Effects of Environmental Temperature Variation on the Spatio-Temporal Shoaling Behaviour of Adult Zebrafish (<i>Danio rerio</i>): A Two- and Three-Dimensional Analysis

Global warming is driving significant changes in aquatic ecosystems, where temperature fluctuations influence biological processes across multiple levels of organisation. As ectothermic organisms, fish are particularly susceptible, with even minor thermal shifts affecting their metabolism, behaviour, and overall fitness. Understanding these responses is essential for evaluating the ecological and evolutionary consequences of climate change. This study investigates the effects of acute (4-day) and chronic (21-day) exposure to three temperature regimes—18 °C (low), 26 °C (control), and 34 °C (high)—on the spatio-temporal shoaling behaviour of adult zebrafish (<i>Danio rerio</i>). Groups of four fish were tested for six minutes in water maintained at the same temperature as their prior acclimation. Shoaling behaviour was assessed by analysing shoal structure—encompassing shoal dimensions and cohesion—as well as spatial positioning. Parameters measured included inter-fish distance, shoal volume, shoal area, homogeneity index, distance to the centroid, and the shoal’s vertical and horizontal distribution. Results revealed complex behavioural changes influenced by both temperature and duration of exposure. At 18 °C, zebrafish showed a marked preference for the bottom zone and exhibited no significant temporal modulation in exploratory behaviour—patterns indicative of heightened anxiety-like responses. In contrast, exposure to 34 °C resulted in increased shoal cohesion, particularly under chronic conditions, and a progressive increase in environmental exploration over the six-minute test period. This enhancement in exploratory activity was especially evident when compared to the first minute of the test and was characterised by greater vertical movement—reflected in the increased use of the upper zone—and broader horizontal exploration, including more frequent occupation of peripheral areas. These findings align with previous research linking thermal variation to neurobiological and proteomic alterations in zebrafish. By elucidating how temperature modulates social behaviour in ectotherms, this study offers valuable insights into the potential behavioural impacts of climate change on aquatic ecosystems.