Immune Responses of Asian Seabass Lates calcarifer to Dietary Glycyrrhiza uralensis

Simple Summary Due to the fact of their low toxicity, small side effects, and little residue, increasing attention has been paid to herbs as environmentally friendly immunostimulants. Results from the present study indicate that adding Glycyrrhiza uralensis to the feed can improve the growth and survival of Lates calcarifer and significantly promote the expression of immune-related genes in the liver and head kidney of Lates calcarifer. The optimum inclusion level of Glycyrrhiza uralensis should be 1–3%. Abstract To understand the impacts of dietary Glycyrrhiza uralensis on the immune responses of Lates calcarifer, the expression of immune-related genes including crp, c-3, c-4, mtor, mlst-8, eif4e, hsp-70, hsp-90, il-8il-8, il-10, tgfβ1, tnf, ifn-γ1, and mxf in L. calcarifer juveniles was evaluated in this study. Fish were fed experimental diets with G. uralensis levels of 0%, 1%, 3%, and 5% for 56 days. The results showed that dietary G. uralensis could improve the growth and survival of L. calcarifer and regulate the immune-related genes’ expression in L. calcarifer. Dietary G. uralensis significantly upregulated the expression level of crp, mtor, hsp-90, c-3, and c-4 genes in the liver of L. calcarifer, while hsp-70 gene expression was nearly downregulated. It did not upregulate the expression of elf4e and mlst-8 in the 1% and 3% inclusion groups, but it was the exact opposite in the 5% inclusion group. G. uralensis significantly affected the expression of il-8, il-10, tnf, ifn-γ1, mxf, and tgfβ1 in the head kidney of L. calcarifer. G. uralensis upregulated the expression of tnf and tgfβ1 consistently, but ifn-γ1 was at a low expression level. The expression of il-8 and il-10 was upregulated in the 1% group, while it was downregulated in the 5% group. The results from the present study indicate that dietary G. uralensis appeared to improve the immune function of L. calcarifer, and the optimum inclusion level should be between 1–3%.


Introduction
Asian seabass Lates calcarifer is widely distributed in the Indo-Pacific region. It is one of the most important marine aquaculture finfish in Australia and Asian countries, and the aquaculture of L. calcarifer increased to 76,842 tons in 2015 [1]. Because of its high nutritional value and rapid growth, L. calcarifer has become one of the main aquaculture species in southern China [2]. However, the outbreak

Experimental Design and Sampling
Before the experiment started, a total of 360 fish were randomly assigned to 12 tanks of 500 L (30 fish/tank) with a flow though system for a 14-day acclimation and fed with experimental diets. After acclimation, the feeding trail started and lasted 56 days. During the experimental period, fish feeding activity and death were observed and recorded, and the water quality was tested. The experiment was conducted in a circulation system and the water exchange rate was a 200% tank volume per day in each tank. The quality of water parameters was measured daily and maintained at a temperature of 27.5 ± 1.3 • C, a salinity of 32.5 ± 0.5% , ammonia nitrogen < 0.01 mg/L, pH 7.5 ± 0.2, and nitrite nitrogen < 0.02 mg/L. The water quality parameters were maintained by adjusting the water exchange rate. Water was run through a mechanical filter and a biofilter and the water exchange occurred only when the water quality parameters exceeded the above ranges. The photoperiod was controlled at 14 h light and 10 h dark. Fish were fed ad libitum twice a day at 09:00 and 15:00. The feces and residual feeds were removed from the experimental tank daily by a siphon method. The residual feeds collected from each tank were dried and weighted. At the end of the feeding trial, fish from each tank were counted for the final survival and were weighted to determine weight gain (WG). All the diets used in each tank were calculated to obtain feed intake (FI). Three fish from each tank were randomly collected after anaesthetizing with overdose of eugenol (7 mg/L eugenol, Shangchi Dental Material Co., Ltd., Changshu, China). Afterwards, fish were dissected on ice. Liver and kidney from each fish were stored in liquid nitrogen until further usage. Other fish growth performance indicators were calculated including specific growth rate (SGR), hepatosomatic index (HIS), and intraperitoneal fat ratio (IPF). The parameters were calculated as: WG = final body weight − initial body weight; FI = (feed consumed per tank/fish) / days, SGR = 100 × ((ln (final body weight) − ln (initial body weight)) /days; HIS = 100 × ((liver weight) / (whole-body weight)); IPF = 100 × ((intraperitoneal fat weight)/(whole-body weight)).

Gene Expression Analysis
The frozen tissue samples were homogenized in liquid nitrogen using a bioprep (Bioprep-24, Hangzhou Allsheng Instruments Co. Ltd., Hangzhou, China). The RNA extraction was performed according to the method described by Fu et al. [18]. The quantity of isolated RNA was determined by measuring their absorbance at 260 and 280 nm using an ND 5000 spectrophotometer (BioTeke Corporation, Beijing, China). Finally, the integrity of RNA was assessed using agarose gel (1%) electrophoresis. The cDNA was synthesized using the PrimeScript ® RT Master Mix (Perfect Real Time, Takara Biomedical Technology (Dalian) Co., Ltd., Dalian, China). The synthesized cDNA samples were stored at −20 • C until further use. The 10 µL of reaction: 2 µL 5 × PrimeScript ® RT Master Mix, RNA 500 ng, some Rnase Freed H 2 O (total volume was 10 µL). Reverse transcription reaction conditions: 37 • C, 30 min; 85 • C, 5 s.
The genes chosen for analysis by qPCR were selected from the L. calcarifer NCBI database (https://www.ncbi.nlm.nih.gov/). The Primer Premier 5 program (Premier Biosoft International, Palo Alto, CA, USA)was used for designing the primers of crp, c-3, c-4, mtor, mlst-8, eif4e, hsp-70, hsp-90, il-8, il-10, tgfβ1, tnf, ifn-γ1, mxf, and β-actin ( Table 2). The direct and indirect effects of these gene transcription and translation products on fish immunity have been confirmed in numerous studies [19][20][21][22][23][24]. The qPCR was performed with the Real-Time qPCR analysis (Hangzhou Longgene Scientific Instrument Co., Ltd., Hangzhou, China) using SYBR Green (Tiangen Biotech Co., Ltd., Beijing, China) [18]. The 20 µL of reaction including 10 µL 2 × Real Universal PreMix, 0.6 µL 10 µM of each primer (10 µM), and 2 µL of diluted cDNA was initially denatured at 95 • C for 10 min and then amplified for 40 cycles (95 • C, 10 s, 58 • C, 20 s, and 72 • C 30 s). Each sample was subjected to qPCR for 3 times. At the end of each RT-qPCR cycle, the melting curve of the primer was analyzed to ensure that only specific products were obtained, and no primer dimer was formed. In addition, a negative control group without DNA a template was set to verify that the PCR process was not contaminated. The relative mRNA expression levels of the target genes were determined by the 2 −∆∆ Ct method and were normalized based on the level of housekeeping gene (β-actin). It has been verified that the reaction efficiency (E) was 90-105% and Pearson's coefficients of determination (R 2 ) > 0.98.

Statistical Analysis
The data were expressed as the mean ± standard deviation (SD). The software SPSS 19.0 (International Business Machines Corporation, Chicago, Illinois, USA) was used for statistical analysis, and one-way ANOVA and Least significant difference (LSD) test were used for inter-group comparison. The level of significance was set at p < 0.05. All data were tested for normality, homogeneity, and independence to satisfy the assumptions of ANOVA.

Results and Discussion
Compared with the control group, the weight gain rate and specific growth rate of each experimental group showed an upward trend, and both showed a significant increase at 5% of the experimental group (p < 0.05, Table 3). The survival rate increased with the increase of G. uralensis content. There was no significant difference in the feed intake and hepatosomatic index among all groups. Intraperitoneal fat ratio decreased with the increase of G. uralensis content, and there was a significant difference between the 5% group and other groups (p < 0.05). Dietary G. uralensis significantly affected the expression of immune-related genes, such as crp eif4e mlst-8 mtor, hsp-70, hsp-90, c-3, and c-4, in the liver of L. calcarifer (p < 0.05, Figure 1). Compared with the control group, the relative expression level of the crp gene in the experimental group was significantly upregulated (p < 0.05), and the expression levels in the 3% and 5% groups were significantly higher than in the 1% group (p < 0.05). The expression levels between the 3% and 5% groups were not significantly different (p > 0.05). The eif4e gene expression levels in the 1% and 3% grade were significantly downregulated (p < 0.05) and was significantly upregulated in the 5% group (p < 0.05). The expression levels of the mlst-8 and mtor genes in the 1% and 3% groups were not significantly different from those in the control group (p > 0.05), but the relative expression levels in the 5% group were significantly upregulated (p < 0.05). G. uralensis had the opposite effect on genes hsp-70 and hsp-90. It almost completely inhibited the expression of the hsp-70 gene, but it had a significant upregulating effect on the hsp-90 gene. G. uralensis had the same effect on genes c-3 and c-4. The relative expression levels of c-3 and c-4 were not significantly different between the 1% group and the control group (p > 0.05) but were significantly upregulated in the other two groups (p < 0.05). Therefore, G. uralensis can promote the expression of most immune-related genes in the liver of L. calcarifer. Moreover, 5% G. uralensis can upregulate the expression of seven immune-related genes other than hsp-70.
G. uralensis significantly affected the expression of kidney immune-related genes including il-8, il-10, tnf, ifn-γ1, mxf, and tgfβ1 in L. calcarifer (p < 0.05, Figure 2). The content of 1% dietary G. uralensis could effectively upregulate the expression of il-8 in the kidney of L. calcarife, but as the proportion of G. uralensis increased, the expression level of il-8 was downregulated significantly (p < 0.05). The expression pattern of il-10 was almost the same as that of il-8; 1-3% dietary G. uralensis could upregulate its expression but in excess, its relative expression was significantly downregulated (p < 0.05). For tnf, its relative expression was significantly upregulated with the increase of G. uralensis content (p < 0.05). The expression of tnf was the exact opposite to the expression of ifn-γ1; G. uralensis appears to have a strong inhibitory effect on ifn-γ1. A small amount of G. uralensis inhibited the expression of the mxf gene, but the expression was significantly upregulated when its content increased (p < 0.05). Dietary G. uralensis significantly upregulated the expression level of tgfβ1 in fish (p < 0.05). The highest expression level of tgfβ1 was observed in fish fed with 1% G. uralensis group (p < 0.05).
In terms of growth, the results of this study show that the addition of G. uralensis to feed has an obvious promoting effect on the growth of L. calcarife juvenile. Similarly, in the white shrimp Litopenaeus vannamei, the specific growth rate of the feed group with glycyrrhizin is significantly higher than that of the control group [36]. Feeding G. uralensis diets significantly increased (p < 0.05) growth performance and antioxidant and immune response in yellow perch Perca flavescens [37]. Glycyrrhetinic acid has been shown to increase the activity of fish digestive enzymes and to increase the expression of tumor necrosis factor (tnf-α) and lipoprotein lipase (lpl) to promote lipolysis for energy, thereby saving more protein for deposition for increased growth performance [15]. In this study, it was also found that the increase of G. uralensis content in the feed reduced the intraperitoneal fat ratio of L. calcarife juveniles. This suggests that dietary supplementation of licorice may enhance protein deposition in juveniles by promoting lipolysis, which might result in increased growth performance. Furthermore, in commercial fish, intraperitoneal fat is usually removed along with the viscera as an inedible portion, and it is generally undesirable, so feeding G. uralensis diets improves the product quality of L. calcarife to some extent [38]. In addition, the survival rate of the juveniles in the experimental group was significantly increased, which was a direct reflection of the effect of licorice on fish immunity. This result is similar to previous reports in yellow croaker [15] and yellow catfish [16]. After nitrite stress, the survival rate of Epinephelus coioides supplemented with fermented G. uralensis is also significantly improved [33].
Meanwhile, dietary G. uralensis supplementation did not decrease but slightly increased the intake of feed in the juveniles. This indicates that G. uralensis is a feasible feed additive. G. uralensis significantly affected the expression of kidney immune-related genes including il-8, il-10, tnf, ifn-γ1, mxf, and tgfβ1 in L. calcarifer (p < 0.05, Figure 2). The content of 1% dietary G. uralensis its expression but in excess, its relative expression was significantly downregulated (p < 0.05). For tnf, its relative expression was significantly upregulated with the increase of G. uralensis content (p < 0.05). The expression of tnf was the exact opposite to the expression of ifn-γ1; G. uralensis appears to have a strong inhibitory effect on ifn-γ1. A small amount of G. uralensis inhibited the expression of the mxf gene, but the expression was significantly upregulated when its content increased (p < 0.05). Dietary G. uralensis significantly upregulated the expression level of tgfβ1 in fish (p < 0.05). The highest expression level of tgfβ1 was observed in fish fed with 1% G. uralensis group (p < 0.05).  Reactive protein (crp) is a phylogenetically highly conserved plasma protein, with homologs in vertebrates and many invertebrates, that participates in the systemic response to inflammation [21]. The crp is capable of specifically binding to and modulating the function of mononuclear phagocytes [39]. In this study, the expression of crp gene in the liver of L. calcarifer was upregulated after fish intake of G. uralensis. The relative expression levels of crp gene were highest in the 3% and 5% groups. Eukaryotic translation initiation factor 4E (eif4e) plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. The gene eif4e exists in both phosphorylated and non-phosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eif4e phosphorylation facilitates its participation in protein synthesis [40]. Our study showed that a small amount of G. uralensis had a certain inhibitory effect on eif4e, but a significant promoting effect was observed when the dietary G. uralensis inclusion level was over 5%.
The mechanistic target of rapamycin (mtor) is the target of a molecule named rapamycin or sirolimus, which is a macrolide produced by Streptomyces hygroscopicus bacteria and that first gained attention because of its broad antiproliferative properties [41]. The mtor kinase nucleates two distinct protein complexes termed mtor-c1 and mtor-c2. The mtor-c1 responds to amino acids, stress, oxygen, energy, and growth factors and is acutely sensitive to rapamycin. The mtor-c2 responds to growth factors and regulates cell survival and metabolism as well as the cytoskeleton [42]. The mammalian lethal with SEC13 protein 8, is the binding protein of the target protein of rapamycin [43] and involved in both mtor-c1 and mtor-c2 [44]. In this study, the expression of mtor was upregulated in the 1% and 5% G. uralensis inclusion groups, and the highest expression level was observed in the 5% inclusion group. Similarly, the expression of mlst-8 was upregulated significantly in the 5% group. It may suggest that 5% G. uralensis could promote the expression of mtor and its binding protein gene mlst-8.
Heat shock proteins (hsp) belong to the family of highly conserved cellular proteins present in all organisms that have been examined [45]. Hsp-70 and hsp-90 are the two main proteins in the heat shock protein family [23]. Hsp-70 is known to assist the folding of nascent polypeptide chains, act as a molecular chaperone, and mediate the repair and degradation of altered or denatured proteins [46]. Hsp-90 is active in supporting various components of the cytoskeleton and steroid hormone receptors [47]. Our results showed that G. uralensis had a consistent inhibitory effect on the expression of hsp-70 gene in the liver of L. calcarifer, while it had the opposite effect on hsp-90, especially in the 5% inclusion group. Complement proteins c-3 and c-4 are also classified as acute phase reactants as their synthesis is upregulated during inflammation. In this study, low levels of G. uralensis did not affect the expression of c-3 and c-4 gene in the liver of L. calcarifer, while higher levels significantly upregulated their expression. This may suggest that higher dose G. uralensis can promote the expression of c-3 and c-4 effectively in L. calcarifer.
Interleukin-8 (il-8) is a chemokine that can activate neutrophils and has endogenous leukocyte chemokine and activation [48]. It is an important pleiotropic cytokine that mediates inflammatory responses and regulates the differentiation and proliferation of some immune cells. It mainly regulates the inflammatory response, which can not only inhibit mononuclear macrophages to release immune medium antigen presentation and cell phagocytosis [49]. In this study, the expression of il-8 gene in the kidney of L. calcarifer in 1% groups was significantly higher than the other groups, and the expression level was downregulated as the content of G. uralensis increased. However, the highest level of il-10 was found in the 3% group. Tumor necrosis factor (tnf), as a cytokine, it not only has cytotoxic effect on tumor cells, but also participates in a variety of pathophysiological processes such as antivirus, anti-infection, coagulation, fever and inflammation, shock, multi-organ failure and malignant fluid. Interferon (ifn) is a broad-spectrum antiviral glycoprotein secreted by recipient cells after viral infection of cells and the body or by nucleic acid bacterial endotoxin cytokinin. Ifn-γ1 is an important member of the ifn family, which also called IIinterferon or immune interferon, mainly involved in inducing major histocompatibility antigen expression and immune regulation effect [50]. The results showed that the expression level of tnf was upregulated with the increase of content of G. uralensis, while the expression level of ifn-γ1 was the opposite.
Transforming growth factor beta (tgfβ) family is a kind of superfamily polypeptide which has the function of regulating cell growth and differentiation, and tgf-β1 is a member of this family [51]. In this study, we could clearly see that the relative expression level of the tgfβ1 gene in the kidney of L. calcarifer with G. uralensis was significantly higher than that of the control group. Myxovirus resistance (mx) is an antiviral protein that can be activated by ifn-I. Mx proteins belong to the dynamin superfamily and contain a tripartite guanosine triphosphate (GTP) binding domain which is essential for the antiviral activity [52,53]. In our study, adding 3-5% G. uralensis could significantly upregulate the expression of mxf gene.

Conclusions
In summary, dietary G. uralensis significantly improved growth performance and promoted the expression of immune-related genes in the liver and the kidney of L. calcarifer. Dietary G. uralensis can significantly upregulate the expression level of crp, mtor, hsp-90, c-3, and c-4 genes in fish liver, and significantly affected the expression of il-8, il-10, tnf, ifn-γ1, mxf, and tgfβ1 in fish kidney. Results from the present study indicated that dietary G. uralensis may improve the immune function of L. calcarifer, and the optimum inclusion level should be 1-3%. Adding G. uralensis to the feed will help to improve the growth, survival, and immunity of L. calcarife.

Conflicts of Interest:
The authors declare no conflict of interest.