State or personality trait: Determinants of boldness and shoal size preference in adult zebrafish

Animals adjust their behavior in response to physiological states to optimize the trade-off between energy acquisition and survival. A key question in animal personality research is whether behavioral variation is driven by consistent physiological state differences or stable personality traits. This study evaluates boldness and shoal size discrimination in adult male zebrafish ( Danio rerio ) across three hunger states: same-day feeding (H0), 24-hour food deprivation (H1), and 48-hour food deprivation (H2). Behavioral assessments were conducted over two testing cycles, with each individual undergoing every test twice for each hunger state within each cycle. The assays comprised the Open Field Test (OFT), Emergence Test (ET), and Social Preference Test (SPT), with the SPT conducted under binary (4 vs. 2 fish) and ternary (4 vs. 2 vs. 1 fish) choice conditions. Hunger significantly reduced emergence latency, but did not affect exploratory behavior in the OFT. Hunger also enhanced preference for larger shoals above chance in the binary SPT during the second testing cycle, though this effect was absent in ternary choices. Testing cycle effects revealed habituation-driven increases in boldness in both the tests and a progressive shift toward larger shoal preferences in the binary SPT. Among all assays, only the OFT showed significant repeatability, making it a reliable measure of boldness. However, it did not show consistent individual differences in plasticity due to hunger. These results highlight the crucial role of labile physiological states, such as hunger and habituation, in driving behavioral variation and underscore the importance of designing studies that disentangle state-dependent behaviors from stable personality traits in animal personality research.