Dissolved oxygen (DO) concentrations in water bodies fluctuate significantly due to global warming, seasonal variations, aquatic plant photosynthesis, water body eutrophication, and aquaculture densities (Breitburg and Levin, 2018; Klaus et al., 2021; Qiu et al., 2024). After reaching a certain water depth, the DO concentration decreases with an increase in vertical height and sedimentation of decomposed organic matter (Zhai et al., 2012; Xie et al., 2022). The DO concentration is an important water quality indicator and an important factor for maintaining life activities of fish. The higher the DO concentration in a water body, the more beneficial it is to the activities of aquatic animals (Jeong et al., 2024). Oxygen deficiencies in nearshore coastal areas are increasing globally, causing irreversible negative effects on the fish farming industry (Diaz and Rosenberg, 2008; Domenici et al., 2017).

DO is not only directly related to fish habitat distribution and environmental homeostasis but also affects fish population structure (Wannamaker and Rice, 2000), behavior (Zhang et al., 2010), growth (He et al., 2022), migration (Boussinet et al., 2024), reproduction (Saha et al., 2022), and disease resistance (Evans et al., 2003). Prolonged hypoxia can lead to fish mortality (Abdel-Tawwab et al., 2019). Normal cells obtain energy through aerobic respiration; however, when the oxygen supply is insufficient, the respiratory mode shifts to anaerobic respiration, which consumes glucose and produces large amounts of lactic acid metabolites (Cuninghame et al., 2017). To adapt to low oxygen stress, fish enhance their anaerobic metabolic patterns to replenish the energy required for life activities (Sun et al., 2021).

Fish often activate various pathways to regulate homeostasis, and in addition to regulating energy metabolism patterns, apoptosis is also activated, which helps fish maintain relative homeostasis under hypoxic stress conditions (Qiang et al., 2020). Reactive oxygen species (ROS), which are small molecules with high activity, have irreversible effects on intracellular proteins, lipids, and DNA. Under hypoxic conditions, accumulated ROS may not be eliminated rapidly, leading to severe cellular damage and enhancing the autophagy signal of organic cells (Scherz-Shouval and Elazar, 2011; Yang et al., 2018). Genomic analyses have shown that continuous hypoxia induces signaling pathways associated with cytoprotection, immune regulation, and energy metabolism in fish (Wang et al., 2023). This indicates that hypoxia can significantly affect the physiological and immune responses of fish, making them more susceptible to diseases (Abdel-Tawwab et al., 2019). Previous studies have found that the hypoxia-inducible factor (HIF) signaling pathway is a key pathway for maintaining oxygen homeostasis in tissues and cells under hypoxia, with the hypoxia-inducible factor (HIF-1α) being a central regulatory gene in this pathway (Bayele et al., 2007; Palazon et al., 2014). A study on Megalobrama amblycephala showed that hypoxia may induce apoptosis by enhancing the HIF pathway and hence activating downstream pathways (Yu et al., 2023). Studies on the function of the HIF-1α gene revealed its involvement in immune regulation of Siniperca chuatsi against diseases (He et al., 2019). Hypoxic stress directly causes apoptosis and alterations in immune function by activating the HIF pathway (Guo and Tan, 2019).

Epinephelus coioides is an economically important farmed fish in Southeast Asia and a major container-farmed fish in China, with advantages of appealing taste and rich nutritional value, making it popular among consumers (Yu et al., 2018). However, with popularization of high-density container aquaculture models, the E. coioides aquaculture industry is facing the problem of low DO concentrations in water bodies (Wen et al., 2016). To address the hypoxia in water bodies caused by high stocking density and high nutrient input, and to elucidate the hypoxia regulation mechanisms of E. coioides, we conducted the study with E. coioides as the research subject. In the study, we examined enzyme activities and gene expressions in serum and the liver to evaluate the changes in energy metabolism, immune response, and apoptosis under low-oxygen conditions [DO: 1.2 ± 0.1 mg/L (hypoxia)] and normal conditions [6.0 ± 0.1 mg/L (normoxia)], providing a theoretical basis for the selection and breeding of new hypoxia-tolerant E. coioides varieties.

Oxygen-sensing signaling pathways enhance tolerance to hypoxia by activating transcription or repressing translation. Fish tended to respond to the stimulation of low DO by enhancing immune defense mechanisms, a finding consistent with studies on Pseudobagrus ussuriensis (Liu et al., 2024) and Larimichthys crocea (Wang et al., 2017). Recently, enormous economic losses have occurred due to the frequent occurrence of hypoxia in marine environments (Sobocinski et al., 2018). Therefore, in this study, we investigated the effects of hypoxic stress on energy metabolism, immune responses, and apoptosis in orange-spotted groupers.

Activity of AST, ALT, and AKP enzymes in extracellular fluid or serum is a sensitive indicator of liver function, and when elevated, it is suggestive of minor cellular damage or stressful tissue injury. They are also key indicators of the nutritional and health status of animals (Palanivelu et al., 2005; Akbary et al., 2018). ALT and AST are positively correlated with the lysis of hepatocytes and generally accepted as relevant stress indicators as biomarkers for the diagnosis of liver function damage (Dassarma et al., 2018). When animals’ liver was damaged and hepatic cells are broken, ALT and AST in hepatic cells would be elevated in the blood (Sheikhzadeh et al., 2011). Activities of AST, ALT, and AKP are susceptible to hypoxia (Li et al., 2022). Hypoxia induces increased AST and ALT activities in Sebastes schlegelii (Jia et al., 2023). As shown in Figure 2 , hypoxic stress resulted in high AST levels in serum, which has also been reported in Hyphessobrycon callistus (Pan et al., 2010), suggesting that AST is more sensitive to hypoxic stress. AKP plays a protective role in physiological functions and immune defense, and elevated AKP activity in serum indicates impaired cell membrane permeability and integrity, leading to cellular damage and host hepatobiliary inflammation (Lin et al., 2015). Consequently, fish function could not be restored to the same level as that of the control group after DO restoration, which was the reason for the high AKP expression in serum at H48 and R24, suggesting that the liver had already suffered a more severe inflammatory injury at this time. The increased AKP activity, which is used as an indicator of immune status, also showed modulation of fish immunity by hypoxia (Ren et al., 2022). These results suggested that E. coioides activated an immune response to hypoxic stress.

Enzyme expression in serum suggested that hypoxia led to a significant immune response in the fish, which was confirmed by the expression of inflammatory factor mRNAs. Interferons (IFNs) and interleukins (ILs) are important factors in the immune system; IFNγ is a type of excreted protein that induces antiviral activity and is involved in apoptosis and cellular immunity regulation (Yang et al., 2017), and IL-8 acts as a chemokine that primarily mediates activity of neutrophils, T-lymphocytes, and basophils in vitro (Whyte, 2007). In zebrafish, hypoxia regulates the migration of neutrophils to the injured site to play an inflammatory response by upregulating the expression of IL8. The long-term effects of hypoxia were regulated by increasing the phosphorylation of ERK (He et al., 2022). In our experiments, IFNγ showed a peak from H0−H6 ( Figure 3A ), and IL-8 showed high expression from H3−H24 ( Figure 3B ), suggesting that the reduction in DO exacerbated the inflammatory response, which is similar to experimental results in Danio rerio (He et al., 2022) and Oreochromis niloticus (Dawood et al., 2021). Unlike IFNγ and IL-8 expression, the MyD88 gene not only showed high expression at H3 and H6 but also showed increased expression after reoxygenation ( Figure 3C ). NF-κB can be activated by MyD88 as a downstream molecule, and we found that NF-κB was significantly expressed throughout the hypoxia phase ( Figure 3D ). NF-κB is a protein complex that controls transcription of many genes and is involved in cellular responses to stimuli such as external environmental stresses, ROS, and cytokines (Moniruzzaman et al., 2018). Notably, NF-κB can induce transcriptional expression of HIF-1α, which is involved in the function of innate immunity and inflammatory response in hypoxic environments (Rius et al., 2008). Our previous studies also found that hypoxia-induced HIF-1α expression (Wu et al., 2023). This result indicated that hypoxia induces the expression of NF-κB, promotes the expression of HIF-1α, and thereby regulates hypoxia stress. Together, these results indicated that hypoxia activated the immune response by upregulating cytokines.

Results from previous transcriptomic analyses of fish livers have shown that hypoxic stress alters the energy metabolic pattern of oxidative phosphorylation in fish, which tends to enhance glycolysis/gluconeogenesis and pyruvate metabolism pathways (Qi et al., 2018; Ding et al., 2020; Lai et al., 2022). During anaerobic glycolysis, sugars are first broken down into glucose, which is then catalyzed by the enzyme LDH to catalyze the primary product (i.e., pyruvate) into lactic acid and produce ATP (Mizock, 1995). The gene HIF-1α, which is closely related to hypoxia, serves as an important factor in glucose metabolism that also activates a variety of enzymes required for glycolysis (e.g., LDA) under hypoxic conditions (Yang et al., 2019). Higher blood glucose levels during hypoxic exposure suggested that glucose metabolism played an important role in energy supply under hypoxic stress ( Figure 4A ). The high LDH expression in serum also showed that anaerobic glycolysis pattern was continued until DO recovery ( Figure 4B ). These results implied that the energy supply under hypoxic conditions may have involved the anaerobic glycolysis pathway.

Under hypoxia, major energy-demanding processes (e.g., protein synthesis) shut down rapidly before expression of proteins required for ATP production is enhanced, indicating that hypoxia triggers metabolic inhibition (Catz and Johnson, 2001). We found that TP in the liver decreased significantly after H6 and increased significantly at R24 ( Figure 5A ). It has been suggested that the total amount of tissue functional proteins in the liver may be affected by hypoxic stress at these two time points (Gracey and Somero, 2001). Because a 6-h period is a critical time for metabolic changes and cellular imbalance, protein inactivation and damage may have occurred during this time, or energy expenditure may have been reduced by shutting down protein synthesis pathways, which was also shown in Trachinotus ovatus (Wang and Wu, 2024). Hepatic glycogen is an important component of energy storage systems. However, the results of the present study showed that hepatic glycogen levels were significantly reduced at H12 ( Figure 5B ). We hypothesize that hepatic glycogen was degraded into glucose to maintain life activities. From H24 until R24, the hepatic glycogen content increased as the fish continued to enhance anaerobic glycolysis for energy supply. This increase was a regulation of energy storage that occurred in fish in response to prolonged hypoxia, and promotion of fish recovery, which was also observed in Carassius auratus (Mandic et al., 2008). Hepatic glycogen degradation is also inhibited in hypoxia-tolerant Trachinotus blochii under hypoxic conditions (Resende and Mauro Carneiro Pereira, 2022). Eirocheir sinensis showed increased liver glycogen levels after 24 h of hypoxic stress (Chen et al., 2023). HK and PK (i.e., the key enzymes of glycolysis) showed high expression during short periods of hypoxic stress ( Figures 5C, D ), which was also observed in Pelteobagrus fulvidraco (Wang et al., 2022). The low HK expression in liver at H24, in conjunction with the glycolytic pathway, suggested that HK acted as the “inlet” of glucose into glycolysis and PK acted as the “outlet” (Ng et al., 2022). The decrease in HK expression implied a tightening of the inlet, and PK expression did not increase. Considering that the aerobic glycolytic pathway was weakened at this point, anaerobic glycolysis becomes the dominant mode of metabolism. These results further suggested that carbohydrates were stored after hypoxic stress to provide energy substrates for different tissues.

Monocarboxylate transporters (MCTs) can help move lactate across cell membranes and thus redistribute carbohydrates (Ngan and Wang, 2009). In the present study, MCT2 expression appeared high during both hypoxic stress and recovery phases, which was related to its involvement in regulation of both aerobic and anaerobic metabolism ( Figure 6A ). GLUT1 (i.e., a anaerobic metabolism marker gene) is a downstream target gene of HIF-1α, and GLUT1 also shows a high expression pattern in Micropterus salmoides (Yang et al., 2017) and Rachycentron canadum (Wang et al., 2019) under hypoxia ( Figure 6B ). The upregulation of GLUT-1 exposed to acute hypoxia effectively increased glucose uptake and maintained energy supplementation in hypoxic conditions (Yang et al., 2017). From H3−H24 in the present study, LDHA expression in liver increased ( Figure 6C ), suggesting that anaerobic glycolysis functioned in liver at this stage, which is similar to the results in M. salmoides (Yang et al., 2019). The enzyme activities and genes discussed above suggested that the hypoxia-induced metabolic pattern shifted from aerobic to anaerobic.

With prolonged hypoxia treatment, the apoptotic index increased, and the expression of apoptotic genes (p53, Bax, Bcl-2, and Caspase-9) was closely related to the degree of tissue necrosis. In black rockfish, hypoxia stress impaired hepatic antioxidant capacity, induced oxidative damage and apoptosis via the p53-bax-bcl2 and the caspase-dependent pathways, but enhanced host immunity by regulating AST and ALT activity and related gene expression to maintain homeostasis (Jia et al., 2023). Hypoxia significantly increased apoptosis in the liver of Pelteobagrus vachelli (Zheng et al., 2021). p53 is associated with the cell cycle and can activate downstream genes to bind to the cell cycle protein–CDK complex and inhibit protein kinase activity, thereby blocking normal cell cycle progression (Imamura et al., 2001; Jones et al., 2005). Hypoxia-inducible factors and p53 regulate each other, and the transcription of these two genes demonstrates the competition law. Initially, with the increasing degree of hypoxia, p53 gradually tended to be enriched from an unstable form and occupied a key position in determining cell fate; however, it was degraded again when sustained high expression of HIF-1α occurred, which is consistent with the experimental process when p53 appeared to have low expression at the first experimental node and was subsequently sustained and elevated ( Figure 7A ). Furthermore, p53 that is fully activated by HIF-1α simultaneously activates the downstream gene Bax, which jointly promotes cell apoptosis (Wang et al., 2019). Bax and Bcl-2 belong to the Bcl-2 family of proteins that are involved in apoptosis regulation. Bcl-2 has antiapoptotic effects and inhibits most cell death types (Hockenbery et al., 1993); however, Bax is pro-apoptotic and a major inhibitor of Bcl-2 (Jin et al., 2011). The Bcl-2/Bax ratio increased under hypoxic stress and reached twofold at H6 until decreasing at R24 ( Figures 7B, C ). When the Bcl-2/Bax ratio decreased, fish exhibited a dominant role of Bax, clearing damaged cells in vivo, as also reported for T. blochii (Gu et al., 2023). When exposed to short-term hypoxia, cells upregulate Bcl-2 expression to inhibit apoptosis; whereas, prolonged hypoxia leads to downregulation of Bcl-2 expression to accelerate the apoptotic process (Shroff et al., 2007). A change in the Bcl-2/Bax ratio reflects the antagonistic effect of the two proteins, which contributes to enhanced apoptosis under environmental stress and, in turn, protects cells from excessive death. In addition, Caspase-9 acts as a downstream gene, causing endoplasmic reticulum stress and mitochondrial damage to activate the Cas signaling pathway and induce apoptosis (Liu et al., 2023). Our results showed that Caspase-9 expression significantly increased after hypoxia treatment, suggesting that hypoxia activated the Cas signaling pathway ( Figure 7D ). Three apoptosis-related genes (i.e., Bax, Bcl-2, and Caspase-9) were upregulated under hypoxia, suggesting that the body activates the Bax-Bcl2 and Cas pathways to promote cells to enter the apoptotic program to adapt to hypoxic stress. Furthermore, the transcriptional expression of Bcl-2 is regulated by NF-κB, which can activate the Bcl-2 promoter to promote gene expression (Catz and Johnson, 2001), which is consistent with the expression pattern of these genes in the present study. This suggested that apoptosis and innate immune responses acted simultaneously under hypoxia to maintain homeostasis.

The results of the present study indicated that environmental hypoxia, followed by reoxygenation, had serious effects on the liver of E. coioides. Hypoxia affected the activities of glycolytic enzymes and immunoenzymes in serum and the liver and induced fluctuating expression of genes involved in energy metabolism, inflammatory response, and cellular regulation in the liver. To adapt to a low-oxygen environment, E. coioides used anaerobic glycolysis as an important metabolic energy supply pathway and activated the immune response and apoptosis to maintain homeostasis.