Page 53 - 《水产学报》2025年第7期

陈庆宇，等 水产学报, 2025, 49(7): 079104

Functional characteristics of zebrafish (Danio rerio) chemical alarm signals
and their effects on behavior

2
1,2
2
CHEN Qingyu , LIN Tingting , LIU Xin , LI Siping 2*
1. Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China;
2. East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China

Abstract: Chemical alarm signals (CAS), released from fish skin upon predator-induced physical damage to provoke fright
behavior, play 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-concentra-
tion CAS exposure remain poorly understood. Using Danio rerio as a model, this study investigated the functional characterist-
ics of CAS and its long-term effects of different concentrations and treatment duration on fish anxiety and exploration behavi-
ors through a series of behavioral assays (fright reaction, novel tank diving, light-dark, and novel object tests). PAS staining res-
ults 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 reveal 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 concen-
tration-dependent CAS effects on fish behavior but also provides a behavioral framework for conditioning hatchery fish prior to
wild release.
Key words: Danio rerio; chemical alarm signals; fright reaction behavior; behaviorial test
Corresponding author: LI Siping. E-mail: lisiping@ecsf.ac.cn
Funding projects: Central Public-Interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences
(2023TD56)

中国水产学会主办 sponsored by China Society of Fisheries https://www.china-fishery.cn
13