Fish collection and management
Test C. ussuriensis, with a body length of 15 ± 1.5 cm and body weight of 30 ± 3.5 g were obtained from the Bohai Coldwater Fisheries Research Station (Heilongjiang Province, P.R. China). Before the start of the experiment, 250 fish were randomly distributed in a semi-recirculation system consisting of five circular polythene tanks (70 cm diameter, 20 cm depth, 75 L water volume) and acclimated to the new rearing environment for 2 weeks. During the experiment and acclimation period, the photoperiod was set at 12 h light: 12 h dark (12 L : 12 D) and the light intensity at 70 lx. Filtered freshwater was used in the trial: dissolved oxygen was 7.8–10.0 mg/L, pH 7.4, water temperature was maintained at 10 ± 0.2 °C, and ammonia-N was < 0.1 mg/L throughout the study. Fish were fed to apparent satiation twice daily (08:00 and 14:00 h), and the amount of bait was 2 % of the mass of the fish. During the acclimation and test period, the water was changed weekly and the amount of water was changed from 30 % to 40 %. The seawater used to change the experimental water was pre-prepared, the salinity difference was < 0.5, and the temperature difference was < 0.5 °C.
Salinity experimental design
To simulate the environmental salinity of estuaries and offshore watersheds, five test groups were designed with salinities of 0‰ (S0; freshwater control), 8‰ (S8), 16‰ (S16), 24‰ (S24), and 32‰ (S32). In the test groups, the salinity was increased by 4‰ every day until the set salinity was reached and the test period was 30 days. Groundwater was used as the test water source, and the salinity was measured with a salinometer (error ≤ 0.5). Each test group had three replicates and a parallel stocking of 15 juvenile fish, with a total of 45 fish. Feeding was stopped 24 h before sampling. For anaesthesia, 100 mg/mL tricaine methanesulfonate (MS-222) was used.
Serum physiological determination
After the fish were anaesthetised, blood was collected from their tail vertebrae on an ice tray; 1 ml of blood was taken from each tail, and nine fish were taken from each group. The blood was centrifuged at 3500 × g at 4°C for 15 min. A fully automatic biochemical analyser (Olympus AU 600, Japan) was used to determine the osmolality, Na+, K+, Mg2+, Ca2+, Cl−, and P+, as well as the activities of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and urea nitrogen (UREA).
Liver and gill samples from the five experimental groups were analysed. Samples were fixed with Bouin’s solution, dehydrated with ethanol, made transparent with xylene, and embedded in paraffin. The samples were cut with a microtome (KD1508), stained with haematoxylin-eosin, and mounted in neutral resin. The prepared sections were observed and photographed using an Olympus CX41 microscope.
Physiological index determination
The livers of six fish from each group were collected and stored at −80°C for enzyme activity analysis, as previously described by Sandstrom et al. (1994). Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) activity were determined out using kits (Nanjing Jiancheng Biological Engineering Research Institute, Nanjing, China). Protein levels in the homogenate were determined using the Coomassie blue method. Serum cortisol levels were measured using commercially available radioimmunoassay equipment (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The growth index was calculated as follows:
SGR = 100% × (ln FW − ln IW)/T
Survival rate = 100% × (NT/N0)
where SGR is the specific growth rate, IW and FW are the initial and final body weights of the juvenile fish (g), respectively, T is the experiment time (d), N0 is the number of tails of C. ussuriensis at the beginning of the experiment, and NT is the number of surviving juvenile fish at the end of the experiment.
RNA isolation and cDNA synthesis
RNA was extracted from the collected liver samples of C. ussuriensis using TRIzol® (Invitrogen) according to the manufacturer’s instructions (1 mL/100 mg tissue). Its concentration was measured using ScanDrop at 260/230 nm (Analytikjena, Germany) and purity was assessed at a 260/280 ratio. Reverse transcription was used to generate cDNA using the PrimeScript RT Reagent Kit (TaKaRa, Japan) according to the manufacturer’s instructions. The obtained cDNA was stored at −20°C until further use as the template in the amplification reaction.
Quantitative real-time PCR was performed using TB Green® Premix Ex TaqTM (TaKaRa, Japan). The conditions were as follows: 95°C for 30 s, 39 two-step cycles at 95°C for 5 s and 60°C for 30 s, followed by 95°C for 10 s, 65°C for 5 s, and 95°C for 5 s. Beta-actin was used as the reference gene. Primers were designed using Primer Premier software 5.0. Primers used for real-time PCR are listed in Table 1. For each cDNA sample, all target and reference genes were independently amplified in triplicates on the same plate and the same experimental run. The melting curve analysis showed that there were no dimers or other non-specific PCR products in any of the reactions performed. Ct values were measured using the CFX96 C1000 touch Thermal Cycler (Bio-Rad, USA), and the value of the target sequence normalised to the reference sequence was calculated as 2−ΔΔCt.
Graphs were plotted using GraphPad Prism 7.0. The least significant difference test and one-way ANOVA were used to analyse the differences between groups. Results are expressed as the mean ± SE. Statistical analysis was performed using SPSS version 13.0. Statistical significance was set at P < 0.05.