Effect of Licorice on Gene Expression Related to the Growth of Asian Seabass Lates calcarifer

work


Introduction
The Asian seabass is a euryhaline fish found mainly in the Western Pacific, being widely farmed in Australia [1].Renowned for its taste, rapid growth, and large size, the Asian seabass has a high economic value and is the primary aquaculture species in China [2].Extensive studies were conducted on the dietary requirements of the Asian seabass, including proteins, fats, carbohydrates, and minerals, as well as the need for additives such as probiotics and antibiotics [3,4].Studies have shown that some traditional Chinese herbs or extracts, such as Allium sativum [5], Cissus quadrangularis [6], Mentha piperita [7], Polygonum chinense [8], can improve the growth performance of Asian seabass.Traditional Chinese herbs, which are rich in nutrients and medicinal components, are characterized by their abundance, multi-targeting properties, diverse pharmacological effects, low toxicity, and minimal residues, making them ideal green additives for aquaculture feed [9][10][11].These additives promote growth, enhance immunity, reduce costs, and improve profitability [12].
In this study, juvenile Asian seabass were fed with feed additives containing licorice, and then their growth performance was measured.The qrt-PCR method was used to study the effects on the level of growth-related genes and muscle growth-related genes.The present research aimed to reveal the mechanisms by which licorice influences the growth of Asian seabass at the molecular level, providing a scientific basis for its application in the aquaculture production of Asian seabass and acting as an important reference for the use of traditional Chinese herbal resources.

Feed Formulation and Experimental Design
Fish and soybean meals were the main protein sources used.Fish oil and wheat flour were the main fat and carbohydrate sources, respectively.A single-factor concentration gradient method was employed to design four levels of licorice feed with mass fractions of 0%, 1%, 3%, and 5%.The basic feed without licorice served as a control group.According to the nutritional requirements of Asian seabass [33], the experimental feeds were formulated to have a protein level of 41% and a lipid level of 17%, with consistency maintained across all experimental groups.The raw materials were ground and sieved through a 40-mesh sieve, mixed thoroughly using a mixer, pelletized using a small pellet machine (pellet diameter 2.0 mm), and stored at −20 • C for later use.The feed ingredients and nutritional components are listed in Table 1.

Experimental Method
Juvenile Asian seabass were bred by the Sanya Tropical Fisheries Research Institute Lingshui Experimental Center (Lingshui, Hainan, China).The fish weighed 13.93 ± 0.87 g and had a length of 8.78 ± 0.39 cm.Three hundred and sixty healthy, active, and responsive Asian seabass with smooth skin, no injuries, and good feeding behavior were randomly classified into four experimental groups, with three replicates per gradient and 30 fish per replicate.The feeding trial was conducted in flow-through seawater culture tanks (800 L) at the Sanya Tropical Fisheries Research Institute Lingshui Experimental Center (Lingshui, Hainan, China).During the experiment, the feeding behavior and mortality of the fish, as well as the water quality were monitored and recorded.The key water quality parameters were 26-29 • C, pH 7.3-7.8,and nitrite < 0.02 mg/L.Feeding was performed at 9:00 AM and 3:00 PM every day, using a satiation feeding method until the fish stopped feeding.
Approximately 1 h after feeding, the tanks were siphoned to remove feces and prevent water pollution.The water was changed at 4:30 PM, with two-thirds of the water being replaced.The experiment lasted for 56 d, after which the fish were weighed for calculated growth performance.12.9 12.9 12.9 12.9 Vitamin premix (Vp) (1)  0.5 0.5 0.5 0.5 Mineral premix (Mp) (2)  0.5 0.5 0.5 0.5 Fish oil (Fo) 13

Experiment Sampling
At the end of the feeding trial, three fish were sampled from each tank (i.e., nine fish per treatment) and placed in seawater containing 7 mg/L eugenol (provided by Changshu Shangchi Dental Materials Co., Ltd., Changshu, China) for anesthesia.Once anesthetized, the fish were quickly dissected to collect the brain tissue for GHRH and GH; the liver tissue for GHR, IGF1, and IGF2; and the muscle tissue for IGF2R, myostatin 1 (MSTN1), and Myosta2tin 2 (MSTN2).The tissues were then placed in cryogenic vials, flash-frozen, and stored at −80 • C.

RNA Extraction
The stored tissues were ground, and the total RNA was extracted using the previous method [34].The concentration of the extracted RNA was detected using an ND 5000 micro-volume spectrophotometer (Beijing Baitake Biotechnology Co., Ltd., Beijing, China) at 260 and 280 nm to analyze the RNA integrity and purity.

qrtPCR Experiment
The extracted RNA was used for cDNA synthesis based on the PrimeScript™ Mix (Takara Bio Engineering Company) protocol and was then stored at −20 • C. The reaction mix for reverse transcription included 2 µL RT Master Mix.The conditions for the reverse transcription reaction were 37 • C for half an hour, followed by 85 • C for 5 s.The growthrelated gene sequences of Asian seabass, including GHRH, GH, GHR, IGF1, IGF2, IGF2R, MSTN1, and MSTN2 were obtained from the NCBI database, and primers were designed with a reference gene as the control (Table 2).The qPCR was performed on an rt-PCR device (Langji Scientific Company).The 20 µL reaction mix included 10 µL 2 × Real PreMix, 0.6 µL 10 µM primers, and 2 µL diluted cDNA.The PCR program was as follows: 95 • C for 15 min; 95 • C for 10 s; 58 • C for 20 s; 72 • C for 30 s; 40 cycles.A melt curve was applied to ensure the specificity of the products and the absence of primer-dimer formation.A no-DNA template control was used to confirm the absence of contamination during PCR.Notes: Hormone-releasing hormone (GHRH), growth hormone (GH), growth hormone receptor (GHR), insulinlike growth factor 1 (IGF1), insulin-like growth factor 2 (IGF2), IGF2 receptor (IGF2R), myostatin 1 (MSTN1), and myostatin 2 (MSTN2).The PCR efficiency of the primers listed in the table has been verified to be 90-110%.

Calculation and Statistical Analysis
The calculation formulas of survival rate (SR), weight gain (WG), feed intake (FI), and specific growth rate (SGR) are as follows: SR (%) = 100 × number of fish at the end of the test/number of fish at the beginning of the test: W t -final average weight (g); W 0 -initial average weight (g); t-the number of days the feeding text lasted.
The relative levels of gene mRNA were detected based on the 2 −∆∆Ct approach, with the β-actin as the reference gene for normalization.Experimental data were presented as mean ± SD.The SPSS 19.0 tool was applied for statistical treatment data.The Shapiro-Wilk test was employed to check the data for normal distribution, and the Levene test was used to assess the homogeneity of variance.Comparisons among the groups were conducted using the ANOVA (LSD test), with a statistical level of 0.05.

Effects of Licorice in Feed on the Growth Performance of Asian Seabass
After adding different levels of licorice to the feed and feeding for 56 days, the survival rate, weight gain rate, body-length growth rate, and specific growth rate of the seabass are shown in Figure 1.The survival rate of the 3% and 5% licorice-supplemented diet group was significantly higher than that of the control group (p < 0.05) (Figure 1A).The survival rate reached the maximum in the 3% licorice-supplemented diet group, and the value was (98.89 ± 1.93) %.The WG was significantly increased in all the licorice treatment groups compared to the control group (p < 0.05) (Figure 1B).The FI was not significant among the groups (p > 0.05) (Figure 1C).The SGR of the 5% licorice-supplemented diet group was significantly higher than that of the control group (p < 0.05) (Figure 1D).mean ± SD.The SPSS 19.0 tool was applied for statistical treatment data.The Shapiro-Wilk test was employed to check the data for normal distribution, and the Levene test was used to assess the homogeneity of variance.Comparisons among the groups were con ducted using the ANOVA (LSD test), with a statistical level of 0.05.

Effects of Licorice in Feed on the Growth Performance of Asian Seabass
After adding different levels of licorice to the feed and feeding for 56 days, the survival rate, weight gain rate, body-length growth rate, and specific growth rate of the seabass are shown in Figure 1.The survival rate of the 3% and 5% licorice-supplemented diet group was significantly higher than that of the control group (p < 0.05) (Figure 1A).The survival rate reached the maximum in the 3% licorice-supplemented diet group, and the value was (98.89 ± 1.93) %.The WG was significantly increased in all the licorice treatmen groups compared to the control group (p < 0.05) (Figure 1B).The FI was not significant among the groups (p > 0.05) (Figure 1C).The SGR of the 5% licorice-supplemented die group was significantly higher than that of the control group (p < 0.05) (Figure 1D).

Effects of Licorice on the Expression of Growth-Related Genes in the Asian Seabass Liver
As shown in Figure 2, adding licorice to the feed significantly affected the level of the GHR and IGF genes in the livers of Asian seabass.According to the comparison result, the relative expression level of the GHR gene in the liver increased significantly after the addition of licorice (p < 0.05), and the influence of licorice on the GHR gene's relative expression level varied with its concentration, with the highest increase of 362% observed in the 5% group, followed by the 1% group, and the lowest in the 3% group.The relative expression level of the IGF1 gene in the livers of the experimental groups significantly increased with the increase in licorice content in the feed (p < 0.05), with the 5% group showing the max level of the IGF1 gene, which was 751% of the control group's level.The IGF2 gene level showed an initial increase, followed by a decrease with increasing licorice content, with significant differences between the two groups (p < 0.05).The 1% group had the highest level, followed by the 3% group, with both levels significantly increased (p < 0.05).The IGF2 level in the 5% group was significantly lower (p < 0.05).
with the increase in licorice content in the feed (p < 0.05), with the 5% group showing the max level of the IGF1 gene, which was 751% of the control group's level.The IGF2 gene level showed an initial increase, followed by a decrease with increasing licorice content, with significant differences between the two groups (p < 0.05).The 1% group had the highest level, followed by the 3% group, with both levels significantly increased (p < 0.05).The IGF2 level in the 5% group was significantly lower (p < 0.05).

Figure 2.
The relative effect of licorice on the level of growth-related genes in the liver tissue.Note: Different letters indicate significant differences.Subscripts 1, 2, and 3 represent GHR, IGF1, and IGF2, respectively.

Effects of Licorice on Level of Muscle Growth-Related Genes in Asian Seabass
As indicated in Figure 3, licorice significantly affected the level of the muscle growthassociated genes IGF2R, MSTN1, and MSTN2 in Asian seabass.With an increase in the licorice content in the feed, the expression level of the IGF2R gene initially decreased and then increased; however, the expression levels of IGF2R in all experimental groups were significantly lower than those in the other group (p < 0.05).Similar to the results of the IGF2R gene, the MSTN1 gene level also initially decreased and then increased, with the lowest level observed in the 1% group.The level increased in the 3% group and was significantly higher compared to the other groups (p < 0.05) and continued to increase in the 5% group, reaching 333% of that in the control group.The addition of licorice to the feed caused a significant reduction in the MSTN2 gene level at all levels (p < 0.05).

Effects of Licorice on Level of Muscle Growth-Related Genes in Asian Seabass
As indicated in Figure 3, licorice significantly affected the level of the muscle growthassociated genes IGF2R, MSTN1, and MSTN2 in Asian seabass.With an increase in the licorice content in the feed, the expression level of the IGF2R gene initially decreased and then increased; however, the expression levels of IGF2R in all experimental groups were significantly lower than those in the other group (p < 0.05).Similar to the results of the IGF2R gene, the MSTN1 gene level also initially decreased and then increased, with the lowest level observed in the 1% group.The level increased in the 3% group and was significantly higher compared to the other groups (p < 0.05) and continued to increase in the 5% group, reaching 333% of that in the control group.The addition of licorice to the feed caused a significant reduction in the MSTN2 gene level at all levels (p < 0.05).

Effects of Licorice on Level of Growth-Related Genes in Brain Tissue of Asian Seabass
The influence of licorice on the level of the growth-related genes GH and GHRH in the brain tissue of Asian seabass is illustrated in Figure 4.After adding licorice, the level of GH decreased significantly (p < 0.05).The level of the GHRH gene exhibited a trend of initially decreasing and then increasing with increasing licorice content.The lowest level

Effects of Licorice on Level of Growth-Related Genes in Brain Tissue of Asian Seabass
The influence of licorice on the level of the growth-related genes GH and GHRH in the brain tissue of Asian seabass is illustrated in Figure 4.After adding licorice, the level of GH decreased significantly (p < 0.05).The level of the GHRH gene exhibited a trend of initially decreasing and then increasing with increasing licorice content.The lowest level appeared in the 3% licorice group, followed by the 5% licorice group, with the highest level observed in the 1% licorice group.However, the expression levels of GHRH in all experimental groups were significantly lower than those in the control group (p < 0.05).

Figure 3.
The relative effect of Glycyrrhiza uralensis on the level of growth-associated genes in the muscle tissue.Note: Different letters indicate significant differences.Subscripts 1, 2, and 3 represent IGF2R, MSTN1, and MSTN2, respectively.

Effects of Licorice on Level of Growth-Related Genes in Brain Tissue of Asian Seabass
The influence of licorice on the level of the growth-related genes GH and GHRH in the brain tissue of Asian seabass is illustrated in Figure 4.After adding licorice, the level of GH decreased significantly (p < 0.05).The level of the GHRH gene exhibited a trend of initially decreasing and then increasing with increasing licorice content.The lowest level appeared in the 3% licorice group, followed by the 5% licorice group, with the highest level observed in the 1% licorice group.However, the expression levels of GHRH in all experimental groups were significantly lower than those in the control group (p < 0.05).

Discussion
We found that adding a proper concentration of licorice in the feed significantly promoted the expression of genes related to GHR, IGF1, and IGF2 in the liver of Asian seabass.The growth of teleost fish is primarily adjusted by the GH/IGF system and other endocrine factors.GH binds to GHR on the surface of target organs, stimulating the liver to secrete IGF [35,36].IGF binds to IGF receptors in target tissues, initiating a series of cellular processes related to growth, such as cell proliferation and differentiation, leading to overall growth [37].In the present research, it was found that adding licorice to Asian seabass feed significantly upregulated the level of GHR.The downstream genes of the GH/IGF axis in the liver, IGF1, and IGF2 showed significant changes, confirming that GH can exert biological effects through IGF1 and IGF2 after binding to GHR [38].When the addition of licorice did not exceed 3%, the levels of IGF1 and IGF2 significantly increased, suggesting that a 3% licorice addition could significantly promote processes related to cell growth.The max level of IGF1 appeared in the 5% licorice group but the significant downregulation of the IGF2 expression indicates that high levels of licorice in Asian seabass feed might affect the functions related to IGF2.
In Nile tilapia (Oreochromis niloticus), GH is expressed only in the pituitary gland, whereas GHR is expressed in various tissues and organs, including muscle, hypothalamus, and thymus, with the highest expression in the liver, indicating that GH has multiple physiological functions [39].In teleosts, MSTN1 primarily inhibits muscle hyperplasia but not hypertrophy, which is mainly achieved through the downregulation of MSTN2 expression [40].This research showed the variable influence of licorice on MSTN1 expression, indicating the stages of rapid muscle hyperplasia in Asian seabass [41].The lower expression of MSTN1 and MSTN2 with 1% licorice suggests reduced inhibition of muscle hyperplasia and promotion of muscle hypertrophy, while the increased MSTN1 expression with 3% and 5% licorice indicates an enhanced inhibition of muscle hyperplasia and the promotion of muscle hypertrophy.
This study found that after adding licorice, the level of GH in the brain samples of Asian seabass was downregulated, and the levels of GHR, IGF1, and IGF2 in the liver tissue were upregulated, possibly due to a delayed regulatory effect of GH on the growth rate and its negative feedback regulation with IGF1 [42].The hypothalamus-secreted GHRH physiologically regulates the generation and release of GH in the pituitary gland, which is an important hormone for regulating fish growth, development, reproduction, and immunity [43].In the present research, the GHRH and GH expression was significantly downregulated and did not show a linear relationship, which may be related to the multifactorial influence on GH secretion that maintains a dynamic balance between promoting and inhibiting factors [44].
The beneficial effects of licorice may be attributed to glycyrice polysaccharides (GPS).Dietary supplementation of GPS has been found to enhance growth performance, body size, and the relative expression of the growth-related gene IGF-1 in broilers [45].Furthermore, it can improve serum and intestinal immune status, promote the expression of immunerelated genes in the spleen, and enhance broiler immunity.The optimal supplemental concentration is 600 mg/kg [46,47].In weaned piglets, GPS supplementation significantly promotes the mRNA expression levels of the IGF-1 gene in the liver, as well as the IGF-1 and IGF-2 genes in the dorsal longus muscle (p < 0.05) [48].Additionally, the dietary addition of GPS improves growth performance, reduces diarrhea rate, enhances humoral immunity, promotes the related growth gene expression, and even exhibits a certain level of resistance against PRRSV infection in piglets.Supplementation with 1000 mg/kg GPS alleviates stress response, reduces diarrhea rate, and improves growth performance by enhancing the intestinal mucosal barrier effect, immune function, and intestinal microflora structure in weaned piglets [49].Moreover, GPS exhibits inhibitory effects on the TLR4/MyD88/NF-κB signaling pathway, thereby reducing the excessive expression of immune and inflammatory, apoptosis, and tight junction protein genes induced by LPS in IPEC-J2 cells [50].Additionally, they enhance the cell's antioxidant capacity and decrease ROS accumulation and the apoptosis rate, ultimately alleviating the inflammatory damage caused by LPS in the IPEC-J2 cells.It also enhances antioxidant capacity, reduces ROS accumulation and cell apoptosis rates, and mitigates inflammatory damage induced by LPS in IPEC-J2 cells.GPS serves as an effective immune enhancer to enhance the integrity of the intestinal barrier [51].The addition of licorice to animal feed can effectively stimulate the growth of terrestrial animals such as chickens and pigs, as well as that of aquatic species such as Asian seabass.Moreover, it exhibits immune-enhancing properties, thereby positioning licorice as a promising feed additive with excellent application prospects.

Conclusions
In conclusion, the inclusion of licorice in the diet of Asian seabass significantly enhances growth performance and survival rates.Specifically, diets supplemented with 3% and 5% licorice showed notable improvements in weight gain and survival compared to the control group.Licorice supplementation positively influenced the expression of growthrelated genes, particularly increasing the GHR and IGF1 levels in the liver.However, higher levels of licorice (5%) resulted in the downregulation of IGF2 expression and complex effects on the IGF2R and MSTN1 levels in the muscle tissue.Additionally, licorice inhibited the expression of GH, GHRH, and MSTN2.These findings suggest that incorporating an optimal proportion of licorice into the diet can effectively enhance the growth and health of Asian seabass in aquaculture.

Figure 1 .
Figure 1.Effects of licorice on survival rate and growth of Asian seabass.(A) survival, (B) weigh gain (WG), (C) feed intake (FI), (D) specific growth rate (SGR); means within rows with the same superscript are not significantly different (p > 0.05), while the different letters mean significant dif ferences (p < 0.05).

Figure 1 .
Figure 1.Effects of licorice on survival rate and growth of Asian seabass.(A) survival, (B) weight gain (WG), (C) feed intake (FI), (D) specific growth rate (SGR); means within rows with the same superscript are not significantly different (p > 0.05), while the different letters mean significant differences (p < 0.05).

Figure 2 .
Figure 2. The relative effect of licorice on the level of growth-related genes in the liver tissue.Note: Different letters indicate significant differences.Subscripts 1, 2, and 3 represent GHR, IGF1, and IGF2, respectively.

J 11 Figure 3 .
Figure 3.The relative effect of Glycyrrhiza uralensis on the level of growth-associated genes in the muscle tissue.Note: Different letters indicate significant differences.Subscripts 1, 2, and 3 represent IGF2R, MSTN1, and MSTN2, respectively.

Figure 3 .
Figure 3.The relative effect of Glycyrrhiza uralensis on the level of growth-associated genes in the muscle tissue.Note: Different letters indicate significant differences.Subscripts 1, 2, and 3 represent IGF2R, MSTN1, and MSTN2, respectively.

Figure 4 .Figure 4 .
Figure 4.The relative effect of Glycyrrhiza uralensis on the level of growth-associated genes in the brain tissue.Note: The letters indicate significant differences.Subscripts 1 and 2 represent GH, and GHRH, respectively.

Table 1 .
Feed formula and the list of ingredients.

Table 2 .
Primer of growth-related genes in barramundi were used in qPCR.