Zebrafish (Danio rerio) Prefer Undisturbed Shoals over Shoals Exposed to the Synthetic Alarm Substance Hypoxanthine-3N-oxide (C5H4N4O2).

As an anti-predation behavior, shoaling enhances survival among prey species by reducing individual predation risk through mechanisms like the dilution effect and collective vigilance. Zebrafish-a highly social and genetically tractable species-are valuable for studying these behaviors. The present study examined zebrafish's social preferences in a 3-chamber open-tank free-swim task, assessing whether visual cues alone could distinguish between an intact and an alarmed shoal exposed to the synthetic alarm substance H3NO. Subjects were allowed to freely associate with either shoal while their behaviors were recorded and analyzed. The results reveal a significant preference for proximity to the intact shoal, indicating zebrafish's ability to visually discern threat levels. Subjects spent nearly twice as much time in the zone near the intact shoal, with reduced freezing and faster movement velocities compared to the alarmed shoal zone. Males exhibited more freezing behavior than females, consistent with sex-specific strategies in threat response. These findings underscore zebrafish's reliance on visual cues for social responding under predatory threat and highlight sex-based differences in threat perception. This research expands the understanding of zebrafish's social dynamics and provides a robust framework for future exploration of the neural mechanisms underlying social behavior and threat assessment in zebrafish.