

(A) CL of Canton-S, w1118, y1, and Oregon-R males. (B) CI of Canton-S, w1118, y1, and Oregon-R males. (C) MD of Canton-S, w1118, y1, and Oregon-R males. For detailed methods, see the background for a detailed description of the mating assays used in this study. (D) CL of group-housed and single-reared Canton-S males. (E) CI of group-housed and single-reared Canton-S males. (F) LMD assays of Canton-S males. In the MD assays, white data points denote males that were group-reared (or sexually naïve), whereas blue data points signify males that were singly reared. The dot plots represent the MD of each male fly. The mean value and standard error are labeled within the dot plot. (G) CL of naive and sexually experienced Canton-S males. (H) CI of naive and sexually experienced Canton-S males. (I) SMD assays of Canton-S males. White data points represent sexually-naïve males and pink data points represent sexually-experienced ones.

behavioral assays of learning in Drosophila which combined with temperature-dependent temporal activation/inhibition methods.

(A) Learning Index (LI) calculation and experimental design for temperature-dependent activation/inhibition of neural circuits in adult Drosophila. This figure illustrates the experimental paradigm for calculating the LI using CI measurements from sham and trained group. The protocol involves rearing flies at 20°C for four days followed by a one-day exposure to 29°C to activate/inhibit temperature-dependent, genetically-encoded neuronal modulators (for example, shits as inhibitor or TrpA1 as activator). (B) CI of training and sham training MB-247-GAL4/UAS-GFP, MB-247-GAL4/UAS-TrpA1 and MB-247-GAL4/UAS-shits flies. (C) CI of different training days (D1: 1 day, D2: 2 days, D3: 3 days) and sham trained Canton-S flies. (D) CI of different training days (D1: 1day, D2: 2 days, D3: 3days) and sham training orbΔ/orbΔQ flies. (E) LI of Canton-S and orbΔ/orbΔQ males. (F) CI of training and sham training rut2080; UAS-rut, rut2080; GAL4ok107, rut2080; GAL4ok107/UAS-rut flies. (G) LI of rut2080; UAS-rut, rut2080; GAL4ok107, rut2080; GAL4ok107/UAS-rut flies.

Setup of mating assay chamber apparatus and architecture of workstation.

(A) Disassembly of the chamber apparatus. Each chamber comprises a roof, two activity areas, and a floor integrated with four pillars. (B) Workstation for the assemble mating assay chambers. Essential required equipments for chamber assembly are shown: a stereomicroscope with an integrated light source, carbon dioxide anesthesia apparatus, a soft brush with feather on the opposite end, chamber apparatus, and fly containing vials. (C) 3D schematic of the mating chamber assembly. Assembly from bottom to top as shown. Briefly, first assemble the floor with one activity area 2 for placing females, then cover the females and separate activity area 1 from activity area 2 using the sex-separation slider. Next, position males and secure the chambers with paper tape. (D) The sex-separation slider is positioned between activity area 1 and activity area 2. (E) The mating chamber assembled with paper tape; the sex-separation slider should be marked with a pen to prevent forgetting to remove it. (F) 3D schematic of single unit of chamber, can hold up to 36 pairs of fruit flies simultaneously. (G) Multiple chambers are set up with four chambers vertically aligned on an LED backlight, allowing the accommodation of 144 pairs of fruit flies across all chambers. (H) Video recording setup. Position a 4K/1080p resolution camera or a smartphone with matching video capabilities above the chambers, turn on the LED backlight, and remove sex-separation slider before recording. (I) Setup for recording mating behavior using a camera mounted on a stand, positioned vertically above chambers arranged on a size-matched LED backlight to ensure uniform illumination. The camera is aligned to capture the entire chamber array, enabling simultaneous recording of mating interactions under controlled lighting conditions.

Schematic representation and behavioral assays of LMD and SMD in Drosophila which combined with temperature-dependent temporal.

(A) Schematic representation of LMD and SMD assays timeline when flies were crossed with heat-sensitive Drosophila cation channel TrpA1 and shits. (B) MD assays of flies expressing the MB247-GAL4 driver together with UAS-GFP. (C) MD assays of flies expressing the MB247-GAL4 driver together with UAS-shits. (D) Schematic representation of LMD and SMD assays timeline when tub-GAL80ts; UAS-KCNJ2/UAS-NaChBac/UAS-TNT or RNAi flies are crossed with specific GAL4 driver. (E) MD assays of flies expressing the MB247-GAL4 driver together with tub-GAL80ts, UAS-mCherry. (F) MD assays of flies expressing the MB247-GAL4 driver together with tub-GAL80ts, UAS-KCNJ2.

Usage of Linux version of DrosoMating.

(A) Typing input speed and input board numbers before selecting analyzing area (upper). Schematic representation of the region selection process (lower). Columns should be selected in a clockwise direction starting from the top left corner.. (B) Selection of reference flies for tracking accuracy. Any three reference flies were chosen for the analysis. (C) The DrosoMating home interface. In brief, “Open videos” to import the recorded mating video. “Run analysis” to initiate the analysis process. The progress bar indicates the status of the analysis. Export raw data by clicking “Export”.