Size-Dependent Salinity Tolerance and Osmotic Regulation in Juvenile Lateolabrax japonicus

Abstract

Salinity is one of the most critical environmental factors for fish, influencing their reproduction, growth, and physiological and metabolic activities. Lateolabrax japonicus is a major commercially important marine fish that has been widely cultured in China. However, there are few reports on the growth tolerance of fish juveniles of different species under various salinity conditions. In this study, we investigated the effects of acute low-salt stress on the survival, plasma osmolality, blood ion concentration, and Na⁺/K⁺-ATPase (NKA) activity in L. japonicus juveniles of two size groups. Our findings revealed no significant difference in survival rates between 5 cm and 10 cm juveniles at salinities of 0.2, 1, 3, 5, 10, 15, and 25. Plasma osmolality and blood ions exhibited a “decrease-increase-stabilization” pattern; 5 cm juveniles stabilized at 6 h at all salinities, whereas 10 cm juveniles required 48 h to stabilize at a salinity of 1, with isosmotic points of 10.91 and 11.00, respectively. Gill and kidney NKA activity followed an “increase-decrease-stabilization” pattern, with 5 cm juveniles achieving stability 12–24 h earlier than 10 cm individuals under low salinities (1, 3, 5). In conclusion, 5 cm L. japonicus juveniles exhibited superior low-salinity tolerance, accelerated osmoregulatory responses, and enhanced adaptation compared to 10 cm juveniles. These findings strongly support the prioritizing of smaller-sized L. japonicus for low-salinity aquaculture practices.

1. Introduction

In response to salinity stress, fish will produce a series of physiological responses that have an important impact on their survival and physiological activities [1,2]. Studies have shown that changes in salinity can significantly affect fish survival rate, growth performance, energy metabolism, nonspecific immune function, and osmoregulatory ability [3,4,5,6]. Fish maintain osmotic pressure homeostasis by regulating physiological parameters to adapt to changes in environmental salinity. Specifically, this manifests as significant changes in key physiological parameters, such as growth performance, respiratory metabolism, enzyme activity, and endocrine levels. This maintains the normal osmoregulatory function of fish, promoting their growth and development and improving their survival rate [7,8].

Salinity changes can affect fish survival, and excessively low or high salinity can impact viability. For example, when juvenile Seriola dumerili were cultured in water with different salinities (5–40) for 72 h, a rise in salinity from 6 to 40 gradually increased their survival rate from 63.3% to 100%. In contrast, all fish under low-salinity stress (below 5) died within 24 h. These results indicated that juvenile S. dumerili cannot tolerate acute salinity stress below 6, further highlighting the limited range of salinity tolerance of juvenile S. dumerili [9]. In cobia (Rachycentron canadum), the survival rate at a salinity of 5 (68.3%) was significantly lower than at salinities of 15 (90%) and 30 (92.5%), indicating that juvenile cobia can be reared more successfully within a salinity range of 15–30 [10]. Fish with different specifications also exhibit varying abilities to adapt to low salinity. For example, in studies on bighead carp (Aristichthys nobilis), it was found that as salinity increased, the mean survival time and median survival time (MST50) of fry decreased. Younger fry were less tolerant of exposure to these salinities than older fry. The median lethal salinity (MLS) after 96 h showed that 35-day-old fry (MLS, 7.6‰) had higher tolerance than 11-day-old (MLS, 2.3‰) and 18-day-old (MLS, 6.0‰) fry, indicating that survival in saltwater depends on their age at initial exposure to low salinity [11]. Therefore, it is necessary to conduct comparative studies on fish of different sizes under low-salinity adaptation.

The osmoregulatory ability of fish determines their salinity adaptation range. The osmoregulatory system of teleost fish is primarily composed of key physiological indicators, such as plasma osmotic pressure [12], ion concentration [13], and NKA enzyme activity [14]. These indicators maintain the osmotic balance in the fish body and internal environmental homeostasis through synergistic actions [15,16]. Among these, plasma osmotic pressure, as an overall regulatory indicator, directly reflects the osmoregulatory status of fish [17]. Upon sudden changes in environmental salinity, teleost fish activate immediate osmoregulatory reactions, characterized by significant increases or decreases in plasma osmotic pressure that correlate positively with salinity levels [18,19,20]. The concentration changes of plasma ions, such as Na⁺, K⁺, and Cl⁻, precisely regulate the osmotic pressure gradient across the cell membrane [21]. The ions levels in the blood, assessed after fish are subjected to saltwater challenge tests, serve as reliable indicators for evaluating the osmoregulatory capacity of fish [22]. In a low-salt environment, the salt concentration of the fish body fluid is higher than that of the external water. This leads to passive water infiltration through the gills and body surface, as well as passive ion loss. To compensate, fish actively absorb Na⁺ and Cl⁻ ions through their gills. The kidneys reduce urination and retain salts. The intestine reduces seawater intake and optimizes ion excretion. These physiological responses help to maintain internal homeostasis [23]. Studies on teleost fish have shown that plasma osmotic pressure and the concentrations of Na⁺ and Cl⁻ exhibit a linear correlation with environmental salinity [24,25,26,27].

The NKA enzyme, an important P-type ATPase, plays a core role in the osmotic regulation process in fish [28]. This transmembrane protein complex, composed of α, β, and γ subunits, is widely distributed in the osmotic regulatory organs of fish, including gill epithelial, kidney tubular, and intestinal mucosal cells [29]. As the primary driving force for ion transport, the activity of the NKA enzyme correlates with osmoregulatory capacity [30]. Studies have shown that the activity of the NKA enzyme exhibits a highly dynamic response to changes in environmental salinity [31]. When fish encounter salinity stress, the activity of this enzyme is upregulated. This is primarily achieved by increasing the expression levels of the enzyme protein and enhancing the activity of individual enzyme molecules. For example, when euryhaline fish are transferred from freshwater to seawater, the activity of the NKA enzyme in gill tissues increases significantly, facilitating the excretion of excessive Na⁺ and maintaining plasma ion concentrations within the physiological range [14]. Alterations in organ-specific NKA activity serve as a key osmotic regulatory response that enables euryhaline teleosts to adapt to fluctuations in salinity. The enhanced NKA activity contributes to efficient osmoregulation and preserves the stability of body fluid homeostasis [32].

Lateolabrax japonicus, commonly known as the Japanese sea bass or seven-star sea bass, belongs to the order Perciformes and the family Lateolabracidae [33]. This commercially important marine fish species is widely cultured in China and can adapt to a wide range of salinities [34,35]. Research on this species has primarily focused on basic biology, aquaculture, genetic breeding, nutritional feed, and disease prevention and control, providing important support for the aquaculture of L. japonicus [34,36,37,38,39]. However, the physiological adaptation mechanisms of L. japonicus in different salinity environments, especially the differences in osmotic regulation capabilities across growth stages, require further research. Therefore, this study aimed to investigate the effects of acute low-salinity environments on the survival rate, osmotic regulation processes, and physiological responses of L. japonicus at two size specifications and analyze the underlying mechanisms. Thus, examining the changes in plasma osmolality, ion concentration regulation, and tissue enzyme activity during salinity adaptation in L. japonicus of different sizes is crucial for revealing the mechanisms of osmotic regulation. By systematically evaluating the tolerance of different-sized L. japonicus to low-salinity environments, this study accumulated physiological and biochemical data on their responses to acute low salinity, providing a scientific basis for mariculture in saline-alkaline water areas. Additionally, screening suitable size specifications for cultivation in saline-alkaline regions will help optimize aquaculture models and improve farming efficiencies.

2. Materials and Methods

2.1. Experimental Materials

L. japonicus used in this experiment was sourced from Jiangsu (Jiangsu Marine Fisheries Research Institute, Nantong, China). Temporary rearing and experimental facilities were provided by the East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (Shanghai, China). The fish were graded based on their length. Two sizes of L. japonicus were selected: 5 cm (body length: 4.95 ± 0.45 cm; body weight: 2.00 ± 0.60 g) and 10 cm (body length: 9.85 ± 0.55 cm; body weight: 15.50 ± 2.50 g). Before the experiment, the fish were transferred to two 1500-L temporary rearing tanks for acclimatization. The culture water was prepared by adding marine crystal (Qingdao Haike General Sea Salt Co., LTD, Qingdao, China) to aerated tap water to ensure that the water quality met the experimental requirements. During the temporary rearing period, the environmental water temperature was maintained at 22.50 ± 2.85 °C, dissolved oxygen was maintained at 7.80 ± 0.30 mg/L, pH at 7.93 ± 0.12, and the ammonia nitrogen concentration was below 0.15 mg/L. Water for each salinity level was prepared 24 h in advance, subjected to aeration, and calibrated to ensure accurate salinity concentrations. To maintain water quality, 80% of the water was replaced daily, and the fish were fed commercial feed twice a day at fixed times to ensure that they remained healthy. Salinity changes were monitored daily, with fluctuations maintained within 0.03.

2.2. Acute Toxicity Experiments

To understand the tolerance limit of different sizes of L. japonicus to low salt, seven salinity gradients were set, 0.2, 1, 3, 5, 10, 15, and 25 (control group). The measured salinities were 0.21, 1.02, 3.02, 4.98, 10.03, 14.03 and 15.02, respectively. Three replicates were set for each gradient, containing 20 fish, and fish mortality was recorded at 12, 24, 48, 72, and 96 h. Fish were considered dead if they were immobile and showed no response upon contact with a glass rod.

2.3. Low Salinity Stress Experiments

The experiment was set up with five salinity gradients: salinity 1 (S1), salinity 3 (S3), salinity 5 (S5), salinity 10 (S10), and salinity 25 (S25, control group), with three replicates per salinity, and 30 fish in each replicate. The measured salinities were 1.01, 3.03, 5.02, 10.03 and 15.01, respectively. The experimental breeding container was a round plastic bucket with a diameter of 100 cm and a volume of 200 L, and the experimental water was prepared as a proportion of sea crystal and filtered tap water, corrected with a salinity meter. During the experiment, nine fish were randomly selected from each group at 0, 6, 12, 24, 48, 72, and 96 h, respectively, and placed on an ice-water mixture with 10 mg/L clove oil (complete anesthesia within 30 s). Blood was collected from the caudal artery using a 1 mL syringe. A portion of fresh blood was used for ion concentration detection. The remaining blood was dispensed in an anticoagulant tube and centrifuged at 4000 r/min in a 4 °C centrifuge for 8 min. The upper plasma was stored in a −80 °C freezer for osmotic pressure detection. After each blood draw, the gill filaments on the second gill arch of the right side, along with the kidney and intestines, were quickly removed. The tissues were rinsed three times with 4 °C physiological saline to remove blood clots and other debris and placed in a 5 mL sterile centrifuge tube. After cryopreservation in liquid nitrogen, the samples were immediately transferred to a −80 °C freezer for storage.

2.4. Measurement of Plasma Osmolality and Blood Ion Concentration

The total osmolality of plasma and water samples was measured using 10 μL of each sample with a vapor pressure osmometer (Vapor Pressure Osmometer Model 5600, ElitechGroup Inc., Logan, UT, USA), and the results were expressed in units of mOsm/kg. Subsequently, the concentrations of Na⁺, Cl⁻, and Ca²⁺ in fresh blood were determined using a blood gas analyzer (SC80, Radiometer Medical Aps, Carlsbad, CA, USA), with the results reported in mmol/L.

2.5. Tissue Enzyme Activity Assay

The gills, kidneys, and intestines of juvenile L. japonicus were used as test materials. According to the weight/volume (W/V) ratio, 0.1 g of tissue was added to 9 times the volume of the homogenization medium. The tissues were homogenized using an Ultra Turrax homogenizer in an ice bath and centrifuged at 12,000 r/min for 10 min at 4 °C. The supernatant of the tissue homogenates was used to measure Na⁺/K⁺-ATPase activity and total protein concentration. Na⁺/K⁺-ATPase activity was determined using the phosphorus determination method with a Na⁺/K⁺-ATPase assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein content was determined using the BCA method with a protein concentration determination kit from the same company.

2.6. Data Analysis

The data obtained were expressed as mean ± standard error (Mean ± SE). The data were processed and analyzed by SPSS 16.0 software (SPSS Inc., Chicago, IL, USA), and all data were analyzed by one-way ANOVA. If the difference was significant, Duncan’s multiple comparison was performed; p < 0.05 indicates a significant difference.

3. Results

3.1. Survival Results

The 5 cm juvenile fish exhibited normal survival across all tested salinity levels (Figure 1). However, mortality was observed in 10 cm juvenile fish at a salinity of 0.20 (

Table 1. Mortality of 10 cm juvenile L. japonicus (n = 420).

Salinity	12 h	24 h	48 h	72 h	96 h
	Number of Deaths/Individuals	Number of Deaths/Individuals	Number of Deaths/Individuals	Number of Deaths/Individuals	Number of Deaths/Individuals
0.2	0	1	1	0	1
1	0	0	0	0	0
3	0	0	0	0	0
5	0	0	0	0	0
10	0	0	0	0	0
15	0	0	0	0	0
25	0	0	0	0	0

), although with no statistically significant difference compared to the control group.

3.2. Osmotic Pressure

The plasma osmolality of juveniles exhibited an initial decrease, followed by an increase and subsequent stabilization after exposure to different salinities (Figure 2). The lowest plasma osmolality was observed 6 h after exposure. Under salinity levels of 1, 3, 5, and 10, the plasma osmolality of 5 cm juveniles progressively stabilized after 6 h of exposure. In contrast, 10 cm juveniles exposed to a salinity of 1 exhibited the lowest plasma osmolality at 24 h, with stabilization occurring after 48 h. However, at salinity levels of 3, 5, and 10, the plasma osmolality of 10 cm juveniles stabilized within 6 h. Figure 2C demonstrates that at 96 h, the isosmotic point of the 5 cm juveniles corresponded to an osmolality equivalent to that of water with a salinity of approximately 10.91. Similarly, as indicated in Figure 2D, the isosmotic point of 10 cm juveniles was equivalent to a salinity of 11.00.

3.3. Blood Ion

As shown in Figure 3A,B, the blood Na⁺ concentration in juveniles exhibited an initial decline, followed by an increase and subsequent stabilization over time, with the lowest Na⁺ concentration observed at 6 h. Under salinity stress levels of 1, 3, 5, and 10, the blood Na⁺ concentration in 5 cm juveniles stabilized after 6 h, and Na⁺ levels increased with rising salinity. For 10 cm juveniles exposed to a salinity of 1, the lowest Na⁺ concentration was recorded at 24 h, followed by stabilization at 6 h. In contrast, under salinity levels of 3, 5, and 10, the blood Na⁺ concentration in 10 cm juveniles stabilized within 6 h and increased consistently with higher salinity.

Figure 3C,D show that the blood Cl⁻ concentration in juveniles initially decreased, followed by an upward trend and eventual stabilization over time, with the lowest Cl⁻ concentration observed at 6 h. Under salinity stress levels of 1, 3, 5, and 10, the blood Cl⁻ concentration in 5 cm juveniles stabilized after 6 h, with Cl⁻ levels rising in response to increased salinity. For 10 cm juveniles at a salinity of 1, the lowest Cl⁻ concentration occurred at 24 h, stabilizing after 48 h. However, at salinity levels of 3, 5, and 10, the Cl⁻ concentration stabilized within 6 h, consistently showing a positive correlation with salinity.

3.4. Tissue Enzyme Activity

As shown in Figure 4A,B, compared to the control group, the gill NKA enzyme activity of juveniles exhibited a dynamic pattern characterized by an initial increase, followed by a decline and eventual stabilization. Except for the salinity of 10, gill NKA enzyme activity in all other salinity groups peaked at 6 h. Under salinity levels of 1, 3, and 5, the gill NKA enzyme activity in 5 cm juveniles stabilized after 12 h, and 10 cm juveniles also reached stabilization after 12 h. In contrast, under a salinity of 10, the gill NKA enzyme activity of both 5 cm and 10 cm juveniles stabilized within 6 h. After stabilization, the gill NKA enzyme activity was lowest at a salinity of 10, and the osmotic regulatory stress exerted by low salinity (1, 3, and 5) was significantly greater than that of high salinity (25).

Similarly, compared to the control group, the kidney NKA enzyme activity of juveniles exhibited an initial increase, subsequent decline, and eventual stabilization (Figure 4). Except for group at a salinity of 10, renal NKA enzyme activity in all other salinity groups reached a maximum at 6 h. Under salinity levels of 1, 3, and 5, the kidney NKA enzyme activity of 5 cm juveniles stabilized after 12 h, whereas that of 10 cm juveniles stabilized after 24 h. At a salinity of 10, kidney NKA enzyme activity in both 5 cm and 10 cm juveniles stabilized within 6 h. Post-stabilization, kidney NKA enzyme activity was lowest at a salinity of 10, with low-salinity environments imposing substantially greater osmotic regulatory stress on juveniles compared to high-salinity conditions.

4. Discussion

The results demonstrate that L. japonicus of varying sizes have good tolerance to low salinity and can survival in brackish water environments, indicating that this species is suitable for brackish water aquaculture. Different sizes of L. japonicus can survive in water with a salinity >0.20. However, Amphiprion ocellaris had a high mortality rate at a salinity of 5, although the survival rates remained ≥95% at other salinities [40]. Lutjanus campechanus exhibited 100% mortality within 72 h at a salinity of 4, with a 96-h median lethal concentration estimated at a salinity of 5.65 [41]. Conversely, Centropomus parallelus showed excellent survival rates (93.3%) at salinities of 5, 15, and 35. These results indicate that different fish species have different tolerances to low salinity [2]. This may be attributed to the different habitats of L. japonicus which often inhabits estuaries and migrates to freshwater for foraging. Therefore, different sizes of L. japonicus may have strong adaptability to fluctuations in salinity.

When the salinity of environmental water changes, fish generally undergo two main stages of adaptation: an initial passive adaptation phase and a later active regulation phase. Euryhaline fish exhibit strong adaptive capabilities to salinity stress. They can regulate the absorption and excretion of key ions, such as Na⁺ and Cl⁻, through the synergistic action of organs, including the gills, kidneys, and intestines, thereby effectively coping with fluctuations in external salinity [42,43]. This active regulatory mechanism enables euryhaline fish to maintain normal physiological functions in a wide range of salinity. Within a certain salinity range, the osmotic pressure of teleost fish dynamically adjusts with changes in environmental salinity, and there is typically a significant positive correlation between them. When external salinity increases, serum osmotic pressure rises accordingly, while a decrease in salinity leads to a corresponding decline in serum osmotic pressure [44]. Plasma osmotic pressure is primarily composed of two components: crystalloid and colloid osmotic pressures [45]. Crystalloid osmotic pressure is mainly determined by the concentrations of inorganic ions, such as Na⁺, Cl⁻, K⁺, and Ca²⁺. These ions maintain a dynamic equilibrium in body fluids through active transport and passive diffusion, collectively sustaining the stability of crystalloid osmotic pressure. Colloid osmotic pressure is primarily provided by macromolecules such as plasma proteins, which play a critical role in regulating water distribution and maintaining fluid balance inside and outside blood vessels. The synergistic effect of crystalloid and colloid osmotic pressures ensures the stability of the plasma osmotic pressure. Studies have shown that changes in the trend of plasma osmotic pressure in euryhaline marine teleosts generally align with the changes in blood Na⁺ and Cl⁻ concentrations [46]. The isosmotic point of euryhaline fish typically ranges between salinities of 10 and 13, reflecting the salinity conditions where internal osmotic pressure is balanced with the external environment [12]. The existence of an isosmotic point indicates that euryhaline fish can reduce energy consumption under specific salinity conditions and maintain internal-external equilibrium without additional osmoregulation. This is highly consistent with the isosmotic points (10.91 and 11.00) of the two L. japonicus sizes used in this study, further verifying the stability of osmoregulatory capabilities.

When salinity decreases, fish passively absorb water due to osmotic pressure differences, leading to a decline in internal ion concentration and osmotic pressure. To cope with this change, fish inhibit hyperosmotic regulatory mechanisms, cease drinking seawater, and excrete excess water by increasing their urine output. Additionally, fish absorb ions, such as Na⁺ and Cl⁻, from ingested seawater and reduce their excretion to maintain internal ion balance and osmotic stability [16]. In low-salinity environments, plasma osmotic pressure and blood Na⁺ equilibrium times were consistent across different-sized juvenile L. japonicus, indicating strong adaptability to low salinity. However, blood Cl⁻ equilibrium times differed, with 10 cm juveniles achieving equilibrium later. This may explain the mortality observed in 10 cm juvenile fish. In water with a salinity of 10, Na⁺ and Cl⁻ concentrations in the blood of the juveniles stabilized after 6 h, suggesting that at a salinity of 10, which is close to the isosmotic point, ions do not need to be replenished via excessive drinking, thereby enabling rapid attainment of ionic equilibrium. In water with salinities below 10, juveniles must drink copiously to replenish ion concentrations and avoid ion imbalances. The divergent patterns in the changes in ion concentration suggest the presence of osmoregulatory pathways beyond Na⁺ and Cl⁻ [47,48]. Collectively, these results indicate that within a certain range of salinity, 5 cm fish can rapidly regulate body fluid to avoid ion imbalances and threats to survival. A more sensitive and rapid osmoregulatory mechanism allows for normal survival in brackish water.

Among the tissues examined in L. japonicus, the activity of NKA was highest in the gills, followed by the kidneys. This indicates that gills play a more critical role in osmoregulation, while the role of the kidney is relatively secondary. This finding is consistent with previous reports. In mammals, the kidney is the primary organ for ion regulation, whereas in fish, the gills serve as the dominant osmoregulatory tissue [49]. As gills are directly exposed to the external water environment, their specialized structure enables the efficient absorption or excretion of ions in water. Compared with the kidneys, the gills exhibit a more sensitive response to salinity changes and are prioritized as tissues for evaluating the severity of salinity stress.

5. Conclusions

In summary, under low-salinity conditions, 5 cm L. japonicus juveniles exhibit more rapid osmotic ion-regulatory capabilities than 10 cm juveniles. Within a certain salinity range, 5 cm juveniles can quickly regulate and maintain their body fluid balance to avoid ion imbalances and survival threats, and can survive in brackish water. This suggests that the osmoregulatory mechanism in L. japonicus is highly sensitive and rapid. The NKA activity in the gills reached equilibrium faster than that in the kidney, implying that the gills may serve as the primary organs for rapid osmoregulation under low-salinity stress. Together, these results demonstrate that 5 cm fish have advantages in osmoregulation, which may be one of the reasons for their higher survival rates. These findings provide a theoretical basis for the development of size-specific transplantation technologies for salt-alkali aquaculture.