Inuence of Salinity on the Survival and Growth of Juvenile Coregonus Ussuriensis Berg

To explore the suitable salinity range of Coregonus ussuriensis Berg, we investigated the effect of induced salinity change in captivity on C. ussuriensis with an initial body weight of 35 ± 1.5 g. After 30 days of salinity acclimation, the survival, growth performance, blood biochemical proles, antioxidative capacity, and tissue structure of juveniles under four salinity conditions (8 ‰ , 16 ‰ , 24 ‰ , and 32 ‰ ) were investigated. Our results revealed that serum penetration, blood glucose, and serum Na + , Cl − , and Mg 2+ gradually increased with increasing salinity until 32 ‰ salinity, when a signicant difference was observed, whereas the K + concentration showed a downward trend. The tissue sections showed that under high salinity (32 ‰ ), the liver and gill tissues of the sh were severely damaged and the vacuolation was serious. The levels of superoxide dismutase, glutathione peroxidase, and serum cortisol gradually increased with increasing salinity. A gene expression analysis showed that the increase in salinity induced higher expression of stress-, growth-, and inammation-related genes (HSP70, Gh and Igf-1, and IL-1β, respectively). The downregulation of stress-related gene expression at 32 ‰ salinity may indicate that this level of salinity exceeded the regulatory capacity of C. ussuriensis. We concluded that C. ussuriensis may survive in an estuary under 0–24 ‰ salinity. Our ndings provide insights into the physiological adaptation of C. ussuriensis to salinity change. These results could improve our knowledge of the stress response and resilience of estuarine sh to hyposalinity and hypersalinity stress. synthesised the The results of the present study demonstrated that as salinity gradually increased peaking at S32 compared with that at S0, indicating that salinity induced a stress response in the sh


Introduction
Salinity is an important environmental factor that affects the distribution and community structure of aquatic species and is closely related to physiological functions, growth performance, and immune regulation in sh farming (Lein et al. 1997;Fang et al. 2019). Owing to the variation in the salinity of aquatic environments, teleost sh have evolved various physiological strategies for adapting to salinity (El-Leithy et al. 2019; Geven et al. 2017). When sh live in an environment with uctuating salinity, various enzymes and transporters participate in the process of salinity adaptation and osmotic adjustment to maintain the body's permeability and ion homeostasis (Pungpung et al. 2007). Tolerance depends on the osmotic adjustment ability of the sh (Rubio et al. 2005). The liver can promote the decomposition of glycogen into glucose to maintain normal blood sugar levels and provide energy for gills and other osmotic adjustment organs under salinity stress (Zhang et al. 2017). However, compared with other osmotic mediators (gills and intestines), there have been fewer studies focusing on the liver. In the present study, the indicators of enzyme activity in the liver were investigated and the physiological responses of juvenile sh were explored under different salinity stress conditions. Salinity usually affects the growth rate of teleost sh because part of the energy used for growth is consumed during osmotic adjustment (Naglaa et al. 2017; Gonzalez et al. 2011). In addition, changes in salinity can cause an increase or decrease in certain blood indicators, such as an increase in cortisol, and these changes can affect oxygen transport in the gills (Yada et al. 2002).
Although the molecular effectors of teleosts under salinity stress have been reported (Mattioli et al. 2017; Whitehead et al. 2012), the in uence of salinity variation on Coregonus ussuriensis Berg (Salmoniformes, Coregoninae) is unclear. Thus, in the present study, we investigated the expression levels of a stress-related gene (heat shock protein 70, HSP70), growth-related genes (growth hormone, Gh and insulin-like growth factor 1, Igf-1), and an in ammation-related gene (interleukin-1β, IL-1β).
C. ussuriensis is mostly distributed in the Heilongjiang Valley in northeast China and the waters of Siberia and Sakhalin in Russia (Bochkarev et al. 2017). In 1998, C. ussuriensis was listed in the "Red Data Book of China's Endangered Animals" (Wang et al. 1998). Owing to its high economic value, C. ussuriensis is considered a promising new sh species for culturing in China. An analysis of otolith growth ring characteristics showed that C. ussuriensis has annual (seasonal) migration characteristics and migration history between rivers and seas or between freshwaters and estuaries (Wang et al. 2019). However, the range of adaptation for salinity in C. ussuriensis has not yet been reported. In the present study, we aimed to explore the adaptation of C. ussuriensis to different salinities through the analysis of physiological and genes expression. Our results provide a reference for supplementing the breeding and release of C. ussuriensis.

Materials And Methods
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 sh were randomly distributed in a semi-recirculation system consisting of ve 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 sh. 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, ve 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 sh, with a total of 45 sh. Feeding was stopped 24 h before sampling. For anaesthesia, 100 mg/mL tricaine methanesulfonate (MS-222) was used.

Serum physiological determination
After the sh were anaesthetised, blood was collected from their tail vertebrae on an ice tray; 1 ml of blood was taken from each tail, and nine sh 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 + , Mg 2+ , Ca 2+ , Cl − , and P + , as well as the activities of glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and urea nitrogen (UREA).

Histological analysis
Liver and gill samples from the ve experimental groups were analysed. Samples were xed with Bouin's solution, dehydrated with ethanol, made transparent with xylene, and embedded in para n. 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 sh 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: where SGR is the speci c growth rate, IW and FW are the initial and nal body weights of the juvenile sh (g), respectively, T is the experiment time (d), N 0 is the number of tails of C. ussuriensis at the beginning of the experiment, and N T is the number of surviving juvenile sh 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 ampli cation reaction.

Real-time PCR
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 ampli ed in triplicates on the same plate and the same experimental run. The melting curve analysis showed that there were no dimers or other non-speci c 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 .

Statistical Analysis
Graphs were plotted using GraphPad Prism 7.0. The least signi cant 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 signi cance was set at P < 0.05.

Results
Survival rate and growth performance In the salinity stress experiment, the survival rate and SGR of the high-salinity group were signi cantly different from those of S0. As shown in Table 2, C. ussuriensis was able to survive normally during the experimental period up to S24 salinity. In the S32 group, the survival rate of C. ussuriensis signi cantly decreased (P < 0.05), with 36 deaths in total and a survival rate of 21.04%. As shown in Table 2, the nal weight of C. ussuriensis was 53.33 ± 5.53 g, and the speci c growth rate was 1.24%. However, in the S32 group, a negative growth phenomenon was observed. The SGR presented a similar pattern of change.

Blood biochemical parameters
The serum physiological indexes of C. ussuriensis under salinity treatment are shown in Table 3. With the increase in salinity, the serum osmolality showed a gradual increasing trend, which was signi cantly different from that of S0 at S32. The change trend in the concentration of Na + and Mg 2+ in serum was basically the same as that of osmolality, whereas the concentration of K + gradually decreased as the salinity gradient increased. The change in Cl − was the most pronounced and was signi cantly different compared with that of S0 at S24. However, the Ca 2+ and P + levels did not signi cantly change. As shown in Table 4, there was no signi cant difference in the glucose levels of C. ussuriensis serum at S24, whereas the glucose concentration signi cantly increased at S32. The concentration of AST gradually decreased with increasing salinity, but the salinity gradient did not signi cantly affect ALT and UREA concentrations.

Observation of tissue sections
Compared with S0, with the increase in salinity, the liver tissues of C. ussuriensis changed to varying degrees (Fig. 1). When the salinity was S0, the hepatocytes were arranged compactly and neatly with clear structures and evident boundaries. With the gradual increase in salinity, the hepatocyte structure had a tendency to shrink, but under S8 and S16 conditions, there was no evident damage. Under S24 and S32 conditions, the liver was characterised by empty vacuoles and fuzzy hepatocyte boundaries. The liver tissue was clearly injured.
At S0, the gill lament epithelium of juvenile C. ussuriensis comprised multiple layers of epithelial cells, including chloride cells and pavement cells. Pavement cells, pillar cells, and blood channels were distributed on the epithelial cells of the gill fragments. As shown in Fig. 2, with the gradual increase in salinity, chloride cells and epithelial cells showed varying degrees of vacuolisation, and gill lament cells had the most severe vacuolisation at S32.

Research on enzyme activity
There was a signi cant difference (P < 0.05) in SOD activity in the increasing salinity gradient. Compared with S0, with the increase in salinity, SOD activity rst increased and then decreased peaking at S24 (Fig. 3A). There was no signi cant difference in CAT enzyme activity during the entire test period (Fig. 3B). The activity of GSP-PX peaked at S32 (Fig. 3C). The serum cortisol content gradually increased with increasing salinity (Fig. 3D).

Gene expression
Different gene expression patterns were observed in the liver at different salinities. As shown in Fig. 4, HSP70 expression in the liver increased 11.46-fold at S8. The relative expression at S32 decreased by 0.75-fold compared with that at S0 (Fig. 4A). The expression of the growth-related gene Gh increased with increasing salinity peaking at S32, which was 16.26-fold higher than that at S0 (Fig. 4B). The expression of Igf-1 was the highest at S16, which was 2.97-fold higher than that at S0, but then showed a downward trend (Fig. 4C). The expression of the in ammation-related gene IL-1β in the liver is shown in Fig. 4. Generally, the expression of IL-1β was 22.10-and 17.79-fold higher at S16 and S24 than that at S0, respectively (Fig. 4D).

Discussion
Arti cial propagation technology for C. ussuriensis is still in its preliminary exploration stage. To supplement the basic biological data of this species and provide theoretical guidance for arti cial breeding, a salinity domestication experiment was carried out on C. ussuriensis. Our results con rmed the previous speculation that the sh has a migration history between rivers and the sea or between freshwaters and estuaries (Wang et al. 2019). Similar to other salmonids, C. ussuriensis has some hypotonic regulatory mechanisms when  (Table 3).
The trend in the osmotic pressure, Na + , Cl − , and Mg 2+ in the C. ussuriensis serum was basically the same as the osmolality trend, whereas the K + concentration gradually decreased as the salinity gradient increased. We Our results indicated that S32 was the salinity threshold for physiological tolerance in C. ussuriensis.
The liver is the main immune organ in teleosts. Although it is not directly involved in osmotic adjustment, it is the main component of glycogen/glucose metabolism, and its metabolism is enhanced during the osmotic adaptation process, which is useful for the osmotic adjustment of biofuels (Vijayan et al. 1996;Peragón et al. 1998). In the present study, salinity had a certain degree of in uence on the liver of juvenile C. ussuriensis. When observed under normal conditions (S0), the liver tissue structure was normal. As salinity increased, liver cells gradually shrunk. When the salinity was S32, the liver was characterised as empty vacuoles (Fig. 2), which could be related to the physiological changes in C. ussuriensis. We consider that salinity had exceeded the liver's physiological regulation ability. The results of the present study demonstrated that as salinity increased, the cortisol concentration gradually increased peaking at S32 compared with that at S0, indicating that salinity induced a stress response in the sh body.
HSP70 plays a key role in a variety of stressful environmental conditions, such as those related to temperature, hypoxia, and salt. Exposure to stressful environments causes the upregulation of gene expression as a protective mechanism. In a salinity stress experiment on blackchin tilapia (Sarotherodon melanotheron), it was found that HSP70 was positively correlated with Na + and K + /ATPase gene expression (Tine et al. 2010). Deane and Woo (2004) also showed that an increase in salinity caused the upregulation of HSP70. In the present study, HSP70 was signi cantly upregulated at S8, which was similar to the results of El-Leithy et al. In summary, combined with the analysis of physiological indicators and the observation of tissue sections, we found that juvenile C. ussuriensis have strong adaptability to moderate salinity (S8-S24), under which they can adapt and grow normally. However, at S32, the sh body produced a stronger stress response to the external environment. Thus, our ndings will provide a theoretical reference for the breeding and release of C. ussuriensis in the future.

Con icts of interest
There are no con icts of interest to declare.

Ethics approval
All experiments were performed in accordance with the European Communities Council Directive (86/609/EEC).
Fish were bred following the guidelines of the Animal Husbandry Department of Heilongjiang Province, P.R.
China. All efforts were made to minimise suffering.   Microscopic section of gill tissue of C. ussuriensis exposed to different salinity. F, lament; L, lamellae; CC, chloride cells; PC, pavemen cells; Pi C, pillar cells; B, blood channel and blood cell