Creation of adgrl3.1 knockout mutant.

A) PCR approach with location and direction of gRNAs (red arrows), and location and direction of primers (large pink and green arrows), on each exon of adgrl3.1. B) Example of genotyping results showing indel mutations for all individual larvae in exon 6, likely indels in exon 8, and partial excisions between exons 6– 8. For each sample a positive control was done using a primer pair in an unrelated genomic location (tenm4 gene). H2O, negative water control; L, DNA ladder; WT, DNA from uninjected wild-type animals.

Details of Exons across the CRISPant mutants.

Experimental design of the adgrl3.1 knockout mutant.

A) Schematic of the experimental workflow. F0 CRISPant-generated adgrl3.1 mutants are created at the one cell stage and raised in a petri dish before being assayed at 7 dpf within the closed-loop virtual reality assay. B) Diagram of the closed-loop visual stimulation system. Individual larvae are tracked in real time within the arena (12cm diameter), with visual gratings updated and projected such that stimuli remained aligned to the animal’s body axis. C) Optomotor response (OMR) stimulus paradigm. Each trial consisted of a 5 second baseline (black screen) followed by 25 seconds of drifting gratings moving either leftward or rightward. Gratings were presented at two contrast levels (0.25 and 0.75 AU) and across multiple spatial frequencies (stripe widths), allowing assessment of contrast- and frequency-dependent visuomotor responses.

Increased locomotor output in adgrl3.1 mutants is driven by altered bout structure.

A-F) Various swim behaviours measured across stripe widths and contrast conditions (Low contrast at 0.25 AU and high contrast at 0.75 AU) for WT (pink and purple colours) and adgrl3.1 mutants (light and dark blue colours). A) Bout speed. B) Bout duration. C) Swim rate (bouts per second). D) Interbout interval (IBI). E) Bout length (distance travelled per bout). F) Total distance travelled. G) Total distance travelled at low (0.25) and high (0.75) contrast for adgrl3.1mutant and control larvae. A – G) n = 62 for adgrl3.1mutant larvae, n = 70 for control larvae. All error bars are mean ± s.e.m. over fish. Asterisks indicate significance *P < 0.05, ***P < 0.001, A-F) 2-way ANOVAs with multiple comparisons and corrected with Tukey test; G) two-tailed t-test with Welch’s correction.

Reduced behavioural variability and subtle vigilance deficits are present in adgrl3.1 mutants.

A) Coefficient of variation (CV) for interbout interval (IBI) and average speed across stimulus conditions to indicate flexibility in motor output. B) Within-trial slope of successful optomotor responses over stimulus conditions. Negative slopes indicate declining performance (vigilance decrement). C) Within-trial delta (Δ) in response success between early and late trial phases across stimulus conditions. Larger positive deltas indicate decline in performance over trials. C) Baseline locomotor behaviour during the pre-stimulus (black screen) period. A – D) n = 62 for adgrl3.1mutant larvae, n = 70 for control larvae. All error bars are mean ± s.e.m. over fish. Asterisks indicate significance *P < 0.05, ***P < 0.001, A-C) 2-way ANOVAs with multiple comparisons and corrected with Tukey test; D) two-tailed t-test with Welch’s correction.