High variability in aggression and habituation to the mirror assay in ornamental Siamese fighting fish Betta splendens

Understanding consistent inter-individual variability in animal behaviour, known as personality traits, is essential for exploring the mechanisms and evolutionary consequences of behavioural diversity. Aggressive behaviour influences survival, resource acquisition, and reproduction, so clarifying individual differences can enhance our understanding of ecological dynamics and improve experimental design accuracy in behavioural studies. In this study, ornamental male Betta splendens, a model organism for aggression research, were analysed for intra- and inter-individual variability in aggressive responses to their mirror image—a standard method for assessing aggression in fish—once per week, and their consistency was evaluated over three consecutive weeks There were significant differences in aggressive behaviour across individuals, with coefficients of variation ranging from 29 to 60%. While most fish exhibited the full suite of aggressive displays, some showed no aggressive behaviour, while others only displayed threat behaviours but did not advance to the attacks. The consistency of individual threat and attack behaviours varied, but repeatability was high overall (intra-class correlation coefficients ≥ 0.5), indicating that individual fish have different levels of aggression. There was habituation to the mirror assay, with aggression decreasing significantly by the second week, though the degree of habituation, a form of learning, varied among individuals in some behaviours. Air-breathing frequency correlated positively with aggression behaviours and can be considered an indicator to infer aggression level in this species. These results indicate that inter-individual variation in aggressive behaviour and habituation to repeated testing using the mirror assay should be considered in aggression studies using B. splendens and potentially in other species.