Antioxidant and Metabolic Response to Acute Acidification Stress of Juvenile Yellowfin Tuna (Thunnus albacares)

Abstract

This study aimed to explore the impact of acute acidification on the antioxidant, metabolic performance, and liver histology of juvenile yellowfin tuna. The experiment subjected juvenile yellowfin tuna to a pH gradient environment of 8.1, 7.6, 7.1, and 6.6 for 48 h. The findings indicate that a seawater pH of 7.1 significantly impacts the antioxidant and metabolic systems of the juvenile yellowfin tuna in comparison to the control group. At pH 7.1, there were observed increases in glutathione reductase (GR), total antioxidant capacity (T-AOC), lactate dehydrogenase (LDH), hexokinase (HK), pyruvate kinase (PK), sodium-potassium ATPase (Na⁺K⁺-ATP), and calcium-magnesium ATPase (Ca²⁺Mg²⁺-ATP). Conversely, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were not significantly different across the treatment groups. However, an increase in transaminases at pH 7.1 suggested potential liver damage, which was further supported by observed structural liver tissue degeneration and hepatocyte vacuolation. In conclusion, under conditions of acute acidification stress, there is a decrease in antioxidant capacity and a suppression of metabolic levels in juvenile yellowfin tuna, leading to oxidative damage. This study lays the foundation for an in-depth understanding of the response mechanisms of juvenile yellowfin tuna in response to seawater acidification as well as healthy tuna farming in the broader context of seawater acidification.

1. Introduction

The pH in aquatic environments is one of the factors affecting the physiological and biochemical functions of fish. Deviations from the optimal pH range directly impact fish’s acid–base balance, respiratory function, circulation, and lipid accumulation [1,2,3]. In recent years, there has been a growing concern for the aquatic environment, highlighting the necessity to focus on the resilience and adaptability of marine species in low pH conditions. However, several factors contribute to the instability of seawater pH. For example, an increase in CO₂ concentration leads to a reduction in seawater pH, with predictions suggesting a decrease of 0.7 pH units by the year 2300 [4]. Additionally, the coastal regions of China, being low-lying, are more susceptible to changes in water body pH due to extreme weather conditions such as typhoons and hurricanes. Human activities also have a more severe impact on the water environment, sometimes even causing extreme disturbances [5,6].

Studies have shown that a decrease in environmental pH leads to an increase in levels of reactive oxygen species (ROS). Antioxidant defenses play a crucial role in neutralizing ROS and protecting fish from oxidative damage [7]. Long-term exposure to low pH environments can damage these antioxidant mechanisms, potentially weakening the fish’s ability to effectively manage oxidative stress [8]. Additionally, acidic waters can lead to a decrease in the pH of fish blood, thereby affecting the binding of hemoglobin to oxygen [9,10]. This impairment restricts oxygen transport, leading to hypoxia in fish. Symptoms include reduced activity, significant declines in metabolic function, decreased appetite, digestive issues, and stunted growth [11]. Ding Zhaokun et al. found that a reduction in water body pH not only affects gene expression but also adversely impacts the energy, protein, lipid, and nucleic acid metabolism of marine organisms [12]. Changes in external environmental conditions can also cause shifts in fish osmotic pressure and serum ion levels, making blood parameters a reliable indicator of fish health under environmental stress [13]. Petochi et al. exposed European sea bass (Dicentrarchus labrax) to low pH environments and discovered that their metabolic rates increased as the water’s pH decreased [14]. Md and colleagues exposed juvenile catfish (Hexanematichthys sagor) to a low pH environment and analyzed their blood for aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, and cortisol. The results showed that exposure to a high-acidity environment can damage the health of the fish, potentially leading to poor growth and reduced survival rates [15]. Although adult fish exhibit a strong adaptability to changes in environmental pH, their juveniles lack effective mechanisms for intracellular pH regulation, making them particularly susceptible to early-life damage caused by acidification [16].

A decrease in water pH poses significant risks to marine ecosystems and threatens the survival and reproductive capabilities of marine fish species [17,18]. The yellowfin tuna, known for its high economic value, is an essential global fishery resource. By 2022, the annual global catch of yellowfin tuna surpassed 1.4 million tons, making it the second most extensively fished tuna species after the skipjack tuna [19]. Understanding how yellowfin tuna manages oxidative stress through its antioxidant system becomes particularly important. Studying the antioxidant and metabolic responses helps to better comprehend the adaptation mechanisms of yellowfin tuna to acidic environments, which is crucial for predicting their survival and reproduction in future marine conditions. Despite the significance of these issues, research on how seawater acidification affects physiological changes in yellowfin tuna juveniles is still limited. This study focuses on juvenile yellowfin tuna as a model organism. Based on predictions from ocean models and experimental results from Bromhead et al., it evaluates the impact of different levels of seawater acidity on the antioxidant and metabolic performance, serum indicators, and liver histology of yellowfin tuna [20,21]. The aim is to provide reference data for the stress response of marine fish to seawater acidification.

2. Materials and Methods

2.1. Experimental Materials

The experimental fish were caught in the sea area near the Xincun port (18°22′31.9″ N, 109°58′28.9″ E). These fish were transported to the Lingshui Experimental Center of Sanya Tropical Fisheries Research Institute (Sanya, China) for a week to acclimate and subsequent acute acidification experiment. A total of 72 healthy juvenile yellowfin tuna of similar size (mean length: 18.21 ± 1.09 cm, mean weight: 307.49 ± 49.38 g) were selected and randomly placed into 12 circular fiberglass tanks (5000 L). To manipulate seawater pH levels, solutions of 1.0 mmol·L⁻¹ NaOH and 1.0 mmol·L⁻¹ HCl [22,23] were used, setting a control pH at 8.1 [24] and treatment pH levels at 6.6, 7.1, and 7.6 [25,26,27,28], with three replications of each condition, and six fish were set up for each replication. The acidification stress test lasted 48 h [29,30], during which time feeding was stopped and seawater acidity was measured using a pH analyzer (Shenzhen Jingxin Microelectronics Co., Shenzhen, China), which was used to monitor seawater pH every two hours to maintain fluctuations within ±0.1 pH units. During the temporary rearing and experimental period, 12 h of light and 12 h of darkness were maintained, the water temperature was kept at 22.5 ± 1 °C, dissolved oxygen levels were maintained above 7.5 mg·L⁻¹, nitrite nitrogen concentrations were kept below 0.05 mg·L⁻¹, and ammonia nitrogen concentrations were also maintained below <0.05 mg·L⁻¹.

2.2. Sample Collection and Processing

After 48 h of acidification stress, the fish were anesthetized with eugenol (Shangchi Dental Material Co., Ltd., Changshu, China), six fish were removed from each bucket, weighed on a scale (to the nearest 0.01 cm), measured for body length with calipers (to the nearest 0.01 cm), and dissected according to the method of Rosseland et al. [31]. Blood samples were drawn from the caudal vein of the fish and collected using a 2 mL syringe precoated with 1% sodium heparin. Blood samples were centrifuged in a cryo-centrifuge (XinAoYi Instrument Co., Ltd., Changsha, China) at 3000 r·min⁻¹ for 10 min, and the serum was collected. The liver was quickly removed, rinsed with pre-cooled saline, blotted dry on filter paper, and collected into a 2 mL sterile cryo-centrifuge tube. The serum and the liver samples for enzyme activity determination were then stored at −80 degrees Celsius.

A total of 0.1~0.2 g of liver tissue was accurately weighed, 9 times volume of 0.9% saline was added, it was mechanically homogenized under ice-water bath condition and centrifuged at 3000 r·min⁻¹ for 10 min, and the supernatant was taken to determine the contents of total protein (TP) content, total antioxidant capacity (T-AOC), glutathione reductase (GR), lipid peroxidation (LPO), lactate dehydrogenase (LDH), hexokinase (HK), pyruvate kinase (PK), sodium-potassium ATPase (Na⁺K⁺-ATP), and calcium-magnesium ATPase (Ca²⁺Mg²⁺-ATP). The serum samples were used to determine alkaline phosphatase (AKP), glutamine transaminase (GPT), glutamic oxaloacetic transaminase (GOT), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TGs), total cholesterol (TCH), and glucose (GLU). All analyses were performed using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). TP was detected using the bicinchoninic acid assay (minimum detection limit: 20 μg·mL⁻¹). T-AOC was detected using the ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical cation decolorization assay (minimum detection limit: 0.5 mmol). GR was detected using the glutathione reductase activity assay (minimum detection limit: 0.1 μ·L⁻¹). LPO was detected using the thiobarbituric acid reactive substances assay (minimum detection limit: 0μ·L⁻¹). LDH was detected using the lactate dehydrogenase activity assay (minimum detection limit: 1 μ·L⁻¹). PK was detected using the pyruvate kinase–lactate dehydrogenase-coupled assay (minimum detection limit: 1.3 μ·L⁻¹). HK was detected using the hexokinase-glucose-6-phosphate dehydrogenase-coupled assay (minimum detection limit: 2.3 μ·L⁻¹). Na⁺K⁺-ATP and Ca²⁺Mg²⁺-ATP were detected using the colorimetric method based on inorganic phosphate detection (minimum detection limit: 0.0026 μmol·mL⁻¹). GLU was detected using the glucose oxidase method (minimum detection limit: 2.2 mmol·L⁻¹). LDL-C and HDL-C were detected using the double reagent direct method (minimum detection limit: 0 mmol·L⁻¹). TG was detected using the glycerol-3-phosphate oxidase-peroxidase method (minimum detection limit: 0.2 mmol·L⁻¹). TCH was detected using the cholesterol oxidase-peroxidase method (minimum detection limit: 0 mmol·L⁻¹). GOT and GPT were detected using the Reitman–Frankel method (minimum detection limit: 0 μ·L⁻¹). AKP was detected using the p-nitrophenyl phosphate method (minimum detection limit: 45 μ·L⁻¹). Determination was carried out using a hybrid microplate reader (BioTek Instruments, Winooski, VT, USA) and a UV–visible spectrophotometer (Shanghai Meppan Instruments Co., Ltd., Shanghai, China) in strict accordance with the operating instructions. Each sample was replicated three times, with negative control and positive control set up for each determination.

Tissue sections were prepared with reference to Yancheva et al. [32]. Juvenile fish livers were collected, preserved in 4% paraformaldehyde (BL 539A, Biosharp, Hefei, China), and then embedded in paraffin. These prepared tissues were then sectioned into 4 µm thick cross sections utilizing a Leica rotary microtome (Leica Instruments Shanghai Co., Ltd., Shanghai, China). The sections were stained with hematoxylin and eosin (H&E) and permanently mounted with a neutral resin. Observations were made with a DMI8 fluorescence inverted microscope (Leica, Wetzlar, Germany), photographs were taken at 400× magnification, and pictures were saved.

2.3. Statistical Analyses

Shapiro–Wilk test [33] was used to examine the normal distribution of the data. The homogeneity of variance was assessed using the Levene test. One-way analysis of variance (ANOVA) and Duncan’s test were employed to analyze the significance of differences between the control group and the treatment group in liver and serum parameters. A p-value of less than 0.05 was considered significant, while equal to or above this threshold was considered insignificant. Data were analyzed using SPSS 25.0 software, expressed as mean ± standard deviation (Mean ± SD), and displayed as bar charts using Origin 2022.

3. Results

3.1. Effect of Acute Acidification Stress on Antioxidant Parameters of Yellowfin Tuna Liver

The change in GR activity exhibited a general downward trend with the decrease in pH, reaching its lowest at pH 7.6 (5.05 ± 1.61μ·gprot⁻¹), which is significantly different from the control group (p < 0.05, Figure 1a). Conversely, T-AOC displayed an upward trend with the decrease in pH, peaking at pH 6.6 (0.84 ± 0.19 mmol·gprot⁻¹), though this did not significantly differ from the control (p > 0.05, Figure 1b). The LPO concentration demonstrated an upward trend with the decrease in pH, achieving its highest level at pH 7.6 (0.04 ± 0.01 μ·gprot⁻¹), having a significant departure from the control (p < 0.05, Figure 1c).

3.2. Effects of Acute Acidification Stress on Metabolic Parameters of Yellowfin Tuna in Liver and Trunk Kidney

The activities of LDH, HK, and PK all peaked at pH 7.1 (LDH: 6.12 ± 0.24 μ·gprot⁻¹, HK: 2.26 ± 0.24 μ·gprot⁻¹, and PK: 14.18 ± 0.97 μ·gprot⁻¹, Figure 2a–c). The LDH and PK activities demonstrated an increasing trend, and a significant difference was observed between the treatment groups and control groups at pH 7.1 (p < 0.05, Figure 2a,c). Na⁺K⁺-ATPase activity showed irregular changes with decreasing seawater pH and was highest at pH 7.1 (0.15 ± 0.01 μ·mgprot⁻¹, Figure 2d), and Ca²⁺Mg²⁺-ATP activity was significantly lower than that of the control group in all treatment groups and reached the lowest level at pH 7.6 (0.06 ± 0.00 μ·mgprot⁻¹, Figure 2e), all of which were significantly different from the control group (p < 0.05).

3.3. Effect of Acute Acidification Stress of Yellowfin Tuna on Serum Indices

The GLU levels initially decreased, then increased, peaking at pH 6.6 (11.47 ± 0.6 mmol·L⁻¹), marking a significant difference from the control group (p < 0.05, Figure 3a). The LDL-C content showed an increasing trend and was highest at pH 6.6 (3.95 ± 1.04 mmol·L⁻¹, Figure 3b), and the HDL-C content was highest at pH 7.6 (15.64 ± 3.00 mmol·L⁻¹, Figure 3c) with no significant difference (p > 0.05) compared with the control group. TG levels were highest at pH 7.6 (0.97 ± 0.10 mmol·L⁻¹, Figure 3d) and TCH levels were highest at pH 6.6 (6.30 ± 0.50 nmol·L⁻¹, Figure 3e) with no significant difference (p > 0.05) compared with the control group. The GOT and GPT levels showed an increasing then decreasing pattern, peaking at pH 7.1 (GOT: 63.83 ± 2.30 μ·L⁻¹, GPT: 19.61 ± 4.69 μ·L⁻¹), with only the GOT level showing a significant difference from the control group (p < 0.05, Figure 3f), whereas the GPT level did not (p > 0.05, Figure 3g). The AKP activity displayed a tendency to increase, reaching the maximum at pH 6.6 (1.37 ± 0.08 Kim’s unit·mL⁻¹), significantly differing from the control group (p < 0.05, Figure 3h).

3.4. Effect of Acute Acidification Stress of Yellowfin Tuna on Liver Histology

In the control group and the pH 7.6 treatment group, hepatocytes displayed a homogeneous cytoplasm, distinct cell boundaries, and a compact arrangement, with normal morphology. The distribution of sinusoidal spaces between hepatocytes was normal (Figure 4a,b). In contrast, hepatocytes in the pH 7.1 treatment group were loosely arranged (Figure 4c). pH 6.6-treated hepatocytes had blurred edges, reduced and displaced nuclei, enlarged sinusoidal spaces, and irregular rounded vacuoles, indicating the presence of fat vacuoles (Figure 4d).

4. Discussion

4.1. Effects of Acute Acidification Stress on the Antioxidant Defense System of Yellowfin Tuna

When organisms encounter external stimuli, they often produce a substantial amount of reactive oxygen species (ROS), leading to various oxidative damages like lipid peroxidation and enzyme deactivation. This effect, known as oxidative stress, occurs if these ROS are not efficiently neutralized [34]. Antioxidant enzymes, such as glutathione reductase (GR) and total antioxidant capacity (T-AOC), play a pivotal role in combating oxidative damage by neutralizing excess ROS and superoxide anion radicals (O₂⁻) [34]. In our research, we observed a reduction in GR activity across all experimental groups compared to controls, aligning with findings in Antarctic fishes (Notothenia rossii and Notothenia coriiceps) exposed to sewage [35]. The decline in GR activity in Antarctic fish was not explicitly explained due to the unpredictable nature of antioxidant enzyme responses to complex contaminant mixtures. Nevertheless, our study utilized natural seawater purified through sand filtration, suggesting the reduced GR activity might stem from the antioxidant system’s failure to efficiently clear accumulated ROS, leading to the impairment or breakdown of crucial antioxidant enzymes. T-AOC represents a comprehensive measure of an organism’s overall antioxidant defense, covering both enzymatic and non-enzymatic systems [36]. Studies have shown that fingerling black sea bream (Acanthopagrus schlegelii) exhibit an increase in serum T-AOC levels under ammonia stress [37]. Similarly, our study detected elevated T-AOC levels in the liver, indicating that organisms bolster their antioxidant defenses in response to stressors like oxidative damage. Our findings elucidate the intricate relationship between oxidative stress and antioxidant defense mechanisms in organisms, emphasizing the critical role of a balanced antioxidant system in minimizing oxidative damage and maintaining health under stressful conditions.

The fish oxidative stress system is not sufficient to scavenge all the oxygen radicals, and the excess free radicals attack the polyunsaturated fatty acids in the biofilm, causing lipid peroxidation and the production of lipid peroxidation [38]. Our study observed an uptick in LPO activity across all experimental groups in comparison to the control group, mirroring findings of rainbow trout [39] (Oncorhynchus mykiss) after heat stress in a similar situation. This increase might be due to the accelerated respiratory rates in fish under acidic stress, leading to heightened oxygen consumption and, consequently, an increase in oxidative stress. This results in elevated levels of reactive oxygen radicals and, thereby, an increase in LPO activity. The observed shifts in the rise in lipid peroxidation activity in our study reflect the stress response and oxidative damage in fish subjected to acidic conditions. Further research is essential to shed light on the mechanisms behind these responses and their potential impact on fish health and welfare.

Blood biochemical markers are indicative of an organism’s metabolic status and the health of its organs and tissues under environmental stress [40]. In our investigation, we focused on serum alkaline phosphatase (AKP), acting as a non-specific phosphohydrolase pivotal for protecting against the intrusion of external pathogens [13]. AKP is intricately involved in the transfer and metabolism of phosphate groups, highlighting its critical role among enzymes that regulate metabolism [41]. We observed an increase in AKP activity at a pH of 6.6, mirroring the trend seen in Sagor catfish [14], suggesting that AKP activity may increase with a sudden drop in seawater pH. This observation underscores the potential impact of heightened seawater CO₂ concentrations on the metabolic frameworks of marine fish. Such species appear to navigate environmental shifts by modulating the activity of key metabolic enzymes within their systems, indicating a sophisticated adaptive mechanism to environmental stressors. This adjustment in enzyme activity reflects a broader biological strategy employed by marine organisms to maintain metabolic equilibrium in the face of changing environmental conditions.

GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase) are critical serum enzymes for assessing animal health, primarily found in cellular mitochondria. Their activity levels serve as crucial indicators of hepatocyte integrity [42]. In our study, we monitored the serum activities of GOT and GPT, which are emblematic of liver damage and have their main residency within the mitochondria of cells. We noted a pattern where these enzyme levels initially increased before decreasing, reaching a zenith at pH 7.1. Significantly, GOT levels were observed to be notably higher than those of GPT, suggesting possible mitochondrial impairment within hepatocytes—a phenomenon mirrored in fingerling black sea bream [37], yellowfin sea bream (Acanthopagrus latus), and Asian sea bass (Lates calcarifer) [43] under ammonia nitrogen stress. This pattern suggests that heightened acidity precipitates liver damage in fish, triggering an acute release of these transaminases into the serum. Tuna, when exposed to acidic conditions, appear to mitigate this effect by downregulating transaminase synthesis, thus diminishing the levels of serum transaminases. This dynamic reflects a complex biological response to environmental stressors, underscoring the delicate balance organisms maintain to preserve physiological homeostasis under adverse conditions.

The liver, an indispensable organ for metabolic processes in fish, is charged with the crucial functions of nutrient metabolism, detoxification of substances, and the breakdown of medications and toxins. However, when there is a discord between the rate of hepatocyte synthesis and their entry into systemic circulation, it can lead to hepatocyte vacuolar degeneration [44]. This condition was notably observed in juvenile blunt snout bream [45] (Megalobrama amblycephala) under acute heat stress as well as in tilapia [46] (Oreochromis niloticus) subjected to cyfluthrin exposure, highlighting a pronounced pathological response of fish livers to acute stress from environmental factors.

In our study, the worsening of liver tissue structure with the increase in seawater pH, characterized by hepatocyte reduction, vacuolation, and nuclear displacement, underscores the escalating adverse effects of seawater acidification on juvenile yellowfin tuna. This is corroborated by alterations in enzyme activities such as GOT and GPT. We propose that the osmotic pressure imbalance in fish within a highly acidic environment (pH 7.1), coupled with the heightened demand for nutrient uptake to sustain osmotic equilibrium and an increased liver burden, contributes to these liver tissue changes. Nonetheless, vacuolar degeneration represents a reversible form of injury, prompting a need for additional research to determine the liver’s capacity to recuperate post-stress alleviation. This insight into the liver’s adaptive and pathological responses to environmental stresses provides a crucial understanding of fish physiology and health, offering a foundation for further investigations into recovery mechanisms and resilience strategies in marine life.

4.2. Effects of Acute Acidification Stress on Metabolic Function of Yellowfin Tuna

The observed decrease in lactate dehydrogenase (LDH) activity in our study might signal a diminished anaerobic capacity and a reduction in tissue glycolysis [47]. Notably, juvenile yellowfin tuna showed an increasing trend in LDH activity with decreasing environmental pH. This pattern is consistent with findings in Mediterranean sea bream [3] (Sparus aurata) and Sagor catfish [15] juveniles and large yellow croaker [48] (Larimichthys crocea), pointing to the possibility that the environmental pH may drop below the optimal range for juvenile tuna, leading to less movement and, consequently, lower rates of oxygen consumption. This reduction in oxygen could prompt a shift from aerobic to anaerobic respiration, as evidenced by the increased LDH activity. LDH plays a crucial role in anaerobic respiration, catalyzing the conversion of lactate to pyruvate and vice versa, depending on oxygen availability. Thus, our findings imply that changes in environmental pH may interfere with the aerobic metabolism of juvenile tuna, triggering an adaptive increase in anaerobic respiration as reflected by the LDH activity. Further investigations are essential to comprehend the full impact of these metabolic adjustments on the health and viability of juvenile tuna under evolving environmental conditions.

Hexokinase (HK) and pyruvate kinase (PK) are critical enzymes in the glycolytic pathway, essential for regulating blood glucose levels [49]. In our study, we found that HK activity tended to increase and then decrease. However, similar to Mediterranean sea bream [3], which exhibited heightened PK activity under environmental stress, our study observed comparable changes. Furthermore, in the Pacific oyster [50] (Crassostrea gigas), both HK and PK activities were found to increase in response to environmental stress, suggesting that variations between species could account for these differences. These findings imply that juvenile tuna may experience shifts in energy distribution patterns, necessitating additional energy to cope with environmental alterations. Yet, the specific mechanisms that regulate these changes remain to be thoroughly investigated and clarified. While HK and PK play fundamental roles in glycolysis and energy metabolism, the distinct responses seen in juvenile tuna under varying environmental conditions highlight the intricacies of metabolic regulation. Further research is crucial to decipher the mechanisms behind these adaptations and their potential effects on the health and sustainability of juvenile tuna.

Na⁺K⁺-ATP and Ca²⁺Mg²⁺-ATP play vital roles as key transmembrane proteins in osmoregulation [51]. The functioning of these proteins often serves as a gauge for assessing the health, metabolic efficiency, and osmoregulatory capabilities of the organism. Notably, the ionic activity in Atlantic salmon smolts [52] (Salmo salar) declines with a decrease in seawater pH. Similarly, juvenile European sea bass [53] exhibit an increase in Na⁺K⁺-ATP activity in low-salt conditions, possibly as an adaptive response to mitigate ionic losses due to reduced salinity. In our research, a reduction in the activities of Na⁺K⁺-ATP and Ca²⁺Mg²⁺-ATP was observed, potentially due to acidification stress affecting the cell membrane’s permeability and, consequently, impairing osmoregulation in fish. These observations indicate that alterations in the activities of Na⁺K⁺-ATP and Ca²⁺Mg²⁺-ATP under acidic conditions could signify disruptions in osmoregulatory processes, underscoring the impact of environmental stressors on fish physiology. Further investigation is necessary to fully understand the mechanisms behind these changes and their implications for fish health and their ability to adapt to shifting aquatic environments.

Sugars, serving as the primary energy substrates, are metabolized into proteins and fats, with triglycerides (TGs) playing a pivotal role in cellular structures as the main form of energy storage [40]. Cholesterol, a precursor for steroid hormones and cellular membranes, is essential for maintaining cell membrane integrity. Its functionality hinges on the balance between low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), where HDL-C facilitates the removal of cholesterol from tissues to the liver for degradation, thus preserving cholesterol equilibrium [54]; conversely, LDL-C distributes cholesterol from the liver to the body’s tissues [55]. Mediterranean gilthead sea bream exposed to temperature stress exhibited a marked increase in blood glucose (GLU) on the fifth day, with TG levels remaining unchanged from the control [56]. In our study, a trend of decreasing GLU levels was noted, with a significant rise at pH 6.6. However, total cholesterol (TCH) and HDL-C remained unaffected. The heightened acidity in seawater may trigger a stress response in fish, leading to the mobilization of hepatic glucose into GLU to fulfill increased metabolic demands and maintain fat equilibrium. The maintenance of TCH levels in tuna, despite acidification stress, could reflect a timely adaptive modulation of LDL-C and HDL-C concentrations. The changes in this column illustrate that under stressful conditions, the body prioritizes rapidly accessible energy sources over those that are energy dense, such as lipids. Initially, the body uses glucose, which mobilizes and utilizes energy more quickly, thus preserving lipid reserves in more severe stressful situations [57]. Nonetheless, significant differences in LDL-C between pH 7.6 and 6.6 and TG levels between pH 7.6 and 7.1 suggest that oxidative stress leads to liver metabolic disturbances under acidic conditions. Our observations underscore the intricate metabolic adjustments in tuna when faced with acidic environments, highlighting shifts in glucose metabolism and lipid regulation. Further studies are required to uncover the precise mechanisms behind these adaptations and their consequences for the health and environmental adaptability of fish.

5. Conclusions

In conclusion, juvenile yellowfin tuna exhibited stress responses to acute acidification, showcasing significant differences in various biomarkers. The key impacts observed were a reduction in antioxidant enzyme activity and a downturn in metabolic function. When faced with highly acidic seawater conditions (pH 6.6), the juvenile yellowfin tuna adapted by utilizing glucose to sustain their metabolism and ensure lipid stability, showing that lipids were the least affected. Moreover, liver analyses revealed hepatocytopenia and vacuolar degeneration, conditions that appeared to intensify with an increase in pH levels. These findings underscore the importance of studying the physiological responses of marine organisms to acidification, providing valuable insights into their adaptive mechanisms and highlighting the potential ecological consequences of seawater acidification on tuna populations. This study provides an experimental model to understand the effects of reduced seawater pH on antioxidant, metabolic function, and organizational structure in tuna. This is critical for the development of predictive models of marine biodiversity as changes in the aquatic environment intensify.