| Considering global climate change, elevated water temperatures have become a significant environmental stressor impacting aquatic organisms. In fish, heat stress can induce metabolic disturbances, thereby impairing growth and survival. The black rockfish (Sebastes schlegelii), a key marine species cultivated in the Yellow Sea and Bohai Sea regions, is valued for its rapid growth, high-quality flesh, and robust resistance to diseases and cold environments. Its primary aquaculture practices include deep-water net cage farming and the use of engineered fencing systems. Currently, stress responses of black rockfish to various factors, including high flow velocities, ocean acidification, hypoxia, low salinity, and acute heat stress, have been studied both nationally and internationally. However, climate change in recent years has led to prolonged periods of high ambient temperatures in China during summer, causing a sustained increase in coastal water temperatures. Consequently, black rockfish mortality rates are elevated during these high-temperature periods, presenting a significant challenge to the sustainable development of the aquaculture industry. Persistent high temperature stress can induce heat stress in fish. The liver, as a core metabolic organ, is particularly sensitive to environmental stressors and plays an important role in regulating the body's homeostasis. Therefore, this study focuses on liver tissue and aims to explore the changes in liver injury and antioxidant enzyme activity under gradient thermal stress, clarify the changes in liver heat shock proteins and key apoptosis-related genes, to provide valuable data and theoretical basis for the healthy breeding and heat tolerance research of black rockfish.
This study focuses on the black rockfish as the research object. A total of 108 fish were used in the experiment, with an average total length of (20.63 ± 0.72) cm and an average body weight of (117.96 ± 10.93) g. Before the experiment, they were temporarily raised for one week, during which the water flow rate was maintained at more than 1000 mL/min, the water temperature was measured at 20 ± 0.5 ℃, the salinity was measured at 30 ± 1 ‰, the dissolved oxygen was greater than 7.8 ± 0.5 mg/L, the pH was 7.6 ± 0.5, and the ammonia nitrogen was less than 0.1 mg/L. The gradient thermal stress experiment was set up in 6 groups, named Control, 23 ℃, 26 ℃, 29 ℃, R1d, R3d, respectively. Each group is equipped with 3 parallel tanks, with 6 fish in each experimental tank(80L). The temperature of the control group was 20 ± 0.5 ℃. Except for the control group, all five experimental groups gradually increased their temperature from 20 ℃. The water temperature was raised by 1 ℃ every 12 hours using a quartz heating rod, and samples were taken at 23 ℃, 26 ℃, and 29 ℃ for 12 hours, respectively; The recovery group was maintained at 29 ℃ for 12 hours, then the quartz heating rod was turned off and the water flow was restored. The water temperature was slowly restored to 20 ℃ through the flow of water, and samples were taken at 1 day and 3 days of recovery, respectively. During the experiment, the temperature changes of the water were monitored and recorded, and the normal dissolved oxygen content in the water was maintained.
The experimental results showed that under gradient thermal stress, the liver of black rockfish underwent significant damage, such as cellular vacuolization and nuclear migration. The increase in temperature leads to cumulative damage to liver tissue, with the most severe damage occurring at 29 ℃. The content of malondialdehyde and the activities of alanine aminotransferase and aspartate aminotransferase significantly increased (P<0.05), reaching their maximum values at 29 ℃ with increasing temperature, which were 26.6 nmol/mg prot, 67.6 U/g prot, and 59.6 U/g prot, respectively. Oxidative damage to the liver induces an increase in antioxidant enzyme activity, with significant increases in superoxide dismutase, catalase, and glutathione peroxidase activities (P<0.05), reaching their maximum values at 29 ℃, which were 178.7 U/mg prot, 3.8 U/mg prot, and 22 U/mg prot, respectively. With the increase of temperature, the expression of liver heat shock protein genes (hsp70a and hsp90a) was significantly up-regulated (P<0.05) to maintain cellular protein stability and repair in the black rockfish. At the same time, gradient thermal stress activated the up-regulation of pro-apoptotic genes (p53, bax, caspase3) expression (P<0.05), while the down-regulation of anti-apoptotic gene (bcl-2) expression (P<0.05), leading to liver cell apoptosis. Tunel staining of liver tissue revealed a significant increase in cell apoptosis rate with increasing temperature (P<0.05).
In summary, under gradient thermal stress, the liver of the black rockfish suffered severe damage and experienced oxidative stress. To resist high temperature stress, the antioxidant enzyme activity in the liver of black rockfish is enhanced to alleviate oxidative damage. Up-regulation of heat shock protein genes (hsp70a and hsp90a) helps repair damaged proteins and maintain cellular environmental stability. However, under high temperature stress, the liver inevitably undergoes cell apoptosis, intensifying with increasing temperature. After the water temperature was restored, the expression of liver damage, oxidative stress, heat shock protein genes, and apoptosis-related genes gradually recovered, indicating that the black rockfish has a certain degree of self-regulation and recovery ability. These results contribute to a deeper understanding of the physiological response mechanism of the liver in black rockfish under high temperature stress, and provide a theoretical basis and data support for sustainable and healthy aquaculture management of black rockfish. |
