Arginine, an amino acid primarily used in protein biosynthesis, is an indispensable amino acid for fish because of their limited ability to endogenously biosynthesize arginine (Andersen et al., 2016). The ability of arginine to impact nutrient metabolism, influence insulin release, modulate immune and antioxidant response, and improve disease resistance in fish has been well demonstrated (Wang Q. et al., 2021). Arginine could stimulate the secretion of insulin-like growth factor 1 (IGF-1) and activate the TOR signaling pathway to promote protein synthesis for the fish body (Levine et al., 2006; Wei et al., 2021). Studies have shown that 3.61% dietary arginine would significantly improve the weight gain and specific growth rate of rice-field eel (Monopterus albus) (Huo et al., 2023). Furthermore, 2.52% dietary arginine would significantly increase the gene expression of Nrf2 in largemouth bass (Micropterus salmoides) (Yu et al., 2023). Like other animals, lack of arginine may interfere with the fish body normal function of riboflavin, which is not conducive to the synthesis of heme and niacin, as well as decrease resistance against pathogen challenges (Huo et al., 2023). At present, the requirements for dietary arginine have been reported for several fish species, including largemouth bass (Yu et al., 2023), hybrid snakehead (Channa maculata ♀ × Channa argus) (Li et al., 2022), spotted seabass (Lateolabrax maculatus) (Cheng et al., 2021), tiger puffer (Takifugu rubripes) (Wei et al., 2021), and rainbow trout (Oncorhynchus mykiss) (Wang et al., 2022). Arginine function may vary with dose, fish species, fish size, fish health status, and environmental conditions (Azeredo et al., 2015; Hoseini et al., 2020). Therefore, the optimum arginine supplementation level and its physiological function in formulated diets should be carefully evaluated for specific fish species, especially carnivorous fish.

Chinese perch (Siniperca chuatsi) is a temperate perch from the order Perciformes and the genus Siniperca. As a highly valued carnivorous fish, China’s S. chuatsi aquaculture output increased from 281,500 tons in 2012 to 374,000 tons in 2021 (CFSY (China Fishery Statistical Yearbook), 2021), with an annual output value of more than 20 billion yuan (Peng et al., 2022). This shows the increasing importance of Chinese perch in China’s aquaculture. In recent years, the requirements and roles of several dietary functional nutrients for Chinese perch were investigated, including exogenous probiotics (Feng et al., 2021; Zhang et al., 2022a), host probiotics (Ji et al., 2023), bile acids (Zhang et al., 2022b), pyridoxine (Xie et al., 2023), zinc (Peng et al., 2023), and feed attractants (Peng et al., 2022). However, the influence of arginine on the physiological function in Chinese perch had not been explored hitherto. To close this gap, this study evaluated the influence of dietary arginine on Chinese perch based on growth performance, antioxidant capacity, intestinal digestive enzyme activity, and gut health. In addition, transcriptome analysis was conducted to examine the influence of arginine on muscle function in Chinese perch. This study would contribute to an in-depth understanding of arginine’s physiological function in Chinese perch.

To further explore the benefits of dietary arginine to important aquaculture species, this study observed that supplementing arginine in diets of S. chuatsi from 2.61% to 3.82% did not have any significant impact on growth performance, indicating that 2.61% dietary arginine satisfied the basic growth requirements of S. chuatsi. Studies on largemouth bass (Yu et al., 2023) and hybrid snakehead (Li et al., 2022) have shown that their growth performance decreases with further increased dietary arginine levels, possibly due to the antagonistic effects between arginine and lysine. In the present study, the HSI, VSI, and CF were not significantly different between the different dietary groups, like rice-field eel (Huo et al., 2023) and golden pompano (Trachinotus ovatus) (Lin et al., 2015). The dietary arginine requirement varies with different fish species, nutrition levels, weights, and breeding densities. The requirement of most fish species was previously reported to range from 1.20% to 1.50% (dry matter) except in Pacific salmon (2.04%, dry matter) (Ball et al., 2007). Further studies later revealed a much higher arginine requirement of over 2.0% in other fish species, especially carnivores (Hoseini et al., 2020; Wang Q. et al., 2021), such as largemouth bass (2.40%–2.63%, dry matter) (Yu et al., 2023), rice-field eel (3.16%–3.63%, dry matter) (Huo et al., 2023), hybrid snakehead (2.95%, dry matter) (Li et al., 2022), and spotted seabass (2.53%, dry matter) (Cheng et al., 2021).

Digestive enzyme activity is an important indicator of how diets affect intestinal health (Roxas, 2008). Although dietary arginine supplementation did not influence the intestinal trypsin and lipase activities, interestingly, amylase activity in S. chuatsi significantly decreased when the dietary arginine supplementation level reached 3.82%. This provides a new perspective on the interaction of l-arginine with digestive enzymes in fish species. Similarly, L-arginine was shown to inhibit the activity of α-amylase by decreasing β-sheets and increasing random coils (Ye et al., 2022).

Lack of balance between reactive oxygen species (ROS) and the antioxidant defense system in animals can lead to oxidative stress (Hoseinifar et al., 2020). To reduce oxidative stress, superoxide dismutase reacts against ROS and catalyze the reaction converting O₂ ⁻ into O₂, and H₂O₂ is further converted into water with CAT or GSH-Px (Zengin, 2021). An increased MDA level is an indication of lipid peroxidation from oxidative stress (Garcia et al., 2020). Our study showed that S. chuatsi fed a 3.37% arginine diet had a significantly increased serum TSOD activity and a decreased MDA content, which indicates that arginine could improve the antioxidant capacity in S. chuatsi. Similar results were reported in juvenile blunt snout bream (1.62% and 1.96% arginine levels) (Liang et al., 2018a) and juvenile Chinese mitten crab (2.7%–3.7% arginine level) (Qi et al., 2019). Arginine has been shown to increase antioxidant capacity by stimulating GSH synthesis and modulating the Nrf2-Keap1 pathway (Liang et al., 2018b).

As a precursor for nitric oxide (NO) synthesis, arginine is very essential in regulating energy metabolism. It also plays a role in several physiological processes like degradation of amino acids and fatty acid and glucose syntheses (Tan et al., 2011; Wang Q. et al., 2021). This study showed that the muscle arginine content of S. chuatsi increased with graded dietary arginine levels. The accuracy of the transcriptome data was confirmed by verification of the expression profiles of selected genes [differentially expressed genes (DEGs)] using qRT-PCR. The clustering of muscle DEGs in arginine biosynthesis and the mTOR signaling pathway supported this result. The mTOR signaling pathway controls many metabolic processes and is regulated by amino acid signals, especially arginine (Gai et al., 2016). Muscle DEGs clustered in the mTOR signaling pathway were observed in fish fed diets with arginine inclusion. Muscle valine, glutamate, tyrosine, and total non-essential amino acid contents were significantly decreased in fish fed 3.82% dietary arginine in this study. Although muscle DEGs clustered in biosynthesis of amino acids were observed with dietary arginine supplementation, the influence of arginine on the metabolism of other amino acids remains unknown. In addition, KEGG pathway analysis showed a dose-dependent arginine function. Different arginine supplementation levels influenced different genes and their functions in S. chuatsi. The PI3K-Akt signaling pathway, MAPK signaling pathway, FoxO signaling pathway, and insulin pathway were clustered under arginine supplementation. Insulin is the core substance of glucose metabolism, and it can strictly control the utilization of glucose. Currently, there are at least two known insulin signal transduction pathways: the PI3K-Akt signaling pathway and MAPK signaling pathway (Guan et al., 2021). PI3K and Akt are two major nodes downstream of insulin receptor substrate 1 (IRS-1), which could regulate the proliferation and differentiation of skeletal muscle myoblasts and modulate the absorption of sugar and amino acids in tissues (Taniguchi et al., 2006). Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt (Yamagata et al., 2008). MAPK signaling could regulate insulin sensitivity to control glucose metabolism (Zhang et al., 2011). In parallel with that, the serum glucose content decreased with arginine supplementation in S. chuatsi. Studies have reported that a 2.31% dietary arginine level resulted in the upregulation of the relative gene expression of IRS-1, PI3K, and Akt in the insulin signaling pathway (improving glycolysis), while a 2.70% dietary arginine level led to inhibition of these genes (high plasma glucose) in juvenile blunt snout bream Megalobrama amblycephala (Liang et al., 2017). Our result showed that an arginine supplementation level of approximately 2.99%–3.82% could lead to serum glucose concentration, which was closely associated with muscle glucose metabolism in S. chuatsi. Lipid metabolism-associated pathways were also influenced by the arginine supplementation level in our study. Similarly, dietary L-arginine supplementation (1%–2%) in tilapia reduced high fat diet-induced fat deposition and fatty acid composition in the liver by regulating the expression of genes for lipid metabolism (Li et al., 2020).

Gut microbes play an essential role in regulating immune response, nutritional status, and metabolic homeostasis (Lai et al., 2020). According to the results from this study, arginine supplementation modulated the bacterial community of Chinese perch. Mycoplasma was the predominant genus in the gut microbes of Chinese perch from the control group, while Cetobacterium dominated in the dietary arginine supplementation groups probably due to the ability of arginine to regulate glucose homeostasis in fish (Wang A. et al., 2021). The low carbohydrate utilization ability of carnivorous fish can lead to postprandial hyperglycemia in high-carbohydrate diets (Hemre et al., 2002). The enhancement of Cetobacterium by arginine can be explored to ameliorate the deleterious effects of high carbohydrate in diets. The enrichment of Cetobacterium could also be observed with additional probiotic treatment (Wang et al., 2020). Cetobacterium is an anaerobic indigenous bacterium present in the gut of most freshwater fish (Tang et al., 2022), and it also acts a sensor of health. The abundance of Cetobacterium would sharply decrease in infected Aeromonas veronii (Liu et al., 2020). Meanwhile, Ofek et al. (2022) also confirmed that the abundance of Cetobacterium was lower in infected tilapia than in healthy tilapia. The enhancement of Cetobacterium by arginine can be explored to ameliorate the deleterious effects of high carbohydrate in diets. Results of network analysis highlighted that adding arginine had a marked impact on the interaction between bacteria taxa, rather than relative abundance. Adding arginine reduced the network complexity by more than 73.1%, which implies microbial interaction. However, it promoted the ratio of positive correlations and led to 100.0% in its network. These results indicated that adding arginine could eliminate microbial competition and Cetobacterium has the potential to produce secondary metabolites to strengthen microbial cooperation due to the weaker carbohydrate metabolism in the gut microbes of carnivorous fish. Cetobacterium might act as the key species in maintaining fish health and providing protection against some pathogenic bacteria. Cetobacterium is a unique species capable of synthesizing vitamin B₁₂ (Qi et al., 2023), and over 80% of microbes require vitamin B₁₂ for their growth (Zhao et al., 2023). Overall, the increase of Cetobacterium might strengthen interactions among gut microbes and improve host resistance to pathogen infections. These results imply that arginine supplementation can proliferate Cetobacterium to improve microbial cooperation and eliminate competition, thus improving the growth of Chinese perch.

Our results revealed that although growth performance was not significantly influenced by dietary arginine supplementation, antioxidant capacity, intestinal digestion, and nutritional metabolism were significantly impacted in Chinese perch. Further transcriptome analysis result showed that graded arginine levels significantly influenced muscle gene expression involved in KEGG pathways of energy metabolism (amino acid, glucose, fatty acid), signaling pathways (mTOR, PI3K-Akt signaling pathway, MAPK signaling pathway, FoxO signaling pathway, and insulin pathway), etc. Microbial analysis showed that adding arginine reduced the network complexity of potential interactions between bacterial taxa and eliminated microbial competition. Based on antioxidant and intestinal health indicators, the optimal dietary arginine requirement for Chinese perch was 2.99%–3.37% of the dry diet (6.08%–6.79% of dietary protein).

The original contributions presented in the study are included in the article / supplementary material. Further inquiries can be directed to the corresponding. The RNA sequencing data has been uploaded to NCBI NO. PRJNA1029502 (https://www.ncbi.nlm.nih.gov/). The high-throughput sequencing data has been uploaded to NCBINO. PRJNA1029319 (https://www.ncbi.nlm.nih.gov/).

The animal study was approved by The Animal Care and Use Committee of Jiangxi Fisheries Research Institute. The study was conducted in accordance with the local legislation and institutional requirements.

LD: Data curation, Investigation, Writing – original draft. JC: Data curation, Investigation, Methodology, Writing – original draft. FH: Formal analysis, Investigation, Writing – original draft. QC: Project administration, Supervision, Writing – review & editing. YL: Supervision, Writing – review & editing. WC: Funding acquisition, Writing – review & editing.