Leucine regulates the mTOR pathway to improve growth and enhance immune responses in Scylla paramamosain challenged with Vibrio parahaemolyticus

Abstract

A 63-d feeding trial was performed to determine the effects of varying dietary leucine levels on the growth, protein metabolism, and disease resistance of Scylla paramamosain against Vibrio parahaemolyticus. A total of 360 juvenile crabs (approximately 50 d after metamorphosis from the megalopa stage and having undergone eight molts; initial weight: 8.65 ± 0.58 g) were randomly assigned to six dietary treatments, with 60 crabs per group, each treatment comprising three replicates of 20 crabs each. Each group was fed one of the six isonitrogenous and isolipidic diets, which were formulated to provide graded leucine levels of 1.81% (control), 2.10%, 2.40%, 2.67%, 2.91%, and 3.18%, respectively. The results showed that crabs fed diets with 2.67% leucine exhibited significantly higher percent weight gain (PWG), molting frequency, and protein efficiency ratio, together with a lower feed conversion ratio, than those fed diets with 1.81%, 2.10%, or 3.18% leucine (P < 0.05). Muscle and hepatopancreas protein contents were significantly higher in the 2.67% and 2.91% leucine groups than in the 1.81% leucine group (P < 0.05). Dietary leucine also altered the muscle amino acid profile and enhanced hepatopancreatic digestive enzyme activities (P < 0.05). At leucine levels of 2.67%–2.91%, leucine significantly upregulated selected mTOR-related genes and increased TOR and p70S6K1 protein levels (P < 0.05). Following V. parahaemolyticus challenge, the 2.91% leucine group showed the highest survival and lower hemocyte apoptosis (P < 0.05). The overall survival difference showed a tendency (log-rank P = 0.056), whereas Cox regression indicated a reduced mortality risk versus the 1.81% leucine group (P = 0.024). Compared with the control, leucine supplementation significantly enhanced antioxidant capacity and innate immune enzyme activities, accompanied by increased expression of antioxidant, immune, amino acid transporter, mTOR signaling, and inflammatory-response genes (P < 0.05). Based on PWG against dietary leucine levels, the optimal dietary leucine requirement was determined to be 2.70% for mud crab under the present crab-apartment recirculating aquaculture system rearing conditions, whereas dietary 2.91% leucine was associated with a significantly reduced mortality risk during the 48-h V. parahaemolyticus challenge period (Cox model).

1. Introduction

Leucine, a branched-chain amino acid (BCAA), stands out among dietary amino acids due to its dual role as both a fundamental structural component of proteins and a key signaling molecule that regulates various metabolic pathways (Plotkin et al., 2021). Unlike most amino acids that primarily serve as building blocks for protein synthesis, leucine uniquely stimulates the mechanistic target of rapamycin complex 1 (mTORC1), a pivotal kinase complex governing cellular growth and anabolic processes. Upon cellular uptake, leucine interacts with intracellular sensors such as sestrin2 and leucyl-tRNA synthetase, thereby relieving the inhibition of mTORC1 and initiating a phosphorylation cascade that promotes protein synthesis (Son et al., 2020). The mTORC1 signaling pathway is particularly critical in tissues with high protein turnover rates, such as skeletal muscle and hepatopancreatic cells, where it supports rapid tissue growth. Beyond its anabolic effects, leucine also plays important roles in energy metabolism. It serves as a substrate for gluconeogenesis and contributes to ketone body formation during prolonged fasting or under low-carbohydrate conditions (Ahmad et al., 2021). Leucine further supports immune function by enhancing lymphocyte proliferation and stimulating antibody production, thereby strengthening adaptive immune responses (Nie et al., 2018). Moreover, it serves as a nitrogen donor for the biosynthesis of glutamate and alanine—key intermediates in amino acid metabolism. The catabolism of leucine produces acetyl-CoA and keto acids, which feed into the tricarboxylic acid (TCA) cycle, especially under nutrient-deprived conditions (Zhang et al., 2017). Importantly, leucine exerts dose-dependent regulatory effects on mTORC1 signaling and muscle homeostasis. Inadequate leucine intake impairs mTORC1 activity and compromises muscle maintenance, while excessive leucine intake may disrupt BCAA balance by competitively inhibiting the intestinal absorption of valine and isoleucine (Castillo and Gatlin, 2018). These multifaceted roles highlight leucine as a critical modulator of growth, metabolism, and health. The dietary leucine requirements of various aquatic animals have been extensively investigated. In teleosts, the determined optimal levels exhibit significant interspecific variation. Among freshwater fishes, the reported leucine requirements range from 1.32% to 2.52% of the diet (Deng et al., 2014; Farhat, 2014; Gan et al., 2016; Ren et al., 2015; Yang et al., 2022). For marine fishes, the requirements are generally higher, spanning from 1.88% to 4.45% (Li et al., 2015; Niu et al., 2021; Sui et al., 2024; Tan et al., 2016). Research on crustaceans has quantified the leucine requirements for key farmed species, which range from 1.70% to 2.50% of the diet (Huo et al., 2017; Millamena et al., 1999; Song et al., 2023; Wei et al., 2023). Quantifying species-specific leucine requirements is critical for formulating nutritionally balanced and cost-efficient aquafeeds. Such precision not only promotes optimal growth performance and feed efficiency but also supports sustainable aquaculture practices by minimizing excess nitrogen excretion and improving feed utilization.

In addition to its fundamental metabolic roles, leucine plays a pivotal role in the regulation of immune function across species (Sun et al., 2023). Through its activation of mTORC1, leucine enhances protein translation and metabolic reprogramming, thereby promoting immune cell proliferation, antibody production, and the regulation of inflammatory responses (Liu et al., 2018). Beyond mTORC1 signaling, leucine modulates immune responses through various metabolic pathways. It serves as a precursor for the synthesis of glutamine and alanine, amino acids critical for lymphocyte energy metabolism and resistance to oxidative stress (Plotkin et al., 2021). Moreover, catabolism of leucine produces ketoisocaproate (KIC) and acetyl-CoA, which feed into the TCA cycle to support immune cell activation (Ananieva et al., 2016). Apart from these metabolic contributions, leucine is implicated in the fine-tuning of inflammatory responses. It can regulate the nuclear factor kappa B (NF-κB) signaling cascade, thereby altering the production of pivotal inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-16 (IL-16), which are crucial for mounting a balanced immune defense (Sun et al., 2023). Additionally, dietary leucine has been demonstrated to enhanced antioxidant capacity in several economically important aquaculture species, including golden pompano (Liang et al., 2018), blunt snout bream (Megalobrama amblycephala) (Tan et al., 2016; Zhou et al., 2020), and rohu (Labeo rohita) (Giri et al., 2016). Despite extensive research on leucine's roles in protein synthesis and immune regulation across aquatic species, the optimal dietary requirement and immunomodulatory functions remain largely unexplored in mud crab. Vibrio parahaemolyticus is a primary bacterial pathogen of mud crab and has been reported to cause milky disease with high mortality in mud crab aquaculture (Zhang et al., 2020). Existing studies in mud crab have mainly focused on characterizing host molecular responses after V. parahaemolyticus infection, including miRNA regulation, transcriptomic profiling, and immunometabolic alterations (Li et al., 2013; Zhang et al., 2019). However, the question of whether a targeted nutritional strategy can enhance disease resilience in mud crab remains insufficiently addressed, particularly regarding amino acids that function not only as substrates but also as nutrient signals. Leucine is a dose-dependent regulator of mTORC1 and has been shown to promote mTORC1-related protein synthesis in crab species (Song et al., 2023). Therefore, combining a graded dietary leucine regimen with a standardized V. parahaemolyticus challenge could provide a mechanism-driven framework linking growth and nutrition with infection resistance, allowing evaluation of whether leucine-mediated nutrient sensing is accompanied by coordinated changes in mechanistic target of rapamycin (mTOR)-associated and immune-related responses.

Mud crab (Scylla paramamosain) is a commercially valuable species of crustacean extensively cultivated in Asian aquaculture systems, prized for its rapid growth, high meat yield, superior flesh quality, and strong adaptability to diverse farming environments, including brackish water ponds and mangrove-based culture systems (Xie et al., 2024). In 2024, the annual production of farmed mud crab reached 162,349 tons, highlighting its significant contribution to regional aquaculture economies (Bureau of Fisheries 2025). However, the sustainability of mud crab aquaculture is increasingly jeopardized by outbreaks of V. parahaemolyticus, a pathogenic bacterium responsible for substantial economic losses due to mortality and growth retardation (Zhang et al., 2019). While supplemental dietary leucine has established benefits for growth performance and immune function in multiple aquatic species, its role in mud crab remains poorly understood. Therefore, this study aims to establish the optimal dietary leucine requirement for promoting growth and protein deposition in S. paramamosain, and to investigate whether leucine supplementation can enhance resistance against V. parahaemolyticus infection. These findings are expected to contribute to the development of nutritionally optimized and disease-resilient feed strategies for sustainable mud crab aquaculture.

2. Materials and methods

2.1. Animal ethics statement

All experimental procedures involving crabs were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Ningbo University (No. SYXK20190005) and were approved by its Animal Ethics Committee. The study adhered to established ethical standards for research involving aquatic organisms.

2.2. Diets design and preparation

Six experimental diets containing 47% crude protein and 10% crude lipid were formulated with graded leucine levels. The leucine contents of the experimental diets were determined using an amino acid analyzer (L-8900, Hitachi High-Tech Corporation, Tokyo, Japan). The measured leucine contents were 1.81% (control), 2.10%, 2.40%, 2.67%, 2.91%, and 3.18%, respectively. The experimental diets were formulated to meet the nutritional requirements of crabs based on the dietary nutrient recommendations summarized by Esmaeili et al. (2024), and the ingredients and nutrient levels of the experimental diets are presented in Table 1. The control diet was formulated to contain Peruvian fish meal, Pacific mackerel fish meal, soybean meal, yeast extract, and krill meal. To achieve a balanced amino acid profile, the diet was supplemented with crystalline amino acids to fulfill the essential amino acid needs of mud crabs. The increased levels of dietary leucine were achieved by adding crystalline leucine, while the corresponding reductions in aspartic acid (Asp) and glycine (Gly) were calculated based on the isonitrogenous principle to maintain constant nitrogen content across all diets. All crystalline amino acids (analytical grade, purity ≥ 98%) were obtained from Shanghai Sanjie Biotechnology Co., Ltd. (Shanghai, China). Dry feedstuffs were ground and passed through a 60-mesh sieve. Subsequently, crystalline amino acids were weighed to 0.0001 g using an analytical balance and thoroughly premixed. Dry feed components were thoroughly blended in a mixer, after which the lipid and water phases were gradually incorporated. The resulting mixture was extruded into noodle-like strands using a pelletizer (F-26, South China University of Technology Machine Factory, Guangzhou, Guangdong, China) and subsequently cut into pellets with a pelletizer (G-250, South China University of Technology Machine Factory, Guangzhou, Guangdong, China) from the same manufacturer. The prepared pellets were approximately 1.5 cm in length, and two diameters (2 and 4 mm) were produced to accommodate the feeding requirements of mud crabs at different growth stages. All feed pellets were stored at −20 °C, with aliquots thawed for immediate use at each feeding event. The amino acid profiles of the six diets are presented in Table 2.

Table 1.

Ingredients and nutrient levels of the experimental diets (%, dry matter basis).

Items	Dietary leucine levels, %
	1.81	2.10	2.40	2.67	2.91	3.18
Ingredients
Peru fish meal	10.00	10.00	10.00	10.00	10.00	10.00
Pacific mackerel fish meal1	10.00	10.00	10.00	10.00	10.00	10.00
Soybean meal	10.00	10.00	10.00	10.00	10.00	10.00
Yeast extract	5.00	5.00	5.00	5.00	5.00	5.00
Krill meal	3.00	3.00	3.00	3.00	3.00	3.00
Wheat flour	25.95	25.95	25.95	25.95	25.95	25.95
EAA permix2	4.60	4.60	4.60	4.60	4.60	4.60
NEAA premix3	9.00	9.00	9.00	9.00	9.00	9.00
Leucine	0.00	0.30	0.60	0.90	1.20	1.50
Gly/Asp4	5.00	4.76	4.52	4.28	4.04	3.81
Fish oil	2.00	2.00	2.00	2.00	2.00	2.00
Soybean oil	2.00	2.00	2.00	2.00	2.00	2.00
Soybean lecithin	2.00	2.00	2.00	2.00	2.00	2.00
Cholesterol	0.50	0.50	0.50	0.50	0.50	0.50
Vitamin premix5	0.50	0.50	0.50	0.50	0.50	0.50
Mineral premix6	1.00	1.00	1.00	1.00	1.00	1.00
Ca(H2PO4)2	2.00	2.00	2.00	2.00	2.00	2.00
Choline chloride	0.30	0.30	0.30	0.30	0.30	0.30
Sodium alginate	2.00	2.00	2.00	2.00	2.00	2.00
BHT	0.20	0.20	0.20	0.20	0.20	0.20
Lutein	0.30	0.30	0.30	0.30	0.30	0.30
Attractant	2.00	2.00	2.00	2.00	2.00	2.00
Cellulose	2.65	2.59	2.53	2.47	2.41	2.34
Total	100.00	100.00	100.00	100.00	100.00	100.00
Nutrients7
Moisture	5.27	5.59	6.21	4.54	5.00	6.73
Crude protein	47.50	47.29	47.63	47.81	47.77	47.43
Crude lipid	9.86	10.39	9.91	10.07	9.89	9.83
Organic matter	92.12	92.13	92.15	92.12	92.16	92.05
Leucine	1.81	2.10	2.40	2.67	2.91	3.18
Gross energy, MJ/kg	20.58	21.04	20.77	21.16	20.49	20.62

EAA = essential amino acid; NEAA = non-essential amino acid; Gly/Asp = glycine/aspartic acid; BHT = butylated hydroxytoluene.

¹Pacific mackerel fish meal was purchased from Zhejiang Fengyu Marine Biological Products Co., Ltd. (Zhoushan, Zhejiang, China). The remaining ingredients were obtained from Ningbo Tech-Bank Feed Co., Ltd. (Ningbo, Zhejiang, China).

²EAA premix (g/100 g, dry matter): arginine, 0.6; lysine, 1; methionine, 0.4; valine, 1.0; threonine, 0.9; phenylalanine, 0.3; histidine, 0.4.

³NEAA premix (g/100 g, dry matter): serine, 2.8; cystine, 0.7; tyrosine, 2.5; proline, 3.0.

⁴Gly/Asp prepared by blending Gly and Asp at a 1:1 weight ratio. All crystalline amino acids (analytical grade, purity ≥ 98%) were supplied by Shanghai Sanjie Biotechnology Co., Ltd. (Shanghai, China).

⁵Vitamin premix purchased from Tech-Bank Feed Co., Ltd. (Ningbo, Zhejiang, China). It provided the following per kg of premix: retinyl acetate, 2,500,000 IU; cholecalciferol, 500,000 IU; all-rac-α-tocopherol, 25,000 IU; menadione, 5.63 g; thiamine, 11.25 g; riboflavin, 9.5 g; ascorbic acid, 95 g; pyridoxine hydrochloride, 10 g; cyanocobalamin, 0.02 g; folic acid, 2 g; biotin, 0.375 g; nicotinic acid, 37.5 g; D-Ca pantothenate, 21.5 g; inositol, 80 g; antioxidant, 0.5 g; corn starch, 696.775 g.

⁶Mineral premix purchased from Tech-Bank Feed Co., Ltd. (Ningbo, Zhejiang, China). It provided the following per kg of premix: FeC₆H₅O₇, 4.57 g; ZnSO₄·7H₂O, 9.43 g; MnSO₄·H₂O (99%), 4.14 g; CuSO₄·5H₂O (99%), 6.61 g; MgSO₄·7H₂O (99%), 238.97 g; KH₂PO₄, 233.2 g; NaH₂PO₄, 137.03 g; C₆H₁₀CaO₆·5H₂O (98%), 34.09 g; CoCl₂·6H₂O (99%), 1.36 g.

⁷Moisture, crude protein, crude lipid, leucine, and gross energy were analyzed. Organic matter was calculated as 100 minus ash content on a dry matter basis.

Table 2.

Amino acid compositions of the experimental diets (%, dry matter basis).

Amino acids	Dietary leucine levels, %
	1.81	2.10	2.40	2.67	2.91	3.18
EAA
Arginine	1.97	1.94	1.96	2.00	1.98	1.93
Lysine	2.47	2.45	2.47	2.52	2.49	2.50
Methionine	0.84	0.81	0.83	0.83	0.83	0.87
Leucine	1.81	2.10	2.40	2.67	2.91	3.18
Valine	1.95	1.95	1.93	2.00	1.99	1.90
Threonine	1.74	1.73	1.75	1.76	1.77	1.68
Isoleucine	1.02	1.00	1.04	1.05	1.05	1.03
Phenylalanine	1.41	1.41	1.36	1.43	1.38	1.46
Histidine	1.01	0.98	0.99	1.01	1.00	0.96
∑EAA	14.22	14.37	14.74	15.28	15.39	15.52
NEAA
Glutamic acid	4.62	4.59	4.64	4.72	4.67	4.62
Aspartic acid	4.22	4.12	4.07	4.01	3.91	3.64
Glycine	3.33	3.20	3.11	3.07	2.95	2.74
Alanine	1.41	1.40	1.40	1.44	1.42	1.47
Serine	3.13	3.11	3.19	3.21	3.17	3.14
Proline	3.14	2.96	3.05	3.07	3.16	3.17
Tyrosine	2.63	2.61	2.57	2.64	2.60	2.59
Cysteine	0.79	0.77	0.74	0.77	0.79	0.76
∑NEAA	23.28	22.77	22.77	22.92	22.67	22.13
TAA	37.50	37.14	37.51	38.20	38.06	37.64

EAA = essential amino acids; ∑EAA = total essential amino acids; NEAA = non-essential amino acids; ∑NEAA = total non-essential amino acids; TAA = total amino acids.

2.3. Animals and feeding management

Mud crabs were obtained from the Ninghai Research Center of the East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (Ningbo, Zhejiang, China). After reaching the megalopa stage, the crabs were transferred to concrete ponds for nursery rearing. Over approximately 50 d, they underwent eight molts and their body weight increased to about 8 to 10 g. In the evening, the juveniles were harvested and immediately transported to Ningbo University (Ningbo, Zhejiang, China). Upon arrival, the crabs were individually acclimated in compartmentalized boxes within a recirculating aquaculture system (Fig. 1). During this one-week period, they were fed a commercial diet (Sagarin Bio-Engineering Co. Ltd., Qingdao, Shandong, China) with 45% crude protein and 8% crude lipid twice daily. After acclimation, 360 healthy crabs (initial weight: 8.65 ± 0.58 g) with intact appendages were randomly distributed to six treatments (n = 60 per group). Each dietary treatment was tested in three replicates, each stocked with 20 crabs housed individually in compartments (0.34 m × 0.22 m × 0.16 m). A CONSORT-style animal-flow summary detailing the number of crabs at each stage (allocation, endpoint sampling, challenge allocation, survival monitoring, and post–challenge sampling) is provided in Table S1. Twice daily (07:00 and 18:00), the crabs were fed a total daily ration equivalent to 8%–10% of their body weight, divided equally between the two meals. Any uneaten feed was subsequently collected 2 h after each feeding to calculate feed utilization efficiency. The 63-d feeding trial was performed using a recirculating aquaculture system (RAS), specifically a “crab apartment” system engineered and supplied by Qingdao Haixing Intelligent Equipment Co., Ltd. (Qingdao, Shandong, China), with one-third of the seawater volume exchanged every day. Throughout the trial, key water quality indicators were routinely measured using a multiparameter water quality meter and maintained within the following optimum ranges: temperature 27.85 ± 1.15 °C, salinity (25.21 ± 0.87)‰, ammonia nitrogen < 0.10 mg/L, and dissolved oxygen ≥ 6.0 mg/L.

Fig. 1.

The culture system used for mud crabs.

2.4. Sample collection

Following the 63-d feeding trial, experimental crabs underwent 12 h of food deprivation before final sampling. Total crab counts and individual body weights were recorded to assess growth performance. Six crabs were randomly selected from every replicate for tissue collection. Prior to hemolymph sampling, each crab was anesthetized by placement on crushed ice for approximately 10 to 15 min until complete immobilization was achieved. Following collection into pre-chilled 1.5-mL microcentrifuge tubes, hemolymph was allowed to clot for 24 h at 4 °C. The clotted samples were then centrifuged at 4 °C, 1200 × g for 10 min using a high-speed refrigerated centrifuge (Eppendorf 5810R, Eppendorf Aktiengesellschaft, Hamburg, Germany), and the resulting supernatant was gently collected for subsequent biochemical analysis. Muscle, hepatopancreas, and intestinal tissues were collected immediately, rinsed with cold physiological saline, and blotted dry. Each tissue was then aliquoted into four portions according to the requirements of subsequent analyses: one appropriately sized portion was stored at −20 °C for proximate composition analysis; a second portion was stored at −80 °C for biochemical assays, including digestive enzyme activities; a third portion was preserved in RNAlater or snap-frozen in liquid nitrogen and stored at −80 °C for RNA extraction and real-time quantitative PCR (RT-qPCR) analysis; and the remaining portion was snap-frozen in liquid nitrogen and stored at −80 °C for protein extraction and Western blot analysis. RNA and proteins were extracted within one week after sampling.

2.5. Vibrio parahaemolyticus challenge and sample collection

Following the feeding trial, the remaining crabs were subjected to V. parahaemolyticus challenge tests. Each of the six dietary treatment groups included three replicates, with 10 crabs per replicate. The V. parahaemolyticus strain was provided by the Crab Genomics and Molecular Genetics Research Team (Ningbo University, Ningbo, Zhejiang, China). A preliminary experiment established the median lethal dose (LD₅₀) as 1 × 10⁷ CFU/mL. For the challenge, crabs received 50 μL bacterial suspension via injection at the fourth walking leg base during challenge trials. Mortality was monitored every 12 h for 48 h post-injection. At 48 h post-infection, three surviving crabs rom each replicate were randomly selected for sampling. Approximately 0.5 mL of hemolymph was collected from the pericardial cavity using a sterile syringe that had been preloaded with an anticoagulant (prepared with 2.996 g sodium citrate, 9.12 g glucose, 7.86 g NaCl, and 1.32 g ethylenediaminetetraacetic acid disodium salt [EDTA-Na₂] in 400 mL purified water, pH 7.0). Samples were immediately centrifuged (500 × g, 4 °C, 10 min) for apoptosis analysis. For hemocyte collection intended for RNA and protein work, the hemolymph was processed as follows: immediately diluted 1:1 with anticoagulant, centrifuged (500 × g, 4 °C, 10 min), and the cell pellet was washed three times with phosphate-buffered saline (PBS) before subsequent analysis. Another aliquot of hemolymph was collected without anticoagulant, allowed to clotted at 4 °C for 24 h, and then centrifuged (1200 × g, 4 °C, 10 min) to obtain supernatant for immune and antioxidant parameter assays.

2.6. Analytical protocols

2.6.1. Chemical composition analysis

Proximate composition of muscle, hepatopancreas and feed samples was analyzed according to AOAC (2006) standard procedures. Briefly, moisture content was measured gravimetrically after oven-drying at 105 °C (method 934.01) using an oven (DHG-9240A, Shanghai Jinghong Laboratory Equipment Co., Ltd., Shanghai, China). Crude protein content was determined using the Dumas combustion method (method 968.06) with a rapid nitrogen analyzer (FP-528, Leco Corporation, St. Joseph, MI, USA) and a nitrogen-to-protein conversion factor of 6.25. Crude lipid content was extracted with petroleum ether using a Soxhlet apparatus (SX-360, Tecator AB, Höganäs, Skåne County, Sweden; method 920.39). Ash content was measured by incineration at 550 °C for 8 h in a muffle furnace (SX-2410, Shanghai Experimental Electric Furnace Co., Ltd., Shanghai, China; method 942.05). Organic matter was calculated as 100 minus ash content on a dry matter basis. Gross energy (GE) of experimental diets was determined in triplicate following ISO 9831:1998 using an automatic isoperibol oxygen bomb calorimeter (Model 6400, Parr Instrument Company, Moline, IL, USA).

2.6.2. Amino acid profile analysis

Fresh muscle samples were freeze-dried for 72 h to remove moisture. The amino acid profile of muscle was analyzed via acid hydrolysis followed by separation and detection with the amino acid analyzer (L-8900, Hitachi High-Tech Corporation, Tokyo, Japan). Specifically, approximately 50 mg of muscle powder was hydrolyzed with 5 mL of 6 mol/L hydrochloric acid at 110 °C for 22 to 24 h under an oxygen-free nitrogen atmosphere to prevent oxidative degradation. After hydrolysis and cooling, the mixture was made up to a final volume of 50 mL with deionized water. A 1 mL aliquot of this hydrolysate was then dried under a stream of nitrogen gas. The residue was reconstituted in 1 mL of 0.02 mol/L HCl, passed through a 0.22-μm membrane filter, and subsequently injected into the analyzer for quantification.

2.6.3. Digestive enzyme activity assay in hepatopancreas

Tissue homogenates were prepared in physiological saline (1:9, w/v), centrifuged (1200 × g, 10 min, 4 °C). The resulting supernatant was collected for subsequent analysis. The assay kits for of pepsin (A080-1-1), trypsin (A080-2-2), lipase (A054-2-1), and amylase (C016-1-2) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The activity of trypsin was analyzed at 253 nm using an ultraviolet spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan). The activities of pepsin, lipase, and amylase were analyzed at their respective absorbance wavelengths using a microplate reader (SpectraMax M2, Molecular Devices, LLC, Sunnyvale, CA, USA).

2.6.4. Immunity and antioxidant parameter analysis in hemolymph

Immune parameters such as lysozyme (LZM), phenoloxidase (PO), acid phosphatase (ACP), and alkaline phosphatase (AKP) were analyzed. The PO assay kit (H247-1-2) was purchased from Shanghai Jianglai Biotechnology Co., Ltd. (Shanghai, China). The activities of LZM (A050-1-1), ACP (A060-1-1), and AKP (A059-2-2) were analyzed using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) in strict accordance with the provided protocols. The specific procedures for measuring these immune enzyme activities were performed as described by Li et al. (2025). The LZM activity was measured using an ultraviolet spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan), whereas the activities of PO, ACP, and AKP were measured using a microplate reader (SpectraMax M2, Molecular Devices, LLC, Sunnyvale, CA, USA).

Additionally, antioxidant markers including total superoxide dismutase (T-SOD; A001-3-2), catalase (CAT; A007-1-1), total antioxidant capacity (T-AOC; A015-2-1), and glutathione (GSH; A006-2-1) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The T-SOD activity and T-AOC and GSH levels were determined using a microplate reader (SpectraMax M2, Molecular Devices, LLC, Sunnyvale, CA, USA), whereas the CAT activity was determined using an ultraviolet spectrophotometer (UV-1800, Shimadzu Corp., Kyoto, Japan). The specific assay procedures for these antioxidant enzymes and products were conducted as described by Yang et al. (2025).

2.6.5. Flow cytometric analysis of hemocyte apoptosis

Hemolymph was collected from mud crabs and immediately mixed with an anticoagulant solution to prevent clotting. Hemocytes were isolated by centrifugation (500 × g, 10 min, 4 °C) and washed twice with PBS (pH 7.4). For experimental samples, hemocytes were resuspended in 500 μL of 1 × Annexin V binding buffer (AP101, MultiSciences Biotech Co., Ltd., Hangzhou, Zhejiang, China). Cells were then stained with Annexin V–fluorescein isothiocyanate (V-FITC) and propidium iodide (PI) following the manufacturer's instructions (5 μL Annexin V-FITC and 2 μL PI per tube), gently mixed, and incubated for 15 min in the dark at room temperature. Samples were analyzed within the 30 min following staining. A Gallios flow cytometer (Model Gallios, Beckman Coulter, Inc., Brea, CA, USA) was used for acquisition, and 10,000 events were recorded per sample. Unstained and single-stained controls (Annexin V-FITC only and PI only) were included for compensation. In addition, a separate positive-control tube was prepared using the manufacturer-provided apoptosis positive-control reagent to verify assay performance and assist with quadrant setting; this positive-control sample was used only for method validation and was not included in dietary group comparisons. Hemocytes were gated on forward scatter (FSC)/side scatter (SSC) to exclude debris, followed by singlet gating to remove doublets. Apoptosis was quantified on Annexin V-FITC versus PI plots and classified as viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+), or necrotic (Annexin V−/PI+).

2.6.6. Gene expression analysis

Each treatment group consisted of three biological replicates, with each replicate comprising two individual samples (resulting in a total of six samples per group). Total RNA was isolated from these samples using TRIZOL reagent (Takara Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning, China), and its purity and concentration were measured. Following cDNA synthesis with a reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China), RT-qPCR was performed on a LightCycler 96 System (Model LightCycler 96, F. Hoffmann-La Roche AG, Basel, Switzerland) using SYBR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) under the thermal conditions of Peng et al. (2025). The amplification protocol is as follows: initial denaturation (1 cycle at 95 °C for 5 min), amplification (55 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 10 s, and extension at 72 °C for 10 s), melting curve analysis (1 cycle with denaturation at 95 °C for 10 s, hybridization at 65 °C for 10 s, and final dissociation at 97 °C for 1 s), and cooling (1 cycle at 37 °C for 30 s). Relative mRNA levels were calculated by the 2^−ΔΔCt method with β-actin as the reference (Livak and Schmittgen, 2001). Primer pairs used for RT-qPCR were either designed using NCBI Primer-BLAST or adopted from published studies. The abbreviations and full names of all genes are provided in supplementary file abbreviations and full names of genes and proteins. Amplification efficiencies for all primers were determined from standard curves, and the resulting amplicon sizes, amplification efficiencies, and slopes are summarized in Table S2. Corresponding standard curves for primer amplification efficiencies are presented in Fig. S1.

2.6.7. Protein extraction and western blot

Total protein was extracted from homogenized tissues using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and quantified with a bicinchoninic acid (BCA) assay kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes (Merck KGaA, Darmstadt, Hesse, Germany), and blocked for 15 min with a rapid protein-free blocking buffer. The membranes were then incubated with primary antibodies overnight at 4 °C, following a previously described protocol (Jin et al., 2024), with minor modifications, including the use of RIPA/BCA-based protein preparation, rapid protein-free blocking for 15 min, and an adjusted primary antibody selection tailored to the signaling targets assessed in this study (Table S3). Following primary antibody incubation, the membranes were washed with Tris-buffered saline containing (TBST) (prepared from a 10 × concentrate with deionized water; Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) for 1 h at room temperature. After extensive washing, the immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) substrate on a gel imaging system (Tanon 5200, Tanon Science & Technology Co., Ltd., Shanghai, China). Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For densitometric analysis, the same rectangular region of interest was applied consistently to corresponding bands within the same membrane, and background signals were subtracted using an adjacent local background area (or an identical rolling-ball setting across lanes when applicable). Target protein signals were then normalized to the corresponding loading control from the same sample and membrane, and the normalized values were expressed relative to the control group (set to 1.0) for statistical analyses. Uncropped original immunoblots images for all proteins analyzed in this study are provided in Figs. S2–S4.

2.7. Statistical analysis

One-way analysis of variance ANOVA was employed to analyze the data, with Tukey's HSD test applied for post-hoc comparisons where a significant main effect was observed (P < 0.05). The validity of ANOVA assumptions was confirmed by testing for normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test). All results are presented as means and standard error of the mean (SEM), and the statistical analyses were conducted using SPSS 29.0 (IBM Corp., Armonk, NY, USA). Survival after V. parahaemolyticus challenge was analyzed using Kaplan-Meier survival curves, with group differences assessed by the log-rank (Mantel-Cox) test. Hazard ratios (HR) and 95% confidence intervals were estimated using a Cox proportional hazards model.

The statistical model for the one-way ANOVA was defined as:

$$Y_{ij}=\mu +{\alpha }_i+{\epsilon }_{ij}\text{,}$$

where Y_ij is the observed value of the response variable; μ is the overall population mean; α_i is the fixed effect of the i-th dietary treatment (leucine level); ε_ij is the random error term associated with the j-th replicate within the i-th treatment group.

The relationships between dietary leucine levels and response variables were evaluated by linear and quadratic regression analyses. Graphs along with regression curve models were constructed using GraphPad Prism version 10 (GraphPad Software, LLC, Boston, MA, USA).

3. Results

3.1. Growth and feed utilization

Effects of dietary leucine levels on growth and feed utilization are shown in Table 3. Survival in all groups exceeded 80%, without significant differences (P > 0.05). Crabs fed the 2.67% leucine diet showed significantly higher percent weight gain (PWG) (P = 0.001) and molting frequency (MF) (P = 0.018) than those fed diets with 1.81%, 2.10%, and 3.18% leucine. Crabs receiving 1.81% and 2.10% dietary leucine exhibited the lowest protein efficiency ratio (PER), which was significantly lower than that of all other treatment groups (P < 0.001). The feed conversion ratio (FCR) of mud crabs fed diets containing 2.40%–3.18% leucine was significantly lower than that of crabs fed the 1.81% leucine diet (P = 0.002). Feed intake (FI) of mud crabs showed no significant difference among the groups (P > 0.05). Quadratic regression of PWG established the optimal dietary leucine requirement of S. paramamosain as 2.70% of the diet in this crab-apartment feeding trial (Fig. 2).

Table 3.

Growth performance and feed utilization of juvenile mud crab fed with experimental diets for 9 weeks.

Items1	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
IBW, g	8.67	8.61	8.31	8.09	9.74	9.00	0.192	0.151	0.230	0.295
FBW, g	26.21c	27.59b	28.15ab	28.99ab	33.55a	29.86ab	0.790	0.031	0.011	0.044
PWG, %	202.1c	220.1bc	238.0ab	258.8a	244.1ab	231.6bc	4.964	0.001	0.013	<0.001
MF	1.59c	1.78bc	1.86ab	2.04a	1.81ab	1.71bc	0.043	0.018	0.208	0.002
Survival, %	86.67	85.00	85.00	86.67	85.00	83.33	1.348	0.099	0.438	0.543
FCR	1.95a	1.84ab	1.69bc	1.55c	1.58c	1.62bc	0.034	0.002	0.007	<0.001
FI, %/d	2.78	3.05	2.50	2.64	2.61	2.66	0.060	0.120	0.168	0.307
PER	1.20b	1.20b	1.42a	1.59a	1.43a	1.41a	0.033	<0.001	0.111	0.010

IBW = initial body weight; FBW = final body weight; PWG = percent weight gain; MF = molting frequency; FCR = feed conversion ratio; FI = feed intake; PER = protein efficiency ratio; SEM = standard error of the mean.

MF = Total molting times/Number of molting crabs.

Survival (%) = (Final crab numbers/Initial crab numbers) × 100.

FCR = Dry feed intake/(Final crab weights + Dead crab weights – Initial crab weights).

FI (%/d) = Dry feed consumed × 2/(Initial crab weight + Final crab weight) × Furation of feeding trial) × 100.

PER = Wet weight gain/(Feed consumed × Dietary protein content).

Data are presented as mean and SEM of three replicates (n = 3). Different superscript letters within a row indicate significant differences (P < 0.05).

¹PWG (%) = (Final crab weights – Initial crab weights)/Initial crab weights × 100.

MF = Total molting times/Number of molting crabs.

Survival (%) = (Final crab numbers/Initial crab numbers) × 100.

FCR = Dry feed intake/(Final crab weights + Dead crab weights – Initial crab weights).

FI (%/d) = Dry feed consumed × 2/(Initial crab weight + Final crab weight) × Furation of feeding trial) × 100.

PER = Wet weight gain/(Feed consumed × Dietary protein content).

Data are presented as mean and SEM of three replicates (n = 3). Different superscript letters within a row indicate significant differences (P < 0.05).

Fig. 2.

Quadratic regression of percent weight gain (PWG) against dietary leucine levels in S. paramamosain. x represents the estimated optimal dietary leucine level corresponding to the maximum PWG.

3.2. Proximate composition in muscle and hepatopancreas

As illustrated in Table 4, crabs fed 2.67% and 2.91% leucine diets exhibited significantly higher muscle protein content (P < 0.001) than other groups, but increasing dietary leucine significantly decreased the muscle lipid levels (P < 0.001). Furthermore, dietary leucine at 2.67%, 2.91%, and 3.18% significantly increased hepatopancreatic protein (P < 0.001) and lipid contents (P < 0.001) relative to the remaining treatments. Different leucine levels did not significantly influence the moisture or ash contents in either muscle or hepatopancreas (P > 0.05).

Table 4.

Proximate compositions in the muscle and hepatopancreas of juvenile mud crab fed with experimental diets for 9 weeks (%, dry matter basis).

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
Muscle
Moisture	73.38	72.67	72.50	71.54	71.25	71.47	0.289	0.208	0.008	0.025
Protein	75.55d	78.47c	82.10b	85.68a	85.47a	80.62bc	0.894	<0.001	0.003	<0.001
Lipid	16.04a	16.23a	15.85a	15.07ab	14.06bc	13.11c	0.294	<0.001	<0.001	<0.001
Ash	5.40	5.31	5.49	5.32	5.38	5.38	0.146	1.000	0.981	1.000
Hepatopancreas
Moisture	74.82	74.77	74.31	73.56	73.83	74.52	0.290	0.818	0.410	0.440
Protein	36.35b	36.71b	38.00b	41.38a	41.72a	41.59a	0.578	<0.001	<0.001	<0.001
Lipid	32.58c	33.15c	34.29b	35.85a	35.87a	35.77a	0.333	<0.001	<0.001	<0.001
Ash	6.55	6.74	6.69	6.81	7.02	7.22	0.197	0.961	0.306	0.585

SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (n = 3). Different superscript letters within a row indicate significant differences (P < 0.05).

3.3. Amino acid profile of muscle

The concentrations of leucine (P = 0.009), glutamic acid (P = 0.001), and Gly (P < 0.001) in muscle were significantly increased with varying leucine levels (Table 5). Elevated dietary leucine levels induced competitive inhibition of BCAA uptake, resulting in significantly reduced muscle concentrations of valine (P = 0.016) and isoleucine (P = 0.009) across treatment groups. Crabs fed the 2.91% leucine diet exhibited significantly higher total amino acid content in muscle than those fed 1.81% and 2.10% leucine diets (P = 0.008).

Table 5.

Amino acid compositions in muscle of juvenile mud crab fed with experimental diets for 9 weeks (%, dry matter basis).

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
EAA
Arginine	7.00	6.78	6.86	6.96	7.41	7.02	0.080	0.294	0.217	0.452
Lysine	5.63	5.40	5.57	5.33	5.56	5.37	0.060	0.673	0.412	0.691
Methionine	1.52	1.63	1.57	1.69	1.55	1.56	0.041	0.907	0.967	0.711
Leucine	4.63b	4.99ab	5.28ab	5.61a	5.56a	5.29ab	0.099	0.009	0.004	<0.001
Valine	2.98a	2.60ab	2.47ab	2.35ab	2.21b	2.10b	0.087	0.016	<0.001	<0.001
Threonine	2.92	2.85	2.93	3.02	2.97	2.78	0.037	0.471	0.789	0.396
Isoleucine	3.16a	2.99a	2.83a	2.81a	2.22b	2.24b	0.105	0.009	<0.001	<0.001
Phenylalanine	2.59	2.65	2.69	2.60	2.77	2.54	0.036	0.532	0.997	0.516
Histidine	1.47	1.43	1.43	1.42	1.54	1.38	0.018	0.098	0.743	0.941
∑EAA	31.91	31.31	31.62	31.79	31.80	30.26	0.200	0.132	0.108	0.109
NEAA
Glutamic acid	10.77b	10.85b	11.47ab	12.30a	12.49a	11.96ab	0.184	0.001	<0.001	<0.001
Aspartic acid	6.08b	6.29ab	6.82a	6.89a	6.88a	6.55ab	0.089	0.006	0.017	<0.001
Glycine	5.45c	6.29b	7.26a	7.40a	7.25a	6.82ab	0.169	<0.001	0.002	<0.001
Alanine	4.15	4.36	4.39	4.25	4.34	4.24	0.056	0.866	0.829	0.615
Serine	2.67	2.64	2.66	2.62	2.74	2.59	0.036	0.920	0.894	0.975
Proline	2.74	2.68	2.82	2.74	2.78	2.62	0.048	0.908	0.711	0.708
Tyrosine	2.50	2.41	2.59	2.47	2.48	2.46	0.037	0.845	0.875	0.904
Cysteine	0.59	0.61	0.56	0.56	0.59	0.64	0.014	0.701	0.515	0.379
∑NEAA	34.94c	36.11bc	38.57ab	39.03a	39.55a	37.88ab	0.445	<0.001	0.002	<0.001
TAA	66.85b	67.43b	70.19ab	70.83ab	71.35a	68.14ab	0.503	0.008	0.065	0.002

EAA = essential amino acids; ∑EAA = total essential amino acids; NEAA = non-essential amino acids; ∑NEAA = total non-essential amino acids; TAA = total amino acids; SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (n = 3). Different superscript letters within a row indicate significant differences (P < 0.05).

3.4. Hepatopancreas digestive enzyme activities

Dietary leucine levels significantly affected hepatopancreatic pepsin, trypsin, and lipase activities of mud crab (Table 6). Crabs fed the diet containing 2.40%–3.18% leucine exhibited significantly higher activities of pepsin (P < 0.001) and trypsin (P = 0.008) compared with those fed the 1.81% leucine diet. In addition, the lipase activity of mud crabs fed diets containing 2.67%–3.18% leucine was significantly higher than that of crabs fed the 1.81% leucine diet (P = 0.005). In contrast, amylase activity (P > 0.05) remained consistent across all leucine treatments.

Table 6.

Digestive enzyme activities in the hepatopancreas of juvenile mud crab fed with experimental diets for 9 weeks.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
Pepsin, U/mg prot	2.33c	3.73b	4.27b	4.18b	5.50a	4.15b	0.141	<0.001	<0.001	<0.001
Trypsin, U/mg prot	692.00c	744.57bc	830.69ab	870.70a	928.18a	829.37ab	20.474	0.008	<0.001	<0.001
Lipase, U/g prot	3.27c	3.20c	3.60bc	4.29ab	4.57a	4.15ab	0.141	0.005	<0.001	<0.001
Amylase, U/mg prot	0.83	0.93	0.88	0.83	0.85	0.91	0.006	0.980	0.427	0.682

SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (two samples of each replicate, n = 6). Different superscript letters within a row indicate significant differences (P < 0.05).

3.5. Antioxidant and immune responses of hemolymph

Crabs fed diets with 2.67% and 2.91% leucine exhibited significantly higher T-SOD activity (P = 0.003) than those fed diets with 1.81% and 2.10% leucine (Table 7). The highest activity of CAT (P = 0.032) in hemolymph was observed in crabs fed diets with 3.18% leucine, which was significantly higher than that in crabs fed the 1.81% leucine diet. The T-AOC (P = 0.030) was significantly elevated in crabs fed diet with 2.67% leucine versus those receiving 1.81% and 2.10% leucine diets. Crabs fed diets with 1.81% and 2.10% leucine had significantly lower concentrations of GSH (P < 0.001) in their hemolymph than those fed the 2.67% leucine diet.

Table 7.

Effects of dietary leucine on antioxidant responses in the hemolymph of juvenile mud crab fed with experimental diets for 9 weeks.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
T-SOD, U/mL	34.35b	37.15b	44.76ab	49.17a	48.58a	41.86ab	1.544	0.003	0.011	<0.001
CAT, U/mL	5.49b	7.41ab	9.52ab	10.28ab	10.35ab	10.59a	0.581	0.032	<0.001	0.001
T-AOC, U/mL	11.71b	12.24b	14.01ab	16.11a	14.02ab	13.35ab	0.443	0.030	0.084	0.011
GSH, μmol/L	32.15b	26.00b	43.43ab	52.19a	42.32ab	44.70ab	2.227	<0.001	0.004	0.004

T-SOD = total superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH = glutathione; SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (two samples of each replicate, n = 6). Different superscript letters within a row indicate significant differences (P < 0.05).

Regarding immune function (Table 8), the activities of PO (P < 0.001) and ACP (P < 0.001) in the hemolymph increased markedly as dietary leucine levels increased from 1.81% to 3.18%. No significant differences were observed in hemolymph LZM and AKP (P > 0.05) activities across dietary leucine treatments.

Table 8.

Effects of dietary leucine levels on immune responses in the hemolymph of juvenile mud crab fed with experimental diets for 9 weeks.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
PO, U/L	87.87c	139.23bc	160.34b	182.05ab	222.61a	232.45a	12.726	<0.001	<0.001	<0.001
LZM, μg/mL	10.19	11.25	10.52	10.88	10.83	11.12	0.398	0.986	0.660	0.904
ACP, U/mL	49.52b	74.95b	69.37b	80.36b	178.36a	249.36a	17.781	<0.001	<0.001	<0.001
AKP, U/mL	14.97	20.16	22.28	26.94	24.18	24.37	2.560	0.865	0.235	0.377

PO = phenoloxidase; LZM = lysozyme; ACP = acid phosphatase; AKP = alkaline phosphatase; SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (two samples of each replicate, n = 6). Different superscript letters within a row indicate significant differences (P < 0.05).

3.6. Protein synthesis-related gene and protein expression levels

The effects of dietary leucine levels on the expression of protein synthesis-related genes and proteins are shown in Fig. 3. Dietary leucine supplementation significantly up-regulated the expression of genes related to branched-chain amino acid transporters (slc7a5 and slc3a2) in the hepatopancreas, intestine, and muscle (P < 0.05). Consistently, the expression of genes such as sesn and 4e-bp1 was significantly down-regulated with varying dietary leucine levels (P < 0.05). However, the expression of genes related to the mTOR signaling pathway (tor, raptor, s6k1, and s6) was significantly up-regulated with varying dietary leucine levels (P < 0.05). In the hepatopancreas, TOR protein expression (P < 0.001) increased with dietary leucine levels and was significantly higher in crabs fed 2.67%–3.18% leucine diets than in those fed 1.81% leucine diets (P = 0.006). In contrast, p70S6K1 protein levels peaked in the 2.67% and 2.91% leucine groups and remained higher than in crabs receiving the 1.81% and 2.10% leucine treatments (P < 0.001). Results of gene and protein expression indicated that an appropriate dietary leucine level promotes leucine transport and activates the mTOR signaling pathway.

Fig. 3.

Effects of dietary leucine levels on expression of genes related to amino acid transporter and mTOR signaling pathway, and protein levels of TOR and p70S6K1 in S. paramamosain. (A) Gene expression of slc7a5 and slc3a2 in hepatopancreas. (B) Gene expression of slc7a5 and slc3a2 in intestine. (C) Gene expression of slc7a5 and slc3a2 in muscle. (D) Gene expression of mTOR signaling pathway in hepatopancreas. (E) Protein expression of TOR and p70S6K1 in hepatopancreas. The gene and protein expression of the control group (1.81%) were set at 1. Data are expressed as mean and standard error of the mean (SEM) with three replicates (gene expression, two samples per replicate, n = 6; protein expression, one sample per replicate, n = 3). Different letters above bars indicate significant differences (P < 0.05).

3.7. Survival after V. parahaemolyticus challenge

Kaplan–Meier survival analysis showed a trend toward differences among dietary leucine groups during the 0 to 48 h period after V. parahaemolyticus challenge (overall log-rank P = 0.056; Fig. 4). Cox proportional hazards modeling (reference: 1.81% leucine) indicated that crabs fed 2.91% leucine diets exhibited a significantly reduced mortality risk (hazard ratio [HR] = 0.386; 95% confidence interval [CI] = 0.169 to 0.882; P = 0.024), whereas the 2.67% leucine group showed a marginal tendency toward reduced risk (HR = 0.496; P = 0.073). Consistent with these analyses, survival probabilities of the 2.91% leucine group remained higher toward the late phase of infection (36–48 h).

Fig. 4.

Effects of dietary leucine levels on Kaplan–Meier survival of S. paramamosain over 0 to 48 h following V. parahaemolyticus challenge. Kaplan–Meier survival curves are shown for mud crabs fed graded dietary leucine levels (1.81%, 2.10%, 2.40%, 2.67%, 2.91%, and 3.18%) and then challenged with V. parahaemolyticus. Each treatment included 30 crabs (three replicates of 10 crabs). Survival status was checked at 12-h intervals (12, 24, 36, and 48 h post-challenge), and deaths were recorded at each inspection; for time-to-event analyses, the event time was assigned as the first inspection time at which mortality was observed (discrete-time approximation due to interval monitoring). Differences among survival distributions were evaluated using the log-rank (Mantel-Cox) test (overall log-rank: P = 0.056).

3.8. Apoptosis in hemocytes and expression of apoptosis-related genes and proteins following V. parahaemolyticus infection

Hemocyte apoptosis was evaluated at 48 h post-challenge with V. parahaemolyticus (Fig. 5 and Table 9). Increasing dietary leucine levels significantly enhanced hemocyte survival rates, as evidenced by reduced apoptotic cell proportions (P < 0.001). Notably, crabs fed the diet containing 2.91% leucine exhibited significantly higher live hemocyte percentages compared to all other treatments (P < 0.001). Conversely, proportions of necrotic cells showed no significant differences across different leucine-supplemented diets (P > 0.05).

Fig. 5.

Effects of dietary leucine levels on hemocyte survival of S. paramamosain at 48 h post V. parahaemolyticus challenge. Flow cytometry dot plots: Q1, necrotic cells; Q2, late apoptotic cells; Q3, early apoptotic cells; Q4, live cells. FITC = fluorescein isothiocyanate; FL = fluorescence.

Table 9.

Effects of dietary leucine levels on hemocyte survival (%) of S. paramamosain at 48 h post V. parahaemolyticus challenge.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
Live cell	28.80d	33.00cd	34.57c	41.30c	57.03a	47.40b	2.348	<0.001	<0.001	<0.001
Apoptosis cell	66.19a	63.31a	52.14ab	46.94bc	34.51c	42.87ab	2.943	<0.001	<0.001	<0.001
Necrotic cell	4.97	3.67	13.33	11.77	8.43	9.76	1.559	0.474	0.263	0.296

SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (n = 3). Different superscript letters within a row indicate significant differences (P < 0.05).

Effects of dietary leucine supplementation on the expression of apoptosis-related genes are shown in Fig. 6A. Crabs fed the 2.67% leucine diet exhibited significantly higher bcl2 (P = 0.030) expression levels compared to those fed the 1.81% leucine diet. In contrast, the pro-apoptotic genes bax (P = 0.001) and caspase 2 (P < 0.001) showed a significant decreasing trend with increasing dietary leucine levels, and their expression was significantly lower in the 2.67%–3.18% leucine groups than in the 1.81% leucine group (P < 0.05), with the lowest mean values observed in the 2.91% leucine group. Moreover, caspase 3 expression was significantly reduced in the 2.40%, 2.67%, 2.91%, and 3.18% leucine groups compared with the 1.81% leucine group (P = 0.002). Based on the protein expression results (Fig. 6B), dietary leucine levels ranging from 2.40% to 2.91% significantly up-regulated the expression of the anti-apoptotic protein BCL2 (P < 0.001) compared to the 1.81% leucine group. Leucine supplementation at 2.91% and 3.18% significantly down-regulated the expression levels of pro-apoptotic proteins, including BAX (P < 0.001), CASPASE-2 (P < 0.001), and CASPASE-3 (P < 0.001) compared to the 1.81% leucine group.

Fig. 6.

Effects of dietary leucine levels on apoptosis-related mRNA and protein expression in hemocytes of S. paramamosain after 48 h V. parahaemolyticus challenge. (A) Gene expression of apoptosis. (B) Protein expression of apoptosis. The gene and protein expression of the control group (1.81%) were set at 1. Data are expressed as mean and standard error of the mean (SEM) with three replicates (gene expression, two samples per replicate, n = 6; protein expression, one sample per replicate, n = 3). Different letters above bars indicate significant differences (P < 0.05).

3.9. Antioxidant enzyme activities and gene expression in hemocytes following V. parahaemolyticus infection

Dietary leucine supplementation significantly enhanced antioxidant capacity in mud crabs following V. parahaemolyticus challenge (Table 10). In terms of antioxidant responses, increasing dietary leucine levels significantly elevated the activities of T-SOD (P < 0.001) and CAT (P = 0.023), with the highest activities observed in the 3.18% and 2.91% leucine groups, respectively. Crabs fed diets with 2.67%, 2.91%, and 3.18% leucine exhibited significantly higher T-AOC (P = 0.008) than the 1.81% leucine diet. Moreover, dietary 3.18% leucine resulted in a significantly higher concentration of GSH (P = 0.009) than that observed in the 1.81% to 2.40% leucine groups.

Table 10.

Effects of dietary leucine levels on antioxidant parameters in the hemolymph of juvenile mud crab challenged by V. parahaemolyticus for 48 h.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
T-SOD, U/mL	55.75c	56.07c	64.19bc	78.73ab	84.93a	89.22a	3.532	<0.001	<0.001	<0.001
CAT, U/mL	10.36b	10.60ab	13.50ab	17.63ab	19.05a	14.95b	1.001	0.023	0.006	0.008
T-AOC, U/mL	3.21b	5.13ab	7.24ab	8.46a	8.07a	8.31a	0.566	0.008	<0.001	<0.001
GSH, μmol/L	26.82b	27.80b	26.27b	32.03ab	31.90ab	44.81a	1.860	0.009	0.002	<0.001

T-SOD = total superoxide dismutase; CAT = catalase; T-AOC = total antioxidant capacity; GSH = glutathione; SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (two samples of each replicate, n = 6). Different superscript letters within a row indicate significant differences (P < 0.05).

The relative expression levels of antioxidant genes following V. parahaemolyticus challenge are shown in Fig. 7A. Crabs fed diets containing 2.91% and 3.18% leucine exhibited significantly higher expression of trx (P = 0.001), cat (P = 0.003), and gpx (P = 0.005) compared to the 1.81% leucine group, while the highest expression of cmnsod was observed in the group receiving the 2.67% leucine diet, which was significantly greater than that in the 1.81% leucine group (P = 0.001).

Fig. 7.

Effects of dietary leucine levels on the expression levels of genes related to immune and antioxidant in hemocytes of S. paramamosain after 48 h V. parahaemolyticus challenge. (A) Expression of antioxidant genes. (B) Expression of immune genes. The gene expression of the control group (1.81%) was set at 1. Data are expressed as mean and standard error of the mean (SEM) from three replicates (two samples per replicate, n = 6). Different letters above bars indicate significant differences (P < 0.05).

3.10. Immune activities and gene expression in hemocytes following V. parahaemolyticus infection

Dietary leucine supplementation significantly enhanced immune capacity in mud crabs following V. parahaemolyticus challenge (Table 11). The PO activity (P = 0.002) in mud crabs fed the 2.67% leucine diet was significantly higher than that in the 1.81% and 2.10% leucine groups, and the highest ACP activity was observed in the group receiving the 2.91% leucine diet, which was significantly greater than that in the 1.81% leucine group (P < 0.001). Furthermore, dietary leucine supplementation significantly enhanced the activities of LZM (P = 0.001) and AKP (P < 0.001), with the highest levels recorded in the 3.18% leucine group.

Table 11.

Effects of dietary leucine levels on immune parameters in the hemolymph of juvenile mud crab challenged by V. parahaemolyticus for 48 h.

Items	Dietary leucine levels, %						SEM	P-value
	1.81	2.10	2.40	2.67	2.91	3.18		ANOVA	Linear	Quadratic
PO, U/L	154.03c	170.20bc	216.51ab	232.96a	227.69ab	216.39ab	8.350	0.002	<0.001	<0.001
LZM, μg/mL	12.89b	16.91ab	17.98ab	18.82a	22.42a	22.49a	0.908	0.001	<0.001	<0.001
ACP, U/mL	116.01c	124.98c	218.24ab	272.41ab	303.27a	188.94bc	18.593	<0.001	0.008	<0.001
AKP, U/mL	24.75e	25.74e	30.80d	37.89c	43.00b	54.05a	2.513	<0.001	<0.001	<0.001

PO = phenoloxidase; LZM = lysozyme; ACP = acid phosphatase; AKP = alkaline phosphatase; SEM = standard error of the mean.

Data are presented as mean and SEM of three replicates (two samples of each replicate, n = 6). Different superscript letters within a row indicate significant differences (P < 0.05).

The relative expression levels of immune genes following V. parahaemolyticus challenge are shown in Fig. 7B. Dietary supplementation with 2.67% to 3.18% leucine significantly up-regulated the expression of propo (P < 0.001), lysozyme (P = 0.002), acp (P = 0.002) and akp (P = 0.003) genes compared to the group receiving 1.81% leucine.

3.11. Expression of mTOR signaling pathway and inflammation-related genes and proteins following V. parahaemolyticus infection

Effects of dietary leucine on the expression of mTOR signaling pathway and inflammation-related genes and proteins in hemocytes of mud crabs following V. parahaemolyticus infection are shown in Fig. 8. Increasing dietary leucine levels significantly up-regulated the expression of slc7a5 (P = 0.027) and slc3a2 (P = 0.013) genes in hemocytes, with the highest expression observed in the 2.91% and 2.67% leucine groups, respectively. In contrast, the expression of sesn (P < 0.001) was significantly down-regulated in crabs fed 2.67% and 2.91% leucine diets compared to the 1.81% and 2.40% leucine groups. Furthermore, compared with the 1.81% leucine group, dietary leucine supplementation significantly increased raptor (P = 0.015) expression in crabs fed the 2.40% and 2.67% leucine diets, tor (P = 0.004) expression in those fed the 2.40 to 3.18% leucine diets, and s6k1 (P = 0.002) compared to the group receiving 1.81% leucine. Protein expression analysis revealed that increasing dietary leucine levels significantly enhanced the expression of p70S6K1 (P < 0.001) and TOR (P = 0.014), with the highest levels observed in the 2.91% leucine group.

Fig. 8.

Effects of dietary leucine levels on the mRNA and protein expression related to mTOR and inflammation in hemocytes of S. paramamosain after 48 h V. parahaemolyticus challenge. (A) Gene expression of mTOR signaling pathway. (B) Gene expression of inflammation. (C) Protein levels related to mTOR and inflammation. The gene and protein expression of the control group (1.81%) were set at 1. Data are expressed as mean and standard error of the mean (SEM) with three replicates (gene expression, two samples per replicate, n = 6; protein expression, one sample per replicate, n = 3). Different letters above bars indicate significant differences (P < 0.05).

Dietary leucine supplementation significantly enhanced the expression of inflammatory factors at both the gene and protein levels in hemocytes of mud crabs after V. parahaemolyticus challenge. The expression levels of tlr4 (P = 0.003), myd88 (P < 0.001), traf6 (P < 0.001), and il-16 (P < 0.001) genes were significantly higher in crabs fed diets with2.67% to 3.18% leucine compared to the 1.81% leucine group. The expression of the relA gene (P < 0.001) exhibited a significant initial increase followed by a decrease with increasing leucine levels, peaking in 2.67% and 2.91% leucine groups. Additionally, dietary leucine at 2.40% to 3.18% significantly up-regulated tnf-α expression (P = 0.002) compared to the 1.81% leucine group. Protein expression analysis revealed no significant differences in total P65 protein levels (P > 0.05) among all dietary treatment groups. However, p-P65 levels (P < 0.001) were significantly higher in the 2.91% and 3.18% leucine groups compared to other treatments.

4. Discussion

To formulate graded leucine diets while keeping dietary nitrogen constant, crystalline L-leucine content was increased at the expense of non-essential amino acids used as nitrogen balancers, mainly Gly and Asp. This is a widely used isonitrogenous strategy in aquatic amino-acid nutrition studies because it maintains overall nitrogen content and minimizes unintended shifts in major nutrients and the essential amino acid profile (Cao et al., 2025). Similar nitrogen-balancing approaches using Gly/Asp (or related non-essential amino acids) have been reported in leucine or other amino-acid dose–response trials in crustaceans and fish, including swimming crab (Portunus trituberculatus), gibel carp, Pacific white shrimp, tiger puffer, and blunt snout bream (Huo et al., 2017; Liang et al., 2018; Wei et al., 2023; Zou et al., 2018). Leucine was tested within an isonitrogenous balancing framework in which Asp and Gly covaried with leucine; therefore, the leucine-specific effects should be interpreted cautiously. Importantly, multiple leucine-responsive markers measured in this study display coherent dose-dependent patterns consistent with amino-acid sensing and mTOR-related signaling, supporting the interpretation that the primary biological signal is attributable to leucine, while future work using inert nitrogen or a balanced non-essential amino acid mixture would further strengthen causal isolation.

A well-balanced amino acid profile is essential for optimizing growth performance in aquaculture species, with leucine recognized as a critical regulator of protein synthesis and a key metabolic signaling molecule (Rehman et al., 2023). In this study, dietary leucine supplementation markedly influenced growth performance, feed utilization, and protein metabolism in S. paramamosain in a dose-dependent manner. Quadratic regression analysis of PWG against dietary leucine levels indicated that the optimal dietary leucine requirement for juvenile mud crab was approximately 2.70% of the diet. It should be noted that this estimate was obtained under controlled crab-apartment (individual-compartment) RAS conditions and may vary under communal pond/cage farming. This estimated requirement is slightly higher than those reported for other crustaceans, such as 2.21% for the swimming crab (Huo et al., 2017), 2.50% for the Chinese mitten crab (Song et al., 2023), and 2.36% to 2.47% for the Pacific white shrimp (Wei et al., 2023). In contrast, the leucine requirements of many freshwater teleosts are markedly lower, including 1.32% for grass carp (Ctenopharyngodon idella) (Deng et al., 2014), 1.65% for fingerling stinging catfish (Heteropneustes fossilis) (Farhat, 2014), and 1.61% for blunt snout bream (Ren et al., 2015). These interspecific differences likely reflect fundamental physiological and evolutionary divergences between crustaceans and teleost fishes, including differences in protein turnover rates, nitrogen metabolism, and amino acid catabolic pathways (Lai et al., 2023). Notably, excessive leucine supplementation (3.18%) resulted in reduced growth performance, suggesting a possible imbalance in BCAA ratios or increased metabolic burden associated with leucine catabolism. Similar growth depression at high leucine levels has been observed in other species, such as largemouth bass (Yang et al., 2022) and Nile tilapia (Gan et al., 2016), and has been attributed to BCAA antagonism or elevated oxidative deamination (Castillo and Gatlin, 2018). These findings highlight the importance of precise leucine formulation in crustacean diets to avoid potential negative consequences of amino acid imbalances or metabolic inefficiencies.

Dietary leucine supplementation at the optimal level significantly enhanced protein retention efficiency, as evidenced by reduced FCR and improved PER. These physiological improvements were corroborated by tissue biochemical composition analyses. Specifically, muscle protein content exhibited a significant quadratic increase, indicating enhanced protein deposition in response to increasing leucine levels. In parallel, the observed decline in muscle lipid content suggests a metabolic shift favoring protein synthesis over lipid accumulation, reflecting an overall improvement in nutrient partitioning efficiency. In the hepatopancreas, elevated protein and lipid contents at dietary leucine levels of 2.67% to 3.18% imply enhanced metabolic function and increased energy reserves. These adaptations are particularly relevant to molting and growth processes in crustaceans, which require substantial energy and nutrient input. Similar leucine-induced enhancements in both protein and lipid deposition have been documented in other aquatic species, including the Pacific white shrimp (Wei et al., 2023), Chinese mitten crab (Song et al., 2023), largemouth bass (Yang et al., 2022), and stinging catfish (Abidi and Khan, 2007). In contrast, Nile tilapia did not exhibit significant changes in tissue composition in response to leucine supplementation (Gan et al., 2016). This interspecific variation may be attributed to the tilapia's omnivorous feeding habits and its robust physiological capacity to maintain protein homeostasis across a broad spectrum of dietary amino acid profiles. Their efficient amino acid recycling mechanisms and adaptive nutrient utilization strategies may reduce the reliance on precise dietary leucine levels, thereby dampening the tissue-level responses observed in other species (Perez et al., 2025).

Amino acid profiling revealed notable metabolic responses to dietary leucine supplementation. Muscle concentrations of leucine, glutamic acid, and Gly increased significantly, suggesting enhanced protein synthesis and potential osmoregulatory benefits associated with these amino acids. This highlights the importance of maintaining balanced BCAA ratios in aquafeed formulations to avoid metabolic inefficiencies and antagonistic effects (Wang et al., 2023). Imbalanced BCAA intake can impair amino acid uptake and utilization, ultimately compromising growth performance and nutrient retention. Interestingly, species-specific responses to dietary leucine have been reported in the literature. For example, significant increases in muscle levels of leucine, isoleucine, and valine were observed in the Chinese mitten crab (Song et al., 2023), whereas tiger puffer (Takifugu rubripes) (Wei et al., 2022) and swimming crab (Huo et al., 2017) exhibited minimal changes in muscle amino acid composition. These interspecific differences likely arise from divergent expression of BCAA transporters (notably slc3a2 and slc7a5) and distinct metabolic priorities between crustaceans and teleosts. Decapod crustaceans, which undergo energetically demanding and periodic molting cycles, may have evolved more efficient mechanisms for BCAA retention and reutilization (Ahmad et al., 2021). This physiological adaptation likely enhances their capacity to direct amino acids toward protein accretion and cuticle regeneration during molting, further underscoring the species-specific importance of dietary BCAA optimization.

Digestive enzyme activities in the hepatopancreas exhibited a bell-shaped response to increasing dietary leucine levels, with peak activities of pepsin, trypsin, and lipase observed at 2.91% leucine inclusion. This enzymatic response closely aligns with the improvements in FCR and PER, suggesting that optimal leucine supplementation enhances the digestive capacity for protein and lipid substrates, thereby facilitating nutrient assimilation and growth. In contrast, amylase activity remained relatively stable across all treatment groups, indicating that leucine exerts minimal influence on carbohydrate digestion. This selective effect on proteolytic and lipolytic pathways is consistent with findings in Channa punctatus (Sharf and Khan, 2023), reinforcing the notion that leucine plays a more prominent role in modulating protein and lipid metabolism than carbohydrate utilization in aquatic species. Integrated analysis of growth performance, tissue composition, amino acid profiles, and digestive enzyme activities supports the conclusion that 2.70% dietary leucine represents the optimal inclusion level for S. paramamosain under the present crab-apartment rearing conditions. At this concentration, metabolic homeostasis is maintained while protein utilization and digestive efficiency are maximized. However, further increases in leucine beyond this threshold led to declines in performance indicators, reductions in digestive enzyme activities, and disruptions in amino acid balance. These changes may reflect metabolic overload, excessive catabolism of surplus amino acids, and possible leucine-induced cytotoxicity, phenomena commonly associated with amino acid imbalance or oversupply (Cao et al., 2023).

Hemolymph antioxidant and immune enzyme activities are critical biomarkers for assessing the physiological status of crustaceans (Xie et al., 2024). Among these, T-SOD and CAT constitute the first line of defense against reactive oxygen species (ROS), with T-SOD catalyzing the dismutation of superoxide anions into hydrogen peroxide (H₂O₂), and CAT subsequently decomposing H₂O₂ into water (Liu et al., 2022). The T-AOC reflects the overall efficacy of non-enzymatic antioxidant systems, while GSH serves as a vital redox regulator (Yuan et al., 2019). A significant upregulation of these antioxidant indicators at moderate dietary leucine levels (2.40%–2.91%) suggests enhanced ROS scavenging capacity, consistent with leucine's conserved antioxidant function across aquatic species. In grass carp, dietary leucine has been reported to elevate SOD1 and glutathione peroxidase (GPx) activities, thereby reinforcing hepatic antioxidant defenses (Zou et al., 2018). Similarly, leucine supplementation partially restored impaired antioxidant systems in Chinese mitten crab fed low-protein diets, particularly by reactivating CAT and glutathione peroxidase pathways (Song et al., 2023). In terms of immune function, PO mediates melanization cascades to combat pathogens (Yuan et al., 2019), ACP contributes to lysosomal degradation, while AKP and LZM play pivotal roles in bacterial cell wall lysis (Shen et al., 2024). In mud crabs, dietary leucine significantly elevated the activities of key immune enzymes such as PO and ACP, underscoring its immunomodulatory potential. These beneficial effects are likely associated with leucine's ability to activate the mTOR signaling pathway, a well-known regulator of immune cell function and inflammatory responses (Sinclair et al., 2013). Furthermore, as a precursor for glutamine synthesis, leucine may support energy metabolism in immune cells and enhance their antimicrobial activity (Nicklin et al., 2009). Collectively, the coordinated enhancement of antioxidant capacity and immune function by dietary leucine highlights its pivotal role in maintaining physiological homeostasis in crustaceans.

The molecular responses observed in this study offer critical mechanistic insights into how dietary leucine enhances growth performance in S. paramamosain. Leucine supplementation significantly up-regulates the heterodimeric amino acid transporter complex slc7a5/slc3a2, which plays an essential role in the cellular uptake of BCAAs across both vertebrates and crustaceans. This finding aligns with previous reports in Pacific white shrimp (Wei et al., 2023) and Chinese mitten crab (Song et al., 2023), where dietary leucine similarly increased BCAA transporter expression and intracellular leucine accumulation. Once internalized, leucine functions as a potent signaling molecule, notably suppressing the expression of the leucine sensor sesn, thereby alleviating its inhibitory effect on the mechanistic mTOR signaling pathway (Kanzaki and Wada, 2023). This leucine-mediated signaling cascade leads to the coordinated up-regulation of tor, raptor, s6k1, and s6, promoting ribosomal biogenesis and phosphorylation of key translational regulators, thereby enhancing the cell's overall translational capacity (Jewell et al., 2015). Simultaneously, the significant down-regulation of the translational repressor 4e-bp1 further facilitates cap-dependent translation initiation (Cangelosi et al., 2022). Importantly, these transcriptional changes were further corroborated by a significant increase in the protein abundance of key signaling components, specifically TOR and its downstream target p70S6K1, supporting the activation of this pathway. Collectively, by enhancing amino acid transport and activating the mTOR signaling pathway, leucine exerts a dual regulatory effect in S. paramamosain, creating an anabolic environment conducive to efficient protein synthesis and ultimately improving growth performance at optimal dietary inclusion levels. Similar activation of the TOR pathway by dietary leucine has been reported in various aquaculture species, including largemouth bass (Yang et al., 2022), Atlantic salmon (Salmo salar) (Lai et al., 2023), turbot (Wang et al., 2023), grouper (Niu et al., 2021), gibel carp (Carassius gibelio) (Zou et al., 2018), hybrid catfish (Pelteobagrus vachelli × Leiocassis longirostris) (Zhao et al., 2020), and grass carp (Deng et al., 2016), where it consistently promotes protein synthesis and muscle growth. However, excessive leucine intake may disrupt metabolic homeostasis and negatively impact growth performance (Calder, 2006). Thus, while leucine's anabolic role in protein metabolism is broadly conserved, its optimal dietary formulation is crucial for maximizing growth potential in commercial crustacean aquaculture.

Survival following bacterial challenge is a critical indicator of host disease resistance (Giri et al., 2016). Kaplan-Meier analysis showed a marginal difference among dietary leucine groups over the 0 to 48 h period post-challenge, and Cox proportional hazards modeling indicated that the 2.91% leucine diet was associated with a significantly lower mortality risk than the 1.81% diet after V. parahaemolyticus challenge, consistent with improved post-challenge survival under optimal leucine supplementation. In line with this survival advantage, hemocyte viability assays revealed significantly reduced apoptosis in the 2.91% leucine group, as reflected by a higher proportion of viable hemocytes and fewer apoptotic cells than in the other dietary treatments. At the molecular level, dietary leucine supplementation increased the expression of the anti-apoptotic gene bcl2 in the 2.67% leucine group, while bax and caspase 2 expression was significantly reduced in the 2.67%–3.18% leucine groups and caspase 3 expression was significantly reduced in the 2.40%–3.18% leucine groups compared with the 1.81% leucine group. These transcriptional responses were generally supported at the protein level, with leucine supplementation (2.40% to 2.91%) significantly increasing BCL2 protein expression and the 2.91% and 3.18% leucine diets significantly reducing the expression of BAX, CASPASE 2, and CASPASE 3 proteins (Tang et al., 2024). These coordinated regulatory effects at both gene and protein levels suggest that dietary leucine modulates the intrinsic apoptotic pathway to maintain hemocyte integrity. Collectively, the findings indicate that leucine enhances crustacean immunity not only by supporting antioxidant and enzymatic defenses, but also by fine-tuning apoptotic gene and protein expression, ultimately strengthening resistance against bacterial pathogens.

Upon V. parahaemolyticus infection, mud crabs initiate a robust immune and antioxidant response to mitigate pathogen invasion and oxidative stress (Zhang et al., 2019). The bacterial challenge elicits a rapid production of ROS as part of the innate antimicrobial defense; however, excessive ROS accumulation can result in cellular oxidative damage if not tightly regulated (Pinegin et al., 2018). This oxidative burst necessitates the up-regulation of key antioxidant enzymes, such as T-SOD, CAT, and GPx, to scavenge ROS, while elevated levels of T-AOC and GSH help sustain intracellular redox homeostasis. Concurrently, the immune system activates critical effector enzymes, such as PO, LZM, ACP, and AKP, which contribute to bacterial lysis, phagocytosis, and melanization processes that encapsulate and eliminate invading pathogens (Chen and Wang, 2019). Dietary leucine supplementation appears to potentiate these defense mechanisms by modulating both antioxidant enzyme activities and the transcriptional expression of associated genes, suggesting that leucine enhances antioxidant defenses at both the enzymatic and transcriptional levels. In many organisms, antioxidant gene programs are classically governed by the nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) regulatory system (Bian et al., 2025; Jaramillo and Zhang, 2013). However, because Nrf2/Keap1-related targets were not directly quantified in the present study, this mechanism is discussed here as a plausible explanation rather than a demonstrated pathway in S. paramamosain. Similarly, the up-regulation of propo, lysozyme, acp, and akp genes, alongside elevated enzyme activity, indicates that leucine may augment immune protein biosynthesis by activating the mTOR signaling pathway, which is a key target of BCAAs in immune regulation. Notably, dietary leucine concentrations ranging from 2.67% to 3.18% appeared to optimize these responses by aligning metabolic demands with signal transduction processes, thereby enhancing pathogen resistance without inducing metabolic stress. This coordinated up-regulation of immune and antioxidant defenses underscores leucine's critical role in fortifying crustacean resilience against bacterial infections.

The results from the V. parahaemolyticus challenge indicate that dietary leucine is associated with improved disease resistance in S. paramamosain, accompanied by concomitant changes in mTOR-related markers and activation of NF-κB signaling (as reflected by p-P65). These findings suggest that optimal leucine supplementation engages both nutrient-sensing and inflammatory signaling programs; however, direct functional coupling between mTOR and NF-κB in crustacean hemocytes remains to be demonstrated and is therefore discussed here as a plausible rather than confirmed mechanism. In hemocytes from crabs fed optimal leucine levels (2.40%–2.91%), the amino acid transporter genes slc7a5 and slc3a2 were significantly up-regulated, consistent with enhanced leucine uptake. Concurrently, suppression of the leucine sensor sesn alleviated its inhibition of the mTOR signaling pathway, leading to enhanced expression of key components (raptor, tor, and s6k1) and a marked elevation of TOR and p70S6K1 protein levels at intermediate leucine levels, particularly in the 2.67%–2.91% groups, whereas the highest leucine diet did not further increase TOR abundance. Such activation of mTOR signaling is important not only for anabolic processes but also for supporting immune cell functional readiness (Patel et al., 2025).

In parallel, NF-κB–related inflammatory responses were enhanced, as evidenced by up-regulation of tlr4, myd88, and traf6 and downstream effectors (il-16 and tnf-α), together with increased p-P65; since P65 phosphorylation is generally associated with elevated NF-κB transcriptional activity, this pattern is consistent with strengthened inflammatory gene induction required for antibacterial defense (Ding et al., 2022; Giri et al., 2016). Although the present data do not establish a causal relationship between the two pathways, mechanistic studies in other systems have shown that mTORC1 can modulate NF-κB activity through functional association with the IκB kinase (IKK) complex and by shaping inflammatory cytokine outputs. Complex and by shaping inflammatory cytokine outputs (Dan et al., 2008; Weichhart et al., 2008, 2015), supporting the plausibility that co-activation of mTOR and NF-κB may contribute to the improved antibacterial phenotype observed here. Similar leucine-responsive TOR activation has also been reported in other decapod crustaceans, including Pacific white shrimp and Chinese mitten crab (Song et al., 2025; Wei et al., 2023), and leucine supply has been associated with improved innate immune indices in swimming crab (Huo et al., 2017; Huang et al., 2020). Moreover, key components of Toll/TLR-MyD88-TRAF6-NF-κB signaling are conserved in shrimp hemocytes and respond to pathogen challenge (Wang et al., 2013; Zhang et al., 2012). Collectively, these findings support the interpretation that dietary leucine acts as an immunometabolic cue that co-engages mTOR signaling and NF-κB–dependent inflammatory/antimicrobial programs, thereby strengthening crustacean resistance to V. parahaemolyticus infection. Overall, this work highlights leucine as a functional immunonutrient in mud crab and provides a working mechanistic framework that warrants further investigation; future pathway-specific modulation experiments may help clarify whether and how mTOR signaling and NF-κB activation interact or synergize to shape hemocyte inflammatory responses.

5. Conclusion

Quadratic regression analysis of PWG in response to dietary leucine levels identified 2.70% as the optimal leucine requirement for S. paramamosain. Leucine supplementation promoted crab growth primarily through activation of the mTOR signaling pathway, thereby enhancing protein deposition in muscle and hepatopancreas. Dietary 2.91% leucine was associated with a significantly reduced mortality risk following V. parahaemolyticus infection (Cox model). This enhanced immunity was associated with coordinated activation of mTOR signaling and increased NF-κB activity, accompanied by up-regulation of the TLR4-MyD88-TRAF6 pathway and enhanced P65 phosphorylation, which likely contributed to elevated pro-inflammatory cytokine expression and strengthened antibacterial responses. These findings highlight the dual role of leucine in supporting growth and enhancing innate immunity in mud crabs.

CRediT authorship contribution statement

Xishuai Cui: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Min Jin: Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. Hongyu Peng: Visualization, Software, Formal analysis. Tiantian Xu: Visualization, Software, Formal analysis. Xiaoyue Li: Visualization, Software, Formal analysis. Shichao Xie: Visualization, Software, Formal analysis. Wenhao Zhan: Visualization, Software, Formal analysis. Yao Deng: Visualization, Software, Formal analysis. Yinqiu Tian: Resources. Zheng Tang: Resources. Tingting Zhu: Resources. Peng Sun: Resources. Qicun Zhou: Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Appendix. Supplementary materials

The following is/are the supplementary material to this article.