Physiological–Biochemical Signatures and Genetic Diversity of Portunus pelagicus Cohorts in Guangdong Coastal Aquaculture

Abstract

This study comprehensively evaluates the phenotypic traits, nutritional profiles, and genetic diversity of three cultured populations of Portunus pelagicus from Guangdong Province, China, namely from Shenzhen (PpSZ), Zhuhai (PpZH), and Chaozhou (PpCZ). We analyzed key nutritional components, including moisture, ash, crude fat, crude protein, total sugar, amino acids, and fatty acids. Notably, significant differences in total sugar content (p < 0.05) were observed among populations, whereas no substantial variation was detected in the other nutritional parameters. PpSZ exhibited significantly higher levels of specific amino acids, especially essential amino acids (p < 0.05). Fatty acid composition revealed a more favorable nutritional profile in PpZH. Analysis of physiological markers such as total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and catalase (CAT) activities demonstrated comparable levels across populations, with no significant differences. Genetic diversity assessment revealed SNP densities of 6.583, 6.16, and 6.08 SNPs/Kb for PpSZ, PpZH, and PpCZ, respectively. The low polymorphism (PIC < 0.25) indicates limited genetic variation within the species. This study provides valuable insights into the biochemical, nutritional, and genetic characteristics of these populations, offering critical implications for the optimization of aquaculture practices and the conservation of genetic resources for this economically significant species.

1. Introduction

The Portunus pelagicus, also known as the flower crab, blue crab, offshore crab, or sand crab, is assigned to the phylum Arthropoda, class Crustacea, order Decapoda, family Portunidae, and genus Portunus. This species is mainly distributed across Japan, the Philippines, Australia, Thailand, the Malay Archipelago, the Indian Ocean, East African waters [1], and the coastal regions spanning Zhejiang, Fujian, Taiwan, Guangdong, Guangxi, and Hainan Provinces in China. Known for its fast growth, strong adaptability, and high-quality meat, P. pelagicus is one of the most economically valuable crab species in China. However, in recent years, wild populations have sharply declined due to overfishing and environmental changes, making artificial aquaculture the primary method for meeting market demand. Guangdong Province, with its favorable natural conditions, has become a major hub for farming this species.

Studying germplasm resources is essential for the sustainable development of aquaculture [2]. By analyzing the quality traits and genetic diversity of farmed populations, we can gain valuable insights for breeding better varieties and improving farming practices. Previous research has shown significant genetic differences among P. pelagicus populations in various regions. For instance, RAPD analysis of P. pelagicus in Thai waters revealed strong genetic differentiation, with each population considered a distinct genetic unit [3]. On the other hand, microsatellite analysis of P. pelagicus along Malaysia’s coast showed low genetic differentiation and signs of inbreeding among populations. Additionally, SNP-based whole-genome analysis of P. pelagicus populations in Vietnam found increased gene flow between central and northern populations, while southern populations had limited genetic exchange [4]. The muscle tissue of P. pelagicus is rich in high-quality proteins, essential amino acids (EAA) like lysine and methionine, and polyunsaturated fatty acids (PUFAs), including EPA and docosahexaenoic acid (DHA), making it highly nutritious. Enzymatic antioxidant activities, such as SOD and CAT, are important indicators of individual health and play a key role in understanding crab farming and environmental adaptation. However, there is still limited systematic research comparing the quality, nutritional composition, and physiological immune characteristics of P. pelagicus from different regions. Advances in molecular biology have made genetic diversity analysis a powerful tool for evaluating germplasm resources. By studying single-nucleotide polymorphisms (SNPs) and polymorphism information content (PIC), we can uncover the genetic structure of farmed populations, providing a scientific basis for conserving and utilizing these resources.

Given the lack of systematic research on P. pelagicus from different farmed populations, especially in China, this study focuses on three farmed populations from Shenzhen (PpSZ), Zhuhai (PpZH), and Chaozhou (PpCZ) in Guangdong Province. We analyzed the conventional nutritional composition, amino acid profiles, and fatty acid composition of muscle tissue to explore quality differences among the populations. In parallel, we quantified critical immune–physiological parameters encompassing principal oxidative stress biomarkers—T-AOC combined with SOD and CAT enzymatic activity levels—to holistically assess the health profiles of P. pelagicus populations. Whole-genome sequencing (WGS) was also used to compare the genetic heterogeneity of these populations and reveal the characteristics of their germplasm resources. The findings of this study not only deepen our understanding of the quality traits and genetic characteristics of P. pelagicus in Guangdong Province but also provide scientific guidance for conserving and utilizing germplasm resources. This contributes to the sustainable development of the P. pelagicus aquaculture industry.

2. Materials and Methods

2.1. Experimental Materials

In the present study, samples of Portunus pelagicus were obtained from aquaculture farms located in three regions along the southeastern coast of China: Shenzhen (PpSZ), Zhuhai (PpZH), and Chaozhou (PpCZ) (Figure 1). At each site, 50 healthy and active individuals were randomly selected and subjected to DNA extraction. From these, 30 individuals with high-quality DNA were further chosen for genomic analysis (

Table 1. Basic information table of investigation points.

Sample Name	Average Nail Width (mm)	Average Nail Length (mm)	Weight (g)	Water Temperature (°C)	Month (Year)	Longitude and Latitude
PpSZ	61.03 ± 1.64	32.42 ± 0.88	117.59 ± 8.98	25	1	22°55′ N, 114°54′ E
PpZH	64.03 ± 1.64	30.23 ±1.34	123.30 ± 8.98	27	1	22°14′ N, 113°37′ E
PpCZ	6.05 ± 0.08	34.25 ± 0.77	158.42 ± 5.65	26	1	22°54′ N, 117°00′ E

). Crabs were captured using dip nets, and during sampling, all sites had similar farming conditions with water temperatures of 25–27 °C. All samples were obtained from a combined farming system integrating pond and factory-based aquaculture, ensuring the representativeness and comparability of the samples.

Upon collection, each crab was immediately labeled, and morphological indicators such as body weight, body length, and carapace width were measured. Body weight measurements were obtained using a precision electronic balance (±0.01 g accuracy), whereas body length and carapace width dimensions were recorded with a digital vernier caliper (±0.1 mm precision). After recording the measurements, the samples were promptly stored in a −20 °C freezer for subsequent analyses. Additionally, portions of muscle tissue were collected and preserved in RNA later solution for molecular biology experiments. All experimental procedures were conducted in strict compliance with animal ethics guidelines to minimize harm to the crabs. The experimental protocols and procedures employed in this study were reviewed and approved by the appropriate institutional ethics committee and regulatory bodies.

2.2. Biochemical Analysis

Proximate analysis of P. pelagicus abdominal muscle tissue systematically quantified major nutritional constituents, encompassing basal physicochemical parameters (moisture content and ash composition) and organic components (protein, lipid, and carbohydrate fractions). Moisture content was measured using the electric constant-temperature drying method (GB 5009.3-2016) [5]. Briefly, 2.0 g of homogenized sample was weighed into pre-dried aluminum dishes and dried at 105 ± 2 °C in a forced-air oven (Model DHG-9070A, Jinghong Ltd., Wenzhou, China) until a constant weight was achieved (typically 6–8 h). Moisture content was calculated as the percentage weight loss relative to the initial sample weight. Crude ash content was quantified via high-temperature combustion (GB/T 6438-92) [6]. Samples (2.0 g) were placed in pre-ashed porcelain crucibles and combusted in a muffle furnace (Model SX2-4-10, Jinghong Ltd., China) at 550 ± 25 °C for 6 h. After cooling in a desiccator, the residual ash was weighed, and crude ash content was calculated as the percentage of residual ash relative to the initial sample weight. The crude protein content was quantitatively analyzed employing the standardized Kjeldahl method (GB/T 6432-94) [7]. Samples (0.5 g) were digested with 15 mL concentrated sulfuric acid and a catalyst tablet (Kjeltec Auto Sampler System, FOSS, Hillerød, Denmark) at 420 °C for 1 h. The digest was distilled with 40% NaOH, and the liberated ammonia was titrated with 0.1 M HCl. Protein content was calculated using a nitrogen-to-protein conversion factor of 6.25. The crude fat content was quantitatively determined through acid hydrolysis following the standardized protocol (GB 5009.6-2016) [8]. Samples (2.0 g) were hydrolyzed with 10 mL 8 M HCl at 70 °C for 40 min. Lipids were extracted with diethyl ether and petroleum ether (1:1, v/v) using a Soxhlet apparatus (Model SOX406, Haineng Ltd., Dongguan, China). The solvent was evaporated, and the residual fat was weighed after drying at 105 °C for 1 h. The total sugar content was quantitatively measured using ultraviolet–visible spectrophotometric analysis according to the national standard protocol (GB/T 9695.31-2008) [9]. Samples (1.0 g) were extracted with 80% ethanol, and the extract was reacted with anthrone reagent at 100 °C for 10 min. Absorbance was measured at 620 nm (Model UV-1800, Shimadzu, Kyoto, Japan), and total sugar content was calculated from a glucose standard curve. Except for moisture determination, all other analyses followed the AOAC (1984) standard methods to ensure the accuracy and reliability of the results. All experiments were conducted under well-controlled laboratory conditions to guarantee data accuracy and reproducibility.

2.3. Nutritional Quality Assessment

This study determined the content of 17 amino acids in accordance with the national standard (GB/T 5009.124-2016) [10], and optimized the pretreatment process for aquatic product characteristics. The specific operation is as follows: take P. pelagicus muscle tissue sample (1.0 ± 0.1 g wet weight), freeze with liquid nitrogen, and dehydrate with freeze dryer (Christ Alpha 1-2 LDplus). Place the freeze-dried sample in a hydrolysis tube, add 15 mL of 6 mol/L hydrochloric acid solution, seal with nitrogen, and hydrolyze at 110 °C constant temperature for 24 h. After filtration with a 0.22 μm microporous membrane, pre-column derivatization was performed using Waters AccQ-Tag derivatization kit, and finally quantitative analysis was completed using Hitachi LA8080 amino acid analyzer. The ion exchange chromatography column (L-8900 type, 4.6 × 60 mm) configured in the instrument realizes amino acid separation under the conditions of gradient elution with mobile phase A (pH 3.45 sodium citrate buffer) and mobile phase B (pH 10.0 borate buffer) at a flow rate of 0.4 mL/min, and the ultraviolet detection wavelength is set at 254 nm. It should be noted that, considering the degradation characteristics of tryptophan in strong acid environment, this study did not include it in the analysis system. Fatty acid detection refers to (GB/T 5009.168-2016) [11], using gas chromatography–hydrogen flame ionization detector (GC-FID) coupled technology. Total lipids were extracted using a Soxhlet apparatus (BUCHI B-811, BUCHI Labortechnik AG, Flawil, Switzerland) with petroleum ether (boiling range 40–60 °C; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for 8 h. The lipid extract was then methylated by adding 0.5 mol/L sodium hydroxide in methanol (Sigma-Aldrich, St. Louis, MO, USA) and heating at 70 °C for 30 min. Subsequently, 14% boron trifluoride in methanol (Sigma-Aldrich, USA) was added, and the mixture was heated at 80 °C for 10 min to complete the methylation. The prepared fatty acid methyl esters (FAMEs) were extracted with hexane and analyzed using an Agilent 7890B gas chromatograph. Chromatographic separation was performed on a DB-23 capillary column (60 m × 0.25 mm × 0.25 μm), with the temperature program set at an initial 140 °C for 2 min, then increased to 240 °C at a rate of 4 °C/min, followed by a 10-min hold. Qualitative analysis was performed by comparing retention times with those of 37-component FAME standards (Supelco CRM47885), and quantitative analysis was performed using a five-concentration-point standard curve (R² ≥ 0.998).

2.4. Antioxidant Enzyme Activity Assay

Systematic assessment of redox regulatory dynamics in the ventral muscle tissue of P. pelagicus, incorporating SOD (EC 1.15.1.1) and CAT (EC 1.11.1.6) kinetic parameters alongside holistic antioxidant potential (T-AOC) quantification, reveals conserved enzymatic patterns. SOD activity was determined using the WST-8 method, defined as the amount of enzyme (U/mg) required to inhibit 50% of the superoxide dismutation reaction per milliliter of reaction solution. The procedures followed the instructions provided with the WST-8 reagent kit (Nanjing Jiancheng Bioengineering Institute, China) [12]. CAT activity was characterized by the enzyme concentration required to degrade 1 μmol H₂O₂/min per milligram of tissue protein, with the unit expressed as U/mg. Measurements followed the CAT assay kit’s protocol (Nanjing Jiancheng Bioengineering Institute, China; Cat. No. A007-1-1). T-AOC reflects the overall antioxidant level contributed by various antioxidants and antioxidant enzymes. It was measured using the ABTS method, following the instructions provided with the T-AOC assay kit (from Nanjing Jiancheng Bioengineering Institute, China; Cat. No. A015-1). All procedures strictly adhered to the assay kit protocols to ensure accurate sample handling and precise reagent distribution. The enzymatic reactions were systematically terminated at predetermined time intervals to facilitate reliable quantification of reaction products and ensure accurate determination of enzymatic activity. Experimental data were meticulously recorded, and variables were strictly controlled during data analysis to ensure the reliability and reproducibility of the results.

2.5. Genetic Diversity

The genetic diversity of P. pelagicus was systematically analyzed. Genomic DNA was isolated from crab abdominal muscle tissue employing the conventional phenol-chloroform extraction protocol. The integrity of extracted DNA was evaluated through 1% agarose gel electrophoresis, while quantification and purity assessment were performed using UV spectrophotometric analysis (Shimadzu, Kyoto, Japan), maintaining a minimum DNA concentration of 12.5 ng/μL. The extracted DNA was fragmented using ultrasonic treatment to meet the requirements for high-throughput sequencing. Library construction followed the Illumina TruSeq protocol, involving DNA end repair, A-tailing, adapter ligation, and PCR amplification. Sequencing library quality control protocols implemented dual validation strategies combining bioanalyzer characterization with qPCR quantification to ensure compliance with next-generation sequencing platform requirements.

High-throughput sequencing was carried out utilizing the Illumina sequencing platform, generating initial sequence data. Primary quality assessment was performed through FastQC analysis to systematically evaluate sequencing quality and detect potential technical artifacts or systematic errors. The acquired sequencing alignment of reads to the reference genome was performed using the BWA software (version 0.7.17) to ensure mapping precision [13]. Genetic variants were identified and characterized through the GATK pipeline, enabling comprehensive detection of single-nucleotide polymorphisms (SNPs) and insertion/deletion variants (indels), with subsequent generation of primary Variant Call Format (VCF) files [14]. To enhance variant detection fidelity, multi-tiered filtering protocols were implemented on primary VCF datasets using VCFtools (v0.1.16), incorporating quality score thresholds (QUAL ≥ 30) and coverage depth parameters (DP ≥ 10) to exclude low-quality variants. Following quality refinement, the curated variant dataset was functionally characterized through computational annotation pipelines utilizing ANNOVAR (v2018Apr16; USC Center for Genetic Epidemiology, Los Angeles, CA, USA), implementing genomic feature prioritization and protein impact prediction algorithms to delineate potential biological consequences [15].

2.6. Data Processing

The experimental data, presented in terms of the mean ± SD, underwent analysis via SPSS 27.0 statistical software. One-way ANOVA was conducted to identify significant differences (p < 0.05), followed by Duncan’s multiple range test for further comparison if significant differences were found [16]. Factor analysis was implemented via SPSS 27.0 for proportional parameters, calculating the extent of contribution and cumulative extent of contribution of each principal component and pinpointing the critical parameters from the nine proportional indices [17]. Nucleotide diversity (π) was calculated using DNASP5.0 software [18]; Observed heterozygosity (Ho) was calculated using PopGene32 software [19]; The software PIC_CALC0.6 was employed to calculate the PIC (polymorphism information content) for each locus in each population [20].

3. Results

3.1. General Nutritional Composition Analysis

The amounts of water, crude fat, crude protein, total sugar, and ash in fresh muscle samples from P. pelagicus populations PpSZ, PpZH, and PpCZ were analyzed. There were no meaningful differences in water, ash, crude fat, and the contents of crude protein among the populations; however, total sugar content exhibited significant differences (p < 0.05, see Figure 2). The water and ash contents showed minimal variation among the groups, with overall values remaining stable. This indicates that the muscle tissues of P. pelagicus have a high water content and a balanced mineral composition. The crude fat content displayed slight fluctuations among the populations but remained relatively low overall, highlighting the low-fat nutritional characteristics of P. pelagicus, aligning with its classification as a high-quality seafood product. Crude protein content varied across populations, ranging from 19.63 to 19.97 g/100 g. The PpSZ population exhibited the highest crude protein content (19.97 g/100 g), which was considerably higher than that of the PpZH and PpCZ populations (p < 0.05). This suggests potential physiological or genetic differences among populations in protein accumulation. Total sugar content showed significant differences among the three populations (p < 0.05). The PpSZ population had the maximal total sugar content, followed by the PpZH population, while the PpCZ population exhibited the lowest levels. This implies potential differences in carbohydrate metabolism and glycogen storage within the muscle tissues of the different populations.

3.2. Amino Acid Profile and Quantification

The analysis identified 17 amino acids in total across the three P. pelagicus populations, providing a detailed evaluation of their amino acid composition (

Table 2. Amino acid profile and quantitative composition (g/100 g on a wet weight basis) in muscle tissue across three Portunus pelagicus populations.

Amino Acids	PpSZ	PpZH	PpCZ
Threonine Thr *	0.48 ± 0.01 a	0.50 ± 0.00 a	0.49 ± 0.01 a
Aspartic acid Asp @	1.09 ± 0.02 a	1.13 ± 0.01 a	1.12 ± 0.03 a
Serine Ser	0.38 ± 0.01 a	0.42 ± 0.00 a	0.43 ± 0.01 a
Leucine Leu *	0.88 ± 0.02 a	0.90 ± 0.00 a	0.84 ± 0.02 a
Glycine Gly @	1.10 ± 0.02 a	1.15 ± 0.01 a	1.22 ± 0.03 a
Methionine Met *	0.24 ± 0.01 a	0.25 ± 0.00 a	0.26 ± 0.01 a
Arginine Arg &	1.31 ± 0.02 a	1.46 ± 0.01 a	1.21 ± 0.03 b
Alanine Ala @	0.66 ± 0.02 a	0.68 ± 0.01 a	0.69 ± 0.02 a
Isoleucine IIe *	0.48 ± 0.02 a	0.44 ± 0.01 a	0.32 ± 0.02 b
Cystine Cys	0.10 ± 0.01 a	0.07 ± 0.01 b	0.09 ± 0.01 a
Glutamic acid Glu @	1.60 ± 0.03 a	1.67 ± 0.02 a	1.81 ± 0.03 a
Tyr tyrosine	0.29 ± 0.01 a	0.34 ± 0.00 a	0.38 ± 0.01 a
Proline Pro	0.44 ± 0.01 a	0.43 ± 0.00 a	0.50 ± 0.02 a
Lys *	0.93 ± 0.02 a	0.97 ± 0.01 a	0.88 ± 0.02 b
Histidine His &	0.23 ± 0.01 a	0.21 ± 0.00 b	0.22 ± 0.01 a
Val *	0.53 ± 0.02 a	0.49 ± 0.00 a	0.38 ± 0.02 b
Phenylalanine Phe *	0.44 ± 0.01 a	0.44 ± 0.00 a	0.43 ± 0.01 a
NEAA	8.13 ± 0.47 a	8.17 ± 0.21 a	7.97 ± 0.28 b
EAA	5.86 ± 0.31 a	5.70 ± 0.13 b	5.55 ± 0.16 c
TAA	15.27 ± 0.84 a	15.13 ± 0.38 a	14.73 ± 0.47 a
DAA	5.97 ± 0.34 a	6.04 ± 0.15 a	5.95 ± 0.17 a
SEAA	1.28 ± 0.07 a	1.27 ± 0.04 a	1.22 ± 0.03 a
EAA/TAA	0.38 ± 0.00 a	0.37 ± 0.00 a	0.37 ± 0.00 a
EAA/NEAA	0.72 ± 0.00 a	0.70 ± 0.00 a	0.70 ± 0.00 a

Note: This study uses the following symbols to denote amino acids: essential amino acids (*), flavor-enhancing amino acids (^@), and semi-essential amino acids (^&). The abbreviations used are EAA (total essential amino acids), NEAA (total non-essential amino acids), DAA (total flavor-enhancing amino acids), TAA: total amino acids, and SEAA (total semi-essential amino acids); (a, b, c) within the same row denote statistically significant differences between groups. Values sharing the same letter are not significantly different (p > 0.05).

). The amino acid profile demonstrated the presence of seven necessary amino acids (EAAs: threonine, valine, methionine, phenylalanine, isoleucine, leucine, and lysine), with tryptophan being undetected, presumably owing to its degradation during the acid hydrolysis process. We also determined the levels of two semi-essential amino acids (histidine and arginine) and eight non-required amino acids (serine, aspartic acid, glycine, glutamic acid, alanine, tyrosine, proline, and cysteine). Among these, glutamic acid, aspartic acid, and glycine were the most abundant, contributing significantly to the composition of flavor-related amino acids (DAAs). This comprehensive analysis highlights the nutritional value of P. pelagicus in terms of its amino acid profile, providing critical data to support aquaculture practices and genetic improvement programs for this economically important species.

3.3. Characterization of Fatty Acid Constituent Profiles and Quantitative Distribution Patterns

Comparative evaluation of fat acid profiles in the three studied populations (PpSZ, PpZH, and PpCZ) demonstrated statistically significant variations in fatty acid composition (p < 0.05), as detailed in

Table 3. Quantitative profile of muscle lipid composition acid composition (mg/100 g fresh weight basis) across three Portunus pelagicus populations.

Fatty Acids	PpSZ	PpZH	PpCZ
C15:0 (Pentadecanoic acid)	3.60 ± 0.02 a	3.40 ± 0.00 a	3.50 ± 0.10 b
C16:0 (Palmitic acid)	64.00 ± 4.30 a	63.60 ± 2.90 a	54.10 ± 3.90 b
C16:1 (Palmitoleic acid)	18.10 ± 1.20 a	18.80 ± 1.00 a	18.40 ± 1.30 a
C17:0 (Heptadecanoic acid)	8.60 ± 0.20 b	11.20 ± 0.60 a	7.20 ± 0.50 b
C18:0 (Stearic acid)	72.50 ± 5.20 a	71.40 ± 3.10 a	55.20 ± 3.90 b
C18:1n9c (Oleic acid)	77.90 ± 5.50 a	55.70 ± 2.40 b	53.20 ± 3.80 b
C18:2n6c (Linoleic acid)	8.10 ± 0.90 b	10.70 ± 0.40 a	5.40 ± 0.20 c
C20:2 (Eicosadienoic acid)	4.80 ±0.40a	3.50 ± 0.10 b	3.60 ± 0.20 b
C20:4n6 (Arachidonic acid)	35.90 ± 2.00 a	37.10 ± 1.40 a	28.50 ± 2.00 b
C22:1n9 (Erucic acid)	11.90 ± 1.00 a	8.80 ± 0.40 c	9.40 ± 1.00 b
C20:5n3 (EPA)	72.00 ± 4.30 b	90.10 ± 3.80 a	57.90 ± 3.90 ab
C22:6n3 (DHA)	72.20 ± 4.30 a	67.70 ± 3.00 b	53.00 ± 3.60 c
Total Unsaturated Fatty Acids	205.00 ± 15.50 a	220.20 ± 11.10 a	155.00 ± 12.30 b
Total Fatty Acid Content	440.00 ± 30.00 a	450.00 ± 30.00 a	350.00 ± 20.00 b
Total Saturated Fatty Acids (ΣSFA)	148.70 ± 0.00 a	149.60 ± 0.00 a	116.50 ± 0.00 b
Monounsaturated Fatty Acids (ΣMUFA)	96.00 ± 0.00 a	74.50 ± 0.00 b	71.60 ± 0.00 b
Polyunsaturated Fatty Acids (ΣPUFA)	309.10 ± 0.00 a	325.90 ± 0.00 a	204.40 ± 0.00 b
DHA ± EPA	144.20 ± 0.00 a	157.80 ± 0.00 a	110.90 ± 0.00 b
n − 3 Series PUFA (Σn − 3PUFA)	144.20 ± 0.00 a	157.80 ± 0.00 a	110.90 ± 0.00 b
n − 6 Series PUFA (Σn − 6PUFA)	116.90 ± 0.00 a	144.10 ± 0.00 a	82.50 ± 0.00 b

Note: Within each row, superscript letters (a, b, ab, c) denote statistically significant intergroup differences at p < 0.05, where values marked with identical superscripts indicate no significant variation.

. Among the fatty acids, C16:0 (palmitic acid) was the most abundant across all groups, ranging from 54.10 to 64.00 mg/100 g, while C20:2 (eicosadienoic acid) had the lowest amount, extending from 36.00 to 48.00 mg/100 g. PpZH exhibited the highest content of C18:2n6c (linoleic acid) at 107.00 mg/100 g, whereas PpCZ showed the lowest at 54.00 mg/100 g. Additionally, the sum of DHA and EPA (DHA ± EPA) in PpZH reached 157.80 mg/100 g, which was significantly higher than that observed in PpSZ and PpCZ, highlighting PpZH’s exceptional enrichment in Omega-3 fatty acids. Further analysis revealed that PpZH had the highest values for total fatty acid content, ΣSFA, and ΣPUFA (450.00 mg/100 g, 149.60 mg/100 g, and 325.90 mg/100 g, respectively), while PpCZ had the lowest values. This indicates that PpZH not only has an advantage in total fatty acid content but also demonstrates a higher proportion of nutritionally important fatty acids, particularly Omega-3 fatty acids, underscoring its potential for higher nutritional value. In contrast, PpSZ exhibited the highest monounsaturated fatty acid (C18:1n9c, oleic acid) content at 77.90 mg/100 g, suggesting its potential for greater antioxidant properties. The marked differences in fatty acid distribution observed within the populations could indicate distinctions in metabolic processes and environmental adaptation strategies. These findings provide a foundation for further nutritional research and health-related applications, emphasizing the importance of fatty acid composition in the assessment of P. pelagicus populations.

3.4. Physiological Indicator Analysis

Physiological parameter analysis revealed distinct profiles across the three P. pelagicus populations. Specifically, the PpSZ population exhibited a T-AOC of 2.68 ± 0.19 μMol/g, with SOD activity measured at 1020.43 ± 118.02 U/g and CAT activity at 876.79 ± 122.33 μMol/L/min. For the PpZH population, The T-AOC was 2.68 ± 0.18 μMol/g, while the SOD activity was ranged from 1021.15 ± 117.42 U/g, and CAT activity was 852.98 ± 122.49 μMol/L/min. For the PpCZ population, the T-AOC was 2.70 ± 0.17 μMol/g, SOD activity was 1024.12 ± 125.33 U/g, and CAT activity was 856.14 ± 113.73 μMol/L/min. Comparative analysis of physiological and biochemical parameters revealed no statistically significant variations across the three populations, as illustrated in Figure 3.

3.5. Assessment of Genetic Diversity in Populations

Based on whole-genome SNP data analysis of the three P. pelagicus populations (

Table 4. Statistical analysis of genetic diversity in three Portunus pelagicus populations.

Population	SNP Number	SNP Density (SNP/Kb)	Nucleotide Diversity (π)	Polymorphism Information Content (PIC)	Observed Heterozygosity (Ho)	Inbreeding Coefficient (FHOM)
PpSZ	6615993	6.583	1.268 × 10−3	0.162 ± 0.127	0.197 ± 0.218	−3.337 × 10−2 ± 4.527 × 10−2
PpZH	6190898	6.16	1.285 × 10−3	0.171 ± 0.126	0.213 ± 0.224	−5.270 × 10−2 ± 2.856 × 10−2
PpCZ	6215622	6.08	1.52 × 10−3	0.166 ± 0.134	0.205 ± 0.191	−5.110 × 10−2 ± 8.120 × 10−3

), the genetic structure and gene flow of the species were revealed. The SNP density (SNP/Kb) was 6.583, 6.16, and 6.08 for PpSZ, PpZH, and PpCZ populations, respectively, with an average of 6.44, indicating a relatively consistent SNP distribution across populations. The nucleotide diversity (π) values were 1.268 × 10⁻³, 1.285 × 10⁻³, and 1.52 × 10⁻³, suggesting a certain extent of genetic diversity between the populations and relatively similar levels of single-nucleotide polymorphism diversity within the species. The polymorphism information content (PIC) was 0.162–0.171, which is below 0.25, indicating relatively low genetic variation and some limitations in genotype diversity within this species. The observed heterozygosity (Ho) was 0.197–0.213, reflecting low heterozygosity among populations, which may be related to the historical genetic characteristics of the species and the close genetic exchange between populations. The inbreeding coefficients (FHOM) were −0.03337, −0.05270, and −0.05110 for PpSZ, PpZH, and PpCZ populations, respectively, indicating a low level of inbreeding and supporting evidence of extensive gene flow among populations.

4. Discussion

4.1. Nutritional Composition Differences in Portunus pelagicus

General nutritional composition is a critical indicator for evaluating the quality of aquatic organisms, as it plays a fundamental role in maintaining their physiological processes and nutritional value. The general nutritional components of aquatic animals primarily include crude protein, moisture, total sugar, crude fat, and ash. Analysis of the general nutritional composition of the three P. pelagicus populations revealed no notable differences in ash content among the populations (p > 0.05), with values reaching from 1.80 to 1.90 g/100 g. Compared to Eriocheir sinensis [21] (1.22~1.50 g/100 g) and Suifenhe crabs [22] (1.03~1.75 g/100 g), the ash content of P. pelagicus was relatively higher. Carbohydrate content is another crucial factor in evaluating the quality of aquatic organisms. Total sugar serves as an essential energy source for aquatic animals, influencing their growth, development, and reproduction. The analysis showed that the PpSZ population had significantly higher total sugar content relative to the PpZH and PpCZ populations (p < 0.05), suggesting that the PpSZ population may have a stronger ability to accumulate carbohydrates, potentially related to its environmental adaptation or metabolic characteristics. High total sugar content may indicate sufficient energy reserves, providing greater energy support during growth. In contrast, the lower total sugar content in the PpZH and PpCZ populations suggests possible differences in carbohydrate metabolism. Crude protein content is a major advantage of P. pelagicus, ranging from 19.20 to 21.00 g/100 g, significantly higher than that of Eriocheir sinensis [23] (16.65–18.84 g/100 g) and Suifenhe crabs [22] (15.04~17.48 g/100 g). The slight differences in crude protein content among the populations, with PpSZ and PpZH showing slightly higher levels than PpCZ, highlight P. pelagicus as an excellent source of high-quality protein. The crude fat content of the three populations (0.70–1.00 g/100 g) was lower than that found in Xishuangbanna river crabs [24] (1.00~1.14 g/100 g) and Eriocheir sinensis [21] (0.98~2.16 g/100 g). This further emphasizes the unique value of P. pelagicus as a healthy aquatic product. Overall, P. pelagicus stands out as a high-protein, low-fat aquatic product. Its low-fat content aligns with the demand for healthy diets, making it particularly suitable for individuals seeking a low-fat, high-protein dietary structure. These nutritional characteristics establish a competitive advantage for P. pelagicus in the aquatic product market.

4.2. Analysis of Amino Acid Composition Differences in Portunus pelagicus

Proteins in living organisms are composed of amino acids, and the nutritional value of these amino acids is largely shaped by their composition and content [25]. This study revealed that the PpSZ population had significantly higher essential amino acid (EAA) content compared to the PpZH and PpCZ populations, indicating that the PpSZ population may have greater potential for protein synthesis and nutrient storage. This advantage could be attributed to its genetic background, farming environment, or physiological state. EAAs are critical for maintaining normal physiological functions and promoting the growth and development of aquatic animals. Higher EAA levels imply greater nutritional value for the PpSZ population, enhancing its market potential. Additionally, according to the FAO/WHO recommended EAA/TAA ratio, which should be ≥0.40, the EAA/NEAA and EAA/TAA ratios for the three populations remained stable (0.37~0.38). These values were higher than those of Scylla paramamosain (0.33) [26] and Portunus trituberculatus (0.35) [27], although slightly below the FAO/WHO benchmark. This indicates that the amino acid composition of P. pelagicus is well balanced and stable, which is significant for optimizing amino acid nutrition and ensuring a balanced protein supply in aquatic animals. Furthermore, studies suggest that the flavor of meat is closely associated with the composition and content of flavor-related amino acids (DAAs), such as glutamic acid, aspartic acid, glycine, alanine, serine, and proline [28]. The high DAA content in the samples, particularly glutamic acid, aspartic acid, and glycine, which were the main components across all three populations, contributes significantly to the unique umami flavor of P. pelagicus. These amino acids enhance the flavor profile, establishing P. pelagicus as a premium seafood product. While the DAA content of the PpCZ population was slightly lower than that of the PpSZ and PpZH populations, the overall differences were not significant, suggesting high consistency in DAA accumulation among the three populations. This further validates the distinctive flavor profile of P. pelagicus as a high-quality seafood product.

In the analysis of non-essential amino acids (NEAAs), glutamic acid was found to be highly abundant in all three populations, playing a key role in flavor formation. Variations in the content of other NEAAs, such as cysteine and proline, may be related to individual metabolic capacity and environmental adaptability, providing a reference for further studies on growth regulation mechanisms. Additionally, differences in the content of semi-essential amino acids (SEAAs), such as histidine and arginine, reflect subtle variations in amino acid metabolism balance among the populations, which may be associated with immune regulation and reproductive physiology. In summary, the amino acid composition of P. pelagicus is comprehensive and balanced, with each population demonstrating unique nutritional strengths. Notably, the PpSZ population excels in EAA and total amino acid content, highlighting its potential value in aquaculture and genetic improvement. These findings provide critical insights for optimizing farming strategies and enhancing the economic value of P. pelagicus, while also offering scientific guidance for consumers seeking high-protein, premium-quality seafood.

4.3. Analysis of Fatty Acid Composition Differences in Portunus pelagicus

The hepatopancreas of decapod crustaceans serves as the primary organ for lipid storage and processing, acting as the metabolic center for lipids [27]. In this study, significant differences were observed in the composition of fatty acids and content among the three populations (PpSZ, PpZH, and PpCZ), likely reflecting variations in genetic characteristics, living environments, and metabolic capacities. Firstly, regarding total saturated fatty acids (ΣSFA), PpSZ and PpZH exhibited similar levels, significantly higher than those of PpCZ. This was primarily attributed to the contributions of C16:0 (palmitic acid) and C18:0 (stearic acid), which were the major saturated fatty acids in all three groups. The C16:0 content in PpSZ was 64.00 ± 4.30 mg/100 g, slightly higher than PpZH’s 63.60 ± 2.90 mg/100 g, while PpCZ had significantly lower levels at 54.10 ± 3.90 mg/100 g. This suggests that PpSZ and PpZH may possess greater capacities for saturated fatty acid metabolism. Moreover, the C16:0 and C18:0 levels in all three populations exceeded those of Chinese mitten crabs (Eriocheir sinensis) [1], which could be closely related to their ecological environments and metabolic demands. It is well known that polyunsaturated fatty acids (PUFAs), particularly Σn − 3PUFA, offer significant health benefits [29], especially in the prevention and treatment of coronary heart disease and cancer [30,31]. Therefore, comparisons among various fatty acids place a greater emphasis on ΣPUFAs [32].

In this study, PpZH demonstrated a clear advantage in PUFA content, with ΣPUFA levels reaching 325.90 mg/100 g, higher than PpSZ’s, 309.10 mg/100 g, and PpCZ’s, 204.40 mg/100 g. This trend was primarily driven by PpZH’s significant enrichment in EPA (90.10 ± 3.80 mg/100 g) and DHA (67.70 ± 3.00 mg/100 g), while PpCZ exhibited significantly lower levels. Moreover, comparative analysis demonstrated significantly higher total polyunsaturated fatty acid (ΣPUFA) content in all three P. pelagicus populations relative to previously reported values for Penaeus monodon [33], Fenneropenaeus merguiensis [34], and Litopenaeus vannamei [35] (p < 0.05). This high PUFA content, especially the substantial levels of n − 3 fatty acids (EPA and DHA), underscores the superior nutritional potential of P. pelagicus. EPA and DHA are widely recognized for their critical roles in promoting cardiovascular health and neurological development, with PpZH showing a marked advantage in the accumulation of these high-value fatty acids. Additionally, the n − 3/n − 6 PUFA ratio varied significantly among the populations. PpSZ had an n − 3/n − 6 ratio of 1.23, PpZH had 1.10, and PpCZ had a relatively higher ratio of 1.34. A higher n − 3/n − 6 ratio is generally associated with better anti-inflammatory and cardiovascular protective effects [36], suggesting that although PpCZ showed lower overall PUFA levels, its optimized fatty acid ratio highlights potential advantages in fatty acid composition. However, the higher ratio in PpCZ may primarily reflect a deficiency in n − 6 PUFAs rather than an enrichment in n − 3 PUFAs. Regarding monounsaturated fatty acids (MUFAs), PpSZ exhibited the highest content (96.00 mg/100 g), significantly exceeding PpZH (74.50 mg/100 g) and PpCZ (71.60 mg/100 g), likely due to its higher levels of C18:1n9c (oleic acid). MUFAs are known for their strong antioxidant properties, and their higher content may enhance PpSZ’s tolerance to oxidative stress.

In summary, the PpZH population demonstrated the highest nutritional potential in terms of PUFA content and enrichment of EPA and DHA. PpSZ showed notable advantages in MUFA content and saturated fatty acid accumulation, while PpCZ, despite lower absolute fatty acid content, displayed potential benefits in optimized fatty acid ratios, particularly in its n − 3/n − 6 balance. These differences are likely associated with the genetic characteristics and ecological adaptability of the populations. Future research should aim to better understand how environmental factors influence fatty acid composition and to uncover the molecular mechanisms behind fatty acid metabolism. These findings will not only help us evaluate the nutritional value of seafood but also guide the development of healthier, functional food products.

4.4. Analysis of Physiological Indicator Differences in Portunus pelagicus

The antioxidant system in organisms has the ability to remove lipid peroxidation products, including antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT). CAT catalyzes the decomposition of hydrogen peroxide (H₂O₂) into water, thereby preventing hydroxyl radical reactions and protecting cells from H₂O₂ toxicity [37]. T-AOC serves as an measure of an organism’s overall antioxidant strength, which reflects the combined activity of enzymes and small molecules. It indirectly shows how well an organism can combat free radicals [38]. This study analyzed the physiological and biochemical indicators of three P. pelagicus populations (PpSZ, PpZH, and PpCZ), including T-AOC, SOD, and CAT activities. The results revealed no marked differences among the three populations in these indicators (p > 0.05), suggesting consistent physiological characteristics in their antioxidant defense systems. Antioxidant capacity is a crucial indicator of an organism’s ability to eliminate reactive oxygen species (ROS) [39], while T-AOC comprehensively reflects the combined effects of endogenous antioxidants (such as glutathione and uric acid) and antioxidant enzymes, serving as an integrated measure of antioxidant capacity [40]. The T-AOC values across the three populations were approximately 2.68 μMol/g, indicating consistent antioxidant substance reserves and ROS elimination capacities. This uniformity may be attributed to their shared marine ecological environment, where stable environmental conditions likely negate the need for significant evolutionary differences in antioxidant capacity. SOD and CAT are two key antioxidant enzymes responsible for converting superoxide radicals and H₂O₂ into non-toxic substances, thereby protecting cells from oxidative stress damage [41]. The SOD activity ranges (1020.43–1024.12 U/g) and CAT activity ranges (852.98–876.79 μMol/L/(min)) among the three populations showed no meaningful differences. This result indicates consistent enzymatic efficiency in ROS elimination across the populations, potentially reflecting similar oxidative stress pressures in their comparable environments. Overall, the results of this study indicate that the three P. pelagicus populations exhibit consistent performance in antioxidant capacity and related enzyme activities. This suggests a degree of conservatism in their strategies for coping with environmental oxidative stress. Such consistency may reflect the stability of their environmental adaptation and metabolic regulation capacities.

4.5. Analysis of Genetic Diversity Differences in Portunus pelagicus

Genetic diversity is a crucial indicator for evaluating the status of genetic resources, as it fundamentally reveals species origin, variation, and evolution [42]. In this study, the genetic diversity levels of three P. pelagicus populations (PpSZ, PpZH, and PpCZ) were analyzed. The results indicate relatively high overall genetic diversity. While our data primarily reflect intra-population genetic variation rather than direct inter-population differentiation measurements, the observed genetic similarities suggest the possibility of gene flow among these populations. The nucleotide diversity (π) values for PpSZ and PpZH populations were 1.268 × 10⁻³ and 1.285 × 10⁻³, respectively, while the PpCZ population exhibited a slightly higher value of 1.52 × 10⁻³. This indicates that P. pelagicus displays some degree of population-level genetic variation. High nucleotide diversity reflects abundant genetic variation, which may result from the species’ long-term adaptation to the complex offshore environment. The SNP density in all three populations was approximately 6 SNP/Kb, further supporting the existence of high genetic diversity within their genomes and relatively small inter-population differences. In terms of polymorphism information content (PIC) and observed heterozygosity (Ho), the PpZH population exhibited the highest values, which could be related to its environmental conditions and population dynamics. In contrast, these indicators were slightly lower in PpSZ and PpCZ populations; however, the overall levels remained relatively high, with PIC values ranging from 0.162 to 0.166 and Ho values ranging from 0.197 to 0.205. Additionally, the inbreeding coefficient (FHOM) was found to be negative across all three populations, ranging from −5.270 × 10⁻² to −3.337 × 10⁻². This phenomenon has been documented in genetic studies [43,44], and its underlying mechanism is primarily related to the selection of the reference population: when the inbreeding coefficient of an individual’s offspring is significantly lower than that of the reference population, FHOM can exhibit negative values. The negative FHOM values suggest relatively high heterozygosity within the populations, aligning with the observed genetic diversity indicators. This result aligns with the observed minimal differences in nucleotide diversity and SNP density among the populations. High genetic diversity is often considered a key factor in enabling populations to adapt to environmental changes [45]. The findings of this study indicate that P. pelagicus possesses high genetic diversity, which may enhance its survival and adaptability in offshore environments. Offshore habitats are often characterized by complex ecological conditions, such as fluctuations in water temperature, salinity, and dissolved oxygen levels. These environmental pressures may drive natural selection, promoting the accumulation of genetic diversity within P. pelagicus populations. Moreover, the relative isolation of offshore environments may help preserve genetic variation within populations. At the same time, the observed genetic similarities suggest potential gene flow among populations, which could play a role in maintaining a stable genetic structure at the population level.

5. Conclusions

This study revealed similar nutritional profiles (moisture, protein, fat, ash) but significant variation in total sugar content among three P. pelagicus populations. All groups contained 17 amino acids, dominated by glutamic acid, aspartic acid, and glycine, with consistent flavor-related amino acid levels supporting their nutritional value. The high activities of antioxidant enzymes (T-AOC, SOD, and CAT) suggest a strong adaptive capacity to environmental challenges. Genetic analyses revealed a high level of diversity, with an average SNP density of 6 SNPs per kilobase. The observed genetic variation was primarily intra-population, and the low genetic differentiation between populations suggests the possibility of gene flow. Additionally, the negative inbreeding coefficients may indicate a close genetic relationship among individuals. These findings highlight the species’ genetic resilience and nutritional value, providing valuable insights for sustainable aquaculture management. Future research should integrate genomic and environmental data to further elucidate the genetic structure and adaptive mechanisms of this species.