Immunological and behavioral biomarkers of Vibrio vulnificus exposure in zebrafish: Implications for aquaculture biosecurity and pathogen surveillance.

Aquatic ecosystems face increasing threats from microbial pathogens, making the early detection of sublethal stress in aquatic organisms critical for environmental and aquaculture biosecurity. In this study, we examined the behavioral and immune responses of zebrafish (Danio rerio) larvae exposed to Vibrio vulnificus, aiming to develop integrated biomarkers for pathogen surveillance and ecological health assessment. Larvae were exposed to varying concentrations of V. vulnificus, with a comprehensive evaluation of behavioral, neurochemical, oxidative, and immune biomarkers. Behavioral analysis revealed significant, dose-dependent disruptions in movement, angular velocity, and turning behavior, especially under dark conditions, indicating compromised sensorimotor function. Neurochemical assays showed reduced acetylcholinesterase (AChE) activity and elevated cortisol levels, reflecting impaired cholinergic signaling and activation of the hypothalamic-pituitary-interrenal (HPI) axis. Concurrently, infected larvae exhibited increased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), along with decreased antioxidant enzyme activity (SOD, CAT, GPX1), indicating oxidative stress. Immune profiling revealed elevated proinflammatory cytokines (IL-1β, IL-6, TNF, TLR1) and suppressed IL-10, suggesting a disrupted inflammatory response. Correlation analysis revealed strong associations between behavioral changes and physiological stress markers, highlighting a systemic interaction between immune, neuroendocrine, and oxidative responses. These findings position zebrafish larvae as a sensitive model for pathogen detection and underscore the utility of multi-endpoint frameworks for pathogen-inclusive environmental risk assessments. This approach has significant implications for aquaculture biosecurity, pathogen monitoring, and the development of early-warning systems for aquatic ecosystem health.