Juvenile tongue soles were provided by Mingbao Aquatic Product Co., Ltd. (Yantai, China) and transferred to the laboratories at the Aoshanwei Research Centre of Ocean University of China. They were acclimated to seawater (30 g/L) in a 1000 L fiberglass tank for at least one week before the experiment started. For the experiment, fish of same age and similar sizes were selected and during the experiment they were fed commercial pellets (Guangzhou Yuequn Technology Co., Ltd, China) to satiation twice daily at 07:00 and 18:00 h. Animal handling procedures were followed according to international guidelines and approved by the Animal Ethics Committee, Ocean University of China, Qingdao, Shandong Province, China.

The effects of three fluctuating frequencies (every 2, 4, or 8 days) and four fluctuation amplitudes (2, 4, 6, or 8 g/L) were compared to an unfluctuating control. The salinity of control was constant i.e. 30 g/L, while treatments S30 ± 2, S30 ± 4, S30 ± 6 and S30 ± 8 (S stands for salinity and values are in g/L) were subjected to different salinity fluctuations. In each amplitude treatment, there were three frequency treatments including D2, D4 and D8 (D stands for days) (Fig. 1). Experimental design was conducted according to Khairnar et al. (2014). Briefly, there were five replicates for each treatment, and each replicate had six juveniles. During the experiment, water quality parameters including temperature, pH and ammonia were monitored daily. Throughout the experiment, photoperiod was set at 14 h light: 10 h dark.

Fish were anaesthetized using 60 mg/L of MS-222 (Sigma Aldrich), and then they individually weighted. Blood was collected from the caudal peduncle using 1 mL syringes that were rinsed with a solution containing 25,000 units of ammonium heparin in 3 mL of 0.6% NaCl. Plasma was separated from cells by centrifuging whole blood (3 min, 10000 g, 4 °C). Then from each fish, the second gill arch on the left side was excised and dried with absorbent paper. Further, other key tissues including kidney, liver and muscles were dissected and frozen in liquid nitrogen and stored at –80°C for further analyses. Frozen samples were thawed at 4°C before analyzed. Samples of 0.2 g of gill, kidney, liver and muscle tissue were homogenized with 1.8 mL of cold sterile normal saline solution (NaCl 0.85%, w/v, pH 7.5) using hand homogenizer. All samples were then centrifuged at 3000 g for 10 min, and supernatants were collected and stored at 4 °C prior to analysis. All enzymatic assays were conducted within 24 h upon extraction.

Protein content of the homogenates was measured according to the method of Spector (1978), using bovine serum albumin as a standard. All assays for enzyme activities were carried out in duplicate and measured using UV 2102PC spectrophotometer (Unico, Shanghai, China).

Acid and alkaline phosphatase. ACP and AKP activity assays were carried out according to Barrett’s (1972) method using a commercial kit (Nanjing Jiancheng Biotech Company, China). Concentration of phenol was measured spectrophotometrically at 520 nm after incubation at 37 °C for 30 min (for ACP), or 15 min (for AKP). ACP and AKP activity was defined as the amount of phenol (mol) produced per milligram of protein.

Superoxide dismutase. SOD activity was determined according to Ji (1991) with an assay kit (Nanjing Jiancheng Biotech Company, China). Assay conditions were 65 μmol phosphate buffer, pH 7.8, 1 μmol hydrochloric hydroxylamine, 0.75 μmol xanthine and 2.3·10⁻³ IU xanthine dismutase. The supernatant (50 μL) had no blank reaction and was incubated in the system for 40 min at 37 °C, and terminated with 2 mL of 3.3 g/L p-aminobenzene sulfonic acid and 10 g/L of naphthylamine. An SOD unit is defined as the amount of enzyme that inhibits the superoxide-induced oxidation (monitored at 550 nm) by 50%.

Catalase. CAT activity was measured according to Goth (1991) with an assay kit (Nanjing Jiancheng Biotech Company, China). The base system including 4.60 μmol phosphate buffers (pH 7.4), 6.5·10^–3 μmol H2O2 and incubated for 1 min at 37°C. The reaction was terminated immediately using 32.4 μmol ammonium molybdate. A CAT unit is defined as catalyzing the use of 1 μmol H₂O₂ per second.

Plasma cortisol. Plasma cortisol was measured by Radio Immuno Assay (RIA) technique according to manufacturer’s instructions (Beijing Beifang Biotech Research Institute, China) and the method used was that of Pickering & Pottinger (1995) modified by Liu et al. (2012).

Statistical analysis of the data was performed with a statistical package (SPSS 16.0, SPSS Inc., Richmond, CA, USA). Values were presented as means ± standard error of the mean. Data for the immune parameters were tested for homogeneity of variances, and then possible differences were tested using one-way ANOVA for immune responses and followed by a Duncan’s multiple comparison to find out the difference between treatments.

During the course of the experiment, the temperature ranged from 18 to 22 °C, pH was around 8.0 and ammonia nitrogen was less than 0.2 mg/L. No mortality, health disturbances or any alterations in behavior were observed in any treatments during the experiment (p>0.05).

There were no siginificant differences in the initial body weights among all treatments (p>0.05). The ANOVA analysis showed that both the frequency and the amplitude of salinity fluctuation had significant effects on the final weights of tongue sole juveniles (p<0.01) (Fig. 2). Further, there was a significant interaction between frequency and amplitude of salinity fluctuations (p<0.01), and the tongue sole exhibited higher specific growth rate at the amplitude of 4–6 g/L at the frequencies of 4 and 8 days than other treatments and the control (p<0.05).

ACP activities in different tissues of a tongue sole in the control was highly tissue-specific having 66.47 ± 0.18, 17.32 ± 0.08, 112.20 ± 0.07 and 199.73 ± 4.62 U/g protein in hepatic, muscular, branchial and renal tissues respectively (Fig. 3). ACP activity was significantly influenced in all the tissues of tongue sole by the amplitude and frequency of salinity (p<0.05). The hepatic ACP activity ranged from 60.29-77.90 U/g protein and it was significantly higher at D4S30 ± 6 and D8S30 ± 6 than other treatments (p<0.05) (Fig. 3A). The muscular ACP activity showed less variation as compared to other tissues and had increasing trend with increasing frequency and amplitude levels compared to control and D8 treatments (p<0.05) (Fig. 3B). The branchial ACP activity at D8S30 ± 4, D4S30 ± 6 and D8S30 ± 6 were significantly higher compared to control and other treatments (p<0.05) (Fig. 3C). The renal ACP activity in juvenile tongue sole were significantly higher at salinity amplitude of 4–6 g/L with the frequencies of 2–8 days compared to control (p<0.05) (Fig. 3D).

AKP activities in hepatic, muscular, branchial and renal tissues for the control were 5.71 ± 0.14, 5.19 ± 0.02, 12.33 ± 0.10 and 41.12 ± ± 0.52 U/g protein respectively (Fig. 4). The hepatic AKP activity at D8S30 ± 4, D4S30 ± 6 and D8S30 ± 6 were significantly higher compared to control and other treatments (p<0.05) (Fig. 4A). The renal AKP activity was significantly higher among the four different tissues and the AKP activity in the branchial tissues were significantly higher than muscular tissues, which were influenced by the amplitude and frequency of salinities (p<0.05) (Fig. 4B). The muscular AKP activity was significantly enhanced at D4S30 ± 4, D4S30 ± 6, D8S30 ± 4 and D8S30 ± 6 as compared with control and other treatments (p<0.05) (Fig. 4C). The renal AKP activities were found significantly higher in D8S30 ± 4 and D8S30 ± 6 than control and other treatments (p<0.05) (Fig. 4D). The increasing trend in AKP activity was observed in renal and branchial tissues with decrease in frequency and amplitude of salinity (up to ± 6 g/L fluctuation levels), while it dropped at higher amplitude ( ± 8 g/L). However, there were no significant differences between branchial and renal AKP activity at ± 4 and ± 6 g/L amplitude at D4 and D8 frequencies (p>0.05).

SOD activity was significantly affected in muscular, renal and branchial tissues of tongue sole (p<0.05) (Fig. 5), whereas the hepatic SOD activities were not found significantly influenced by the fluctuating frequency and the amplitude of salinity compared to the constant salinity (Fig. 5A, p>0.05). The renal SOD activity was significantly higher among the four different tissues, and muscular tissue had the lowest SOD activity (p<0.05) (Fig. 5D). The muscular SOD activity was significantly higher at salinity amplitude of 4–6 g/L at the frequencies of 2–8 days compared to control (p<0.05) (Fig. 5B). The renal SOD activity followed the same trend as in muscular tissues, and was significantly higher at D4S30 ± 6 and D8S30 ± 6 in all the treatments. The branchial SOD activity was significantly influenced by treatments D4S30 ± 4, D4S30 ± 6, D8S30 ± 4 and D8S30 ± 6 as compared to control and other treatments (p<0.05) (Fig. 5C) and showed a tendency to increase with the increase of amplitude from ± 2 to ± 6 g/L. However at ± 8 g/L amplitude, fluctuation was lowest (p<0.05).

CAT activity in different tissues of tongue sole in control was highly tissue-specific having 24.95 ± 0.18, 8.29 ± 0.02 and 16.08 ± 0.53 U/g protein in hepatic, branchial and renal tissues respectively (Fig. 6). However, renal CAT activity showed no significant effect on frequency and amplitude of salinity fluctuations (p>0.05) (Fig. 6C). Muscular CAT activity was not recorded, as in muscle CAT activity is low or even absent. The hepatic CAT activity was significantly higher than in branchial and renal tissues, and the branchial tissue had the lowest CAT activity (p<0.05). The hepatic CAT activity tended to increase with increase in amplitude fluctuations except ± 8 g/L (Fig. 6A). The branchial CAT activity was significantly influenced in control (S30 ± 0), D4S30 ± 6 and D8S30 ± 6 compared to other treatments (p<0.05) (Fig. 6B).

The plasma cortisol levels observed in all treatments ranged between 30.49 and 52.29 nmol/L. Cortisol levels were significantly affected in lower (S30 ± 2) and higher (S30 ± 8) salinity fluctuations. However, plasma cortisol levels remained low in moderate salinity fluctuations (S30 ± 4 and S30 ± 6) and control (30 gL) (p<0.05) (Fig. 7).