Trajectory parameters of real flies and computer-generated data (grouping codes given below the graph)

Both A. Meander (turning angle divided by speed) and B. stripe deviation are similar in fly and computer-generated data. Red line denotes 45°, the mean value for computer-generated data. C–D. Centrophobism score for sitting (C) or for moving (D) is positive only for fly data. E. The distance traveled is different between the three types of data. Bars represent means and error bars standard errors, asterisks denote significant differences after a MANOVA analysis, n = 20 in each group

Transition plots for the different groups of data

The relative frequency of the fly passage at each position is plotted (red denotes a frequency above the 95% quantile value, dark blue means flies were rarely present. White indicates that none of the flies ever transitioned through this position). A. Computer-generated data (here correlated walk, but Lévy-walk transition plot is nearly identical) B. Endogenous locomotion. C. Buridan's paradigm with narrow stripes (11°). D. Buridan's paradigm with wide stripes (20°). n = 20 in each group

Correlation plot for the different groups of data

The order of the variables was set by clustering them for endogenous locomotion data (A, lower left part). Only significant correlations are shown. Each matrix is divided into two halves representing the correlation in different groups as indicated below and on their side. Movements smaller than 0.8 mm were discarded in A and C, but not in B, Tm -“Thresholds for movement”. Positive correlations are represented by green dots, negative correlations by violet dots. The size and color of the dots represent the correlation coefficient, as indicated. A. Correlation matrices of endogenous locomotion (lower left half-matrix) and computer generated correlated walk (upper right half-matrix). B. Same as in A without discarding movements smaller than 0.8 mm C. Correlation matrices of fly data in Buridan's paradigm, with narrow (lower left half) and wide (upper right half) stripes. Highlights in A and B: Small red squares: The number of pauses is not correlated with the total activity time in the real fly data. Elongated red rectangles: The median speed is correlated with the total activity time (TT) in the fly data. Large red squares: The duration of activity bouts (TT but not ST) correlates with the total activity time (ST and TT) in the fly data. Highlights in A and C: Small purple squares: The number of walks is not correlated with stripe deviation. Small red squares in C: The angle deviation and stripe deviation are correlated only in the narrow stripes situation. Elongated purple rectangle in C: The median speed correlates with the duration of activity periods in the narrow stripes situation. n = 20 flies for each group

Inexpensive Hardware for Buridan's paradigm

The fly walks on a 117 mm platform surrounded by a water moat. The arena is homogenously illuminated, while stripes can be positioned on the inside of the arena wall. The fly is filmed from above and each frame is then treated by the tracking software

Reduced state space of fly locomotion behavior after PCA separates the three experimental conditions

A. 2D scatter plot showing each fly as a dot, and the mean and standard error of the factor loadings as bars. Abscissa is the first principal component (PC1). Ordinate is PC2 on the upper panel and PC3 on the lower panel. B. Snapshot of the corresponding 3D representation plot. Each dot corresponds to a fly coordinate, bars are means and standard errors, and the ellipsoids represent the 80% confidence interval (calculated from the covariance using the loadings on the three first principal components)

Fly trajectory metrics for endogenous locomotion (gray bars), Buridan's paradigm with narrow stripes (white bars) or wide stripes (striped pattern)

A. In the presence of visual targets, the fly shows more walks between the stripes than in their absence. B. Median stripe deviation is different in the three groups. Red line denotes the value for random walks. C. Centrophobism during pauses is still present in all three groups. D. Centrophobism while moving is eliminated by narrow stripes. E. Median speed is not significantly affected by visual targets. F. The number of pauses is lower in the wide stripe condition as compared to the two other conditions. Asterisks denote significant differences after a MANOVA analysis. Bars represent means and error bars standard errors, n = 20 in each group

Software schematics

The experimenter enters information (in red) about the fly and the platform (semi-automatically) into the tracker application (BuriTrack). The tracker saves this information along with a time stamp in an XML file. Online analysis of the video leads to the extraction of the position of the fly over time, which is directly saved to the data file. The analysis software (CeTrAn) then reads a text file indicating the path to the XML file and the fly grouping information. It then automatically imports the data, transforms it into an easily workable class of data (ltraj) and performs the analyses following different variables the experimenter can set (in red). As outputs, CeTrAn writes R workspaces (before and after the analysis), a csv file of the computed parameters and pdf files where those metrics are plotted against the group factor

Activity calculations using different computations give similar results for endogenous locomotion and computer-generated data (grouping codes given below the graph)

Pause-activity patterns were determined using either a speed- (left, labeled 1) or a time threshold (right, labeled 2). A. Total activity time represents the time the animal is considered active. A2 was set to be similar in the computer-generated data. B. Duration of activity periods. Inset represents the same calculation as in B2 but considers only activity bouts leading to a displacement of 1 cm or more. C. Duration of the pause periods. D. Number of pauses. Bars represent means and error bars standard errors, n = 20 in each group