Thinking outside the boxes: analyzing the current landscape of popular behavioral tests for adult zebrafish

lab animal Comment https://doi.org/10.1038/s41684-026-01694-w Thinking outside the boxes: analyzing the current landscape of popular behavioral tests for adult zebrafish Allan V. Kalueff, Adam Michael Stewart, Murilo S. de Abreu & Matthew O. Parker Zebrafish are commonly tested in various behavioral assays. To better understand such practices, we surveyed active global zebrafish neuroscience labs, asking to rank such assays based on their perceived utility. Overreliance on few well-established assays and their inconsistent nomenclature call for further innovation and standardizing of adult zebrafish neurobehavioral testing. Zebrafish (Danio rerio) have rapidly become a widely used model organism in biomedical research and drug discovery, owing to their high genetic and physiological homology with humans1. Their translational utility is especially evident in behavioral neuroscience2, with the number of Pubmed items on zebrafish brain and behavior doubling in the past decade. Zebrafish are increasingly utilized to model brain disorders3 and study complex behaviors4,5. However, this research depends critically on providing appropriate testing conditions6, accurate identification of relevant behaviors7,8, the use of validated protocols9, high resolution of behavioral tracking10, robust data interpretation11 and reproducibility of findings across and within laboratories12,13. There has been a recent growing effort to optimize zebrafish neurobehavioral research, including testing different housing environments and procedures14, as well as behavioral endpoints7. However, these assays themselves represent a critical bottleneck. It is therefore timely to assess the current landscape of adult zebrafish behavioral assays and to identify the limitations and opportunities for its advancement. Current landscape To better understand practices and stimulate discussion around behavioral assay use, the International Zebrafish Neuroscience Consortium (ZNRC) has conducted a targeted survey, which was distributed in June 2025 to nearly 50 active zebrafish laboratories worldwide, both members and non-members. The survey posed a single question, asking laboratories “to list and rank their top 10 adult zebrafish behavioral assays by a general utility for the field”, with a specific request to focus on individual behavioral tests (e.g., novel tank, mirror test), rather than broader constructs (e.g., anxiety/fear) or contexts (e.g., predator exposure). The results were collected 4 months later and analyzed, scoring the frequencies of mentioning each test (and its respective rank, from 1 to 10), expressed as % of total number of responders for each rank. Overall, twenty active laboratories responded, offering a snapshot of commonly used assays and highlighting both expected trends and less conventional choices (Table 1). Nearly 80% responders identified Lab Animal | Volume 55 | March 2026 | 65–68 Check for updates the novel tank test as the most useful behavioral assay in adult zebrafish, with another 16% listing it as the second most useful test. The open field test was ranked second by 35%, followed by the light-dark test (11%). Other assays frequently appearing in the top five include the T/Y-maze and the shoaling tests (16% each). The shoaling test was listed as the fourth most useful by 22% responders, followed by the T/Y-maze and the social preference test (11% each). The fifth position in the ranking was shared by the mirror exposure test, the light-dark box test and the T/Y-maze (11% each). Cumulative analyses of these and other tests are summarized in Table 1. Other tests listed among top 5 by the responders included the plus maze test, conditioned aversion test and the zebrafish tail immobilization (ZTI) test. Tests ranked 6-10, based on their utility, included the social preference and the novel object tests (33% each), the T/Y-maze and mirror test (28% each), the shoaling test and the open field test (22% each), the ZTI test and conditioned place preference (CPP) (16% each), as well as conditioned place aversion test and dyadic aggression test (6% each). Less frequently used tests, mostly ranked as 6-10 in the list, included the inhibitory avoidance test (4 labs), the plus-maze test (2 labs) and the 5-choice serial testing test (1 lab). Three unclear entries involved aggression display, aggression and swimming test. The predator exposure test was mentioned twice and was excluded due to its ‘context’ nature (i.e., listed as an experimental manipulation in a well-established test rather than a test on its own). Overall, this informal audit reveals widespread reliance on a small core of broadly applicable tests, alongside more domain-specific assays used by subsets of laboratories. Given the field’s heavy reliance on novelty-based behavioral tests (Table 1), this also raises important questions about the breadth, diversity and conceptual foundations of behavioral tools currently in use across the field. Potential implications These survey results (Table 1) suggest that the field is dominated by a few widely used, general-purpose behavioral assays (novel tank test > open field, light-dark box > T/Y-maze and shoaling test), and several domain-specific assays. This likely reflects their broad applicability, relative ease of use, and sensitivity to pharmacological, genetic and environmental manipulations. However, their ubiquity also highlights a lack of diversity in behavioral paradigms, which may constrain the range of the central nervous system (CNS) domains explored in zebrafish research. Notably, the novel tank test stands out as the clear front-runner. Its popularity is understandable, as the test offers a rapid, single-trial measure of locomotion and anxiety-like behavior and is highly sensitive to a wide array of experimental manipulations14,15. However, its broad scope (capturing activity, neophobia, freezing, exploration and even social behavior) complicates interpretation. Furthermore, its multidimensionality may also hinder reproducibility across labs, given the many intrinsic (e.g., personality, sociality and stress responsiveness) and extrinsic (e.g., lighting, tank geometry and water characteristics) factors that influence 65 lab animal Comment Table 1 | Top most useful adult zebrafish behavioral assays ranked by active zebrafish laboratories responding to the survey Behavioral assay Test specifics Main targeted domains Number of responders The novel tank test Assesses the natural tendency for protective bottom swimming (geotaxis), initially avoiding a more aversive, top area of an unfamiliar tank. Over time, fish gradually habituate and begin to explore the top areas of the tank. Locomotion and noveltyevoked anxiety 17 The open field test Evaluates spontaneous locomotor activity and protective peripheral (thigmotaxis) vs. more aversive ‘central’ swimming in a novel arena. Locomotion and noveltyevoked anxiety 10 The light-dark box test Examines the (...truncated)