Fig. 2

a Schematic of the automated behavior tracking system for 96-well and 24-well chambers. Mutants are derived from heterozygous intercrosses and all animals are randomized and individually placed in each well without prior knowledge of their genotypes. See recording of animals in 96-well (Supplementary Movie 1) and in 24-well (Supplementary Movie 2) housings. b Representative time plot of locomotor tracking in 96-well chamber. 14-h light cycle and 10-h dark cycle (14 h L: 10 h D) was applied. Light turns on at 7 am and turns off at 9 pm each day, and the “light off” event triggers the expected hyper visuomotor response (sudden rapid increase in swimming activity, arrow). nlrc3l mutants (red graph) consistently show a significant average decrease in spontaneous swimming. c Calculations of different daytime locomotor metrics from three independent behavior tracking sessions. One of these sessions is represented in time plot in b. d Representative swimming traces of a day and a night timepoint from 24-well tracking. Genotypes were determined after completion of experiment and added back to the traces. Traces show typical inter-individual variation not readily associated with any genotype. Activity is highly suppressed at night. Teal traces mark large movements (≥0.5 cm/s) and magenta traces mark small movement (<0.5 cm/s). e–h Behavior tracking using the large-arena 24-well platform and associated quantifications from two independent behavior tracking sessions. See associated Supplementary Movie 3. i Diagram illustrating locomotor pattern analysis based on a 3 × 3 grid system. Traces of swimming trajectories were recorded at every 10-minute interval over the entire 72-hour tracking. Traces corresponding to d2 and d3 timepoints were scored. See Methods for details on the calculations. j, k Heatmaps combined with hierarchical clustering of perimeter and center scores do not show any segregation of fish based on genotype, indicating similar patterns of locomotion between nlrc3l mutants and their siblings. c, e–h Number in parenthesis is n, number of individual animals analyzed. Scatter box-and-whisker plots show minimum and maximum; each data point represents an individual animal. Daytime locomotion is measured by the total swimming distance per 10-min interval. Daytime activity is number of bouts of swimming detected per 10-min interval. All metrics were averaged over d2 and d3 timepoints per fish. d2, 14-h light period on day 2; d3, 14-h light period on day 3; d4, partial light period on day 4. Scale bar, 6 h (hours). ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; all p-values are FDR-adjusted. Statistical significance was calculated using multiple comparisons test after one-way ANOVA test, or the Brown-Forsythe and Welch ANOVA test for groups with unequal standard deviations.