cupido/docs/experimental_design.md

Experimental Design: Barrier-Opening Social Interaction Assay

Overview

This document describes the experimental design of a Drosophila behavioral tracking experiment conducted as part of the Cupido project. The experiment uses a barrier-opening assay to measure social interaction patterns between trained and untrained flies.

Date: July 15, 2025 Species: Drosophila melanogaster, Canton-S (CS) wild-type strain

Assay Description

The barrier-opening assay places two flies in each Region of Interest (ROI), separated by a physical barrier. After a configurable delay from the start of recording, the barrier is manually opened, allowing the flies to interact socially. The primary behavioral metric is the distance between the two flies over time following barrier opening, which serves as a proxy for social engagement.

Experimental Groups

• Trained: Flies that received prior social experience before the assay.
• Untrained: Socially naive flies with no prior social experience.

Note

: The exact training protocol (duration, conditions, group sizes) should be documented separately. "Trained" refers to flies with prior social experience; "untrained" refers to socially naive individuals.

Equipment and Recording Parameters

Parameter	Value
Resolution	1920 x 1088 pixels
Frame rate	25 fps
Video codec	H.264
Quality	28q
ROIs per session	6 (each containing a pair of flies)
Tracking output	SQLite databases (one per session)

Machines and Recording Sessions

Three ethoscope machines were used for tracking, with a fourth (Machine 139) having metadata but no tracking data.

Machine	Session Start Time	Barrier Opening (s)	Status
ETHOSCOPE_076	16:03:10	52	OK
ETHOSCOPE_076	16:31:34	25	OK
ETHOSCOPE_145	16:03:27	42	OK
ETHOSCOPE_145	16:31:41	20	OK
ETHOSCOPE_268	16:32:05	75	OK
ETHOSCOPE_139	16:31:52	Not recorded	DATA MISSING

Total sessions: 6 (5 with tracking data, 1 missing)

ROI-to-Group Mapping

Each session contains 6 ROIs. The assignment of trained/untrained groups to ROIs varies across sessions.

Machine	Session	ROI 1	ROI 2	ROI 3	ROI 4	ROI 5	ROI 6
076	16:03:10	Trained	Untrained	Trained	Untrained	Trained	Untrained
076	16:31:34	Trained	Trained	Trained	Untrained	Untrained	Untrained
145	16:03:27	Trained	Trained	Trained	Untrained	Untrained	Untrained
145	16:31:41	Trained	Trained	Trained	Untrained	Untrained	Untrained
268	16:32:05	Untrained	Untrained	Untrained	Trained	Trained	Trained
139	16:31:52	Trained	Trained	Trained	Untrained	Untrained	Untrained

Sample Sizes

Group	ROIs (total)	ROIs (with data)	ROIs (missing)
Trained	18	15	3 (Machine 139)
Untrained	18	15	3 (Machine 139)
Total	36	30	6

Tracking Database Schema

Each recording session produces a SQLite database file containing tables ROI_1 through ROI_6. Each table has the following columns:

Column	Type	Description
id	INTEGER	Row identifier
t	INTEGER	Timestamp in milliseconds from start of recording
x	REAL	Horizontal position in pixels
y	REAL	Vertical position in pixels
w	REAL	Width of detected object (pixels)
h	REAL	Height of detected object (pixels)
phi	REAL	Angle/orientation of detected object
is_inferred	INTEGER	Whether the position was inferred (not directly detected)
has_interacted	INTEGER	Whether an interaction was detected

Known Issues and Data Caveats

1. Machine 139 missing data: Metadata entries exist for ETHOSCOPE_139 (session 16:31:52) in the metadata CSV, but no corresponding tracking database file is present and no barrier opening time was recorded. This accounts for 6 missing ROIs (3 trained, 3 untrained). The cause needs investigation.

2. Time unit mismatch between files: The tracking databases store time (t) in milliseconds, while 2025_07_15_barrier_opening.csv stores barrier opening times in seconds. The analysis pipeline converts barrier opening times to milliseconds for alignment.

3. Machine name type inconsistency: The metadata CSV stores machine identifiers as integers (e.g., 76, 145, 268), while 2025_07_15_barrier_opening.csv also stores them as integers (e.g., 076, 145, 268). String conversion with zero-padding is required when matching between files and when constructing tracking database filenames (e.g., ETHOSCOPE_076).

Source Files

File	Description
2025_07_15_metadata_fixed.csv	ROI-to-group mapping (trained/untrained)
2025_07_15_barrier_opening.csv	Barrier opening times per machine/session
*_tracking.db	SQLite tracking databases (one per session)