Temperature is a major environmental factor influencing fish behaviour and physiological function. In the present study, adult zebrafish were exposed to 18 °C for 21 days and compared with controls maintained at 26 °C to evaluate the behavioural effects of chronic low-temperature exposure. Behaviour was assessed using four individual-based paradigms—the novel tank diving test, light–dark preference test, social preference test, and mirror biting test—covering locomotion, exploration, social behaviour and anxiety-related responses. Chronic exposure to 18 °C was associated with a behavioural profile characterised by increased immobility, reduced exploratory activity, enhanced bottom-dwelling, and reduced active engagement with the mirror stimulus, whereas social preference towards conspecifics was largely preserved. Comparison with behavioural responses previously reported after 4 days of exposure to 18 °C suggests that several behavioural differences are still detectable after 21 days, indicating that the behavioural profile of fish maintained at 18 °C remained distinct from that of controls within the time window examined. These findings should be interpreted in light of the absence of independent tank-level replication. Previous studies conducted under comparable thermal conditions have reported molecular changes in the brain and in other neural-derived structures, such as the eye. Although these molecular findings were not generated in the present experiment and cannot be directly linked to individual behavioural endpoints, they provide a broader context for interpreting the behavioural effects of prolonged low-temperature exposure. Overall, the present findings show that chronic exposure to 18 °C is associated with persistent differences in locomotor, exploratory, and stimulus-directed behaviours, while social preference is comparatively preserved.

Brain-derived neurotrophic factor (BDNF) is a key regulator of neural development, plasticity, and behaviour. Recent work has enabled the generation of a viable adult bdnf^−/− zebrafish line, which provides a unique opportunity to investigate how complete loss of bdnf affects social behaviour. Here, we examined three-dimensional shoaling behaviour in adult male and female AB wild-type and bdnf^−/− knock-out zebrafish to determine whether the extensive molecular and behavioural alterations previously observed in individual-based assays extend to collective contexts. The bdnf^−/− shoals showed no differences in group structure, as inter-fish distance, shoal volume, shoal area, distance to the centroid, and homogeneity index were comparable to wild-type groups. Vertical spatial distribution was also largely preserved, although bdnf^−/− fish shifted toward the upper regions of the tank earlier during the trial. By contrast, horizontal distribution revealed a clear genotype effect: bdnf^−/− shoals spent more time in peripheral regions and displayed a pronounced early peak in peripheral occupancy. These findings indicate that bdnf loss does not impair shoal formation or cohesion but selectively modulates specific components of spatial exploration. The results also highlight a dissociation between the vertical and horizontal axes of behaviour, as well as between individual- and group-based phenotypes, underscoring the importance of social context in shaping the behavioural consequences of BDNF deficiency.