Functional characterization of chemical alarm signals and their dose-dependent behavioral effects in zebrafish (Danio rerio)

Chemical alarm signals (CAS), released from fish skin upon predator-induced physical damage to provoke fright behavior, plays a critical role in enhancing individual survival in natural environments and is a potential tool for behavioral domestication of hatchery-reared fish prior to restocking. However, the behavioral impacts of prolonged and high-concentration CAS exposure remain poorly understood. Using Danio rerio as a model, this study investigated the functional characteristics of CAS and its long-term effects of different concentrations and treatment duration on fish anxiety and exploration behaviors through a series of behavioral assays (fright reaction, novel tank diving, light-dark, and novel object tests). PAS staining results revealed that D. rerio alarm substance cells are localized in the epidermal middle layer with colorless cytoplasm and blue-purple nuclei. In the fright reaction behavior test, skin extract (5.5 mg/mL of stock concentration) significantly increased the duration of initial freezing (244 and 0 s, respectively), total freezing time (244 and 0 s, respectively), and bottom-dwelling (300 and 144 s, respectively) compared to the distilled water control (P<0.05). Dilution experiments demonstrated that 10-fold, 30-fold, and 50-fold dilutions (0.11-0.55 mg/mL) retained CAS efficacy, whereas 100-fold dilution as 0.055 mg/mL concentration markedly reduced freezing and bottom-dwelling behaviors (P<0.05), as no significance compared to distilled water control (P>0.05). CAS stored at 25 °C remained effective for less than 4 h (P>0.05), with significant behavioral attenuation after 6-8 h (P<0.05). The experimental results of long-term continuous stimulation (10 and 20 d) with different dilution (10-fold, 50-fold dilutions, and stock) factors in D. rerio demonstrated that prolonged stimulation duration significantly enhanced anxiety-like behaviors as the latency for entering the top area gradually increased (P<0.05). Moreover, higher concentrations of skin extract (e.g. 5.5 mg/mL stimulation for 20 d) induced greater reductions in exploratory behavior and more pronounced anxiety-like responses in D. rerio (P<0.05). This results reveals that while utilizing the behavioral response characteristics of CAS for stock enhancement behavior conditioning, the long-term effects of high concentrations on fish anxiety and exploratory behaviors must also be considered to avoid counterproductive outcomes. Overall, this study not only advances understanding of concentration-dependent CAS effects on fish behavior but also provides a behavioral framework for conditioning hatchery fish prior to wild release.