Ontogeny of zebrafish behaviors: comparative evaluation of locomotor, social and anxiety parameters in larval, juvenile and adult stages

lab animal Article https://doi.org/10.1038/s41684-026-01712-x Ontogeny of zebrafish behaviors: comparative evaluation of locomotor, social and anxiety parameters in larval, juvenile and adult stages Check for updates Barbara D. Petersen1,2, Gabriel Rodrigues2, Kirya Liriel    2, Lana Ferreira2 & Carla D. Bonan    1,2,3 Zebrafish are a prominent model for investigating behavior and development. However, most behavioral studies have primarily focused on larval and adult stages. The juvenile stage—a critical period of neural and behavioral maturation—has been insufficiently explored, partly because standardized methods for evaluating behavior in different ages are not available. Here we present a behavioral platform adapted for cross-stage assessments and provide an initial characterization of juvenile zebrafish behavior. Locomotion, anxiety, social interaction and scototaxis were evaluated. Our findings reveal developmental stage-specific differences in juvenile zebrafish locomotion, such as increased mobility and reduced erratic movements, along with a steady and progressive increase in social preference from late larval to the adult stage. Scototaxis reversal was also found to be one of the first major behavior transitions, occurring early in the larval stage. These results establish juveniles as a transitional phase with distinct phenotypes and support previous hypotheses that a behavioral metamorphosis accompanies morphological changes of juveniles in these species. Furthermore, this study establishes a foundation for longitudinal behavioral analyses, with the standardization of tests enabling further studies on neurodevelopmental disorders, pharmacological interventions and behavioral ontogeny. Zebrafish are a vertebrate model species, disseminated in laboratories from the 1970s onward1 and extensively used in a variety of research fields, such as genetics, pharmacology, developmental biology and neuroscience2–5. As an animal model, zebrafish are an excellent alternative for early developmental studies due to their short embryogenesis6, the ability to evaluate behavior as early as 17 h post-fertilization7, their capacity to absorb water-soluble compounds8,9 and transparent embryos that allow in vivo phenotype observations10. Adult zebrafish maintain some of these characteristics, such as the possibility to expose them to compounds directly in the tank water, while also displaying a variety of behavioral phenotypes11 and functional homology in neuronal circuits compared to mammals12,13. By contrast, other developmental stages of zebrafish, such as the juvenile and elderly stages, remain underrepresented and understudied in the current literature. The juvenile stage, the primary focus of our investigation, corresponds to adolescence in mammals14,15. This is a sensitive period, marked by extensive physical, physiological, cerebral and behavioral changes16. When atypical processes occur during the changes in neural systems underlying cognitive function, social interaction, emotional control, risk–reward balance and motivation during adolescence, psychiatric conditions such as depression, anxiety disorders and psychosis may emerge or worsen. This is particularly relevant when these abnormal changes are associated with an increased propensity of risk-taking, novelty seeking and other behaviors typical of this developmental stage16–21. Nevertheless, the relationship between the changes that occur during adolescence and the development of psychopathologies is complex and not linear. In addition, methodological limitations in studying this issue in mammals remain a substantial challenge16. In this scenario, modeling adolescence in alternative animal models, such as zebrafish, can be useful not only for testing new interventions and unraveling disease mechanisms22,23, but also because comparative studies between species can reveal evolutionary conserved functions, mechanisms and targets, thus facilitating the elucidation of central aspects of diseases24,25. 1 Programa de Pós-Graduação em Medicina e Ciências da Saúde, Escola de Medicina, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil. 2Laboratório de Neuroquímica e Psicofarmacologia, Escola de Ciências da Saúde e da Vida, Porto Alegre, Brazil. 3Programa de Pós-Graduação em Biologia Celular e Molecular, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Brazil. e-mail: Lab Animal | Volume 55 | May 2026 | 172–180 172 https://doi.org/10.1038/s41684-026-01712-x Table 1 | Anatomical parameters for determining life stages Developmental stage Description Early larval stage, 4.5 mm, 7 dpf •Absence of defined rays in the fins Late larval stage, 7.6 mm, 21 dpf •Posterior lobe of the swim bladder inflated •Defined rays and incomplete pigmentation in the anal, caudal and dorsal fins •Anterior lobe of the swim bladder inflated •Absence of defined rays in the pelvic fin •Pelvic fin with defined rays and incomplete coloration Early juvenile stage, 9.8 mm, ~30 dpf •Incomplete resorption of the fold between the pelvic fin and cloaca •Incomplete scaling •Complete pigmentation of the pelvic fin Late juvenile stage, 13 mm, ~45 dpf •Absence of fold between the pelvic fin and cloaca (complete resorption) •Complete scaling •Animals with complete sexual dimorphism Adult stage, 26 mm, >90 dpf •Male: slim body, pinkish coloration •Female: bulging body in the ventral region, silvery coloration The juvenile stage of zebrafish’s life cycle is poorly standardized in literature. First, animals classified as juveniles can be found in the literature ranging from as early as 6 days post-fertilization (dpf) to as late as 70 dpf (refs. 26,27). Other studies suggest the juvenile stage encompasses the period between ~30 dpf and ~90 dpf (refs. 14,28). These discrepancies can be partly explained by the influence of factors, such as temperature, population density, stress levels and others, which can affect developmental rates in this species and delay metamorphosis14. This has led to the proposal of using ecological and anatomical hallmarks as more reliable criteria for determining life stages, rather than relying only on dpf14,29,30. Second, innate behaviors, which directly affect animal survival and serve as a functional reading of neural activity31–33, show noticeable differences between larval and adult zebrafish; however, the timeline of when exactly these changes occur has not yet been elucidated. In this context, the transition from the larval to the juvenile stage has been proposed to involve a behavioral metamorphosis, marked by enhanced reactivity to stimuli34. This hypothesis remains untested, partly because the current methods for evaluating behaviors in zebrafish are not standardized to allow comparisons between life stages35. Therefore, this study aims to develop a behavioral analysis platform that allows direct comparisons between the (...truncated)