1. Introduction

The Scylla paramamosain, commonly known as the mud crab, is an economically significant species in China, celebrated for its high nutrition value, succulent meat, short growth cycle, and good adaptability [1]. This species is predominantly found along China’s southeastern coastal regions, especially in the provinces of Zhejiang, Fujian, Guangdong, and Guangxi [2]. Beyond China, the mud crab’s range extends across the Pacific and Indian Oceans into tropical and subtropical areas, including Thailand, the Philippines, Malaysia, and Indonesia [3, 4]. As of 2023, China’s marine farming area dedicated to mud crab cultivation covered 24,809 hectares, with the largest farming regions located in Zhejiang, Fujian, and Guangdong provinces [5]. In the same year, the total output of mud crab from marine farming reached 157,012 metric tons, reflecting a 1.52% increase from the previous year, 2022 [5]. The mud crab aquaculture industry remains a crucial component of China’s aquatic supply chain.

Significant progress has been made in several areas of mud crab aquaculture, including the overwintering of parental crabs, refinement of breeding techniques, identification of critical control points in larval incubation, standardization of artificial breeding protocols, and the development of exemplary aquaculture models [6–8]. Notably, the “Dongfang No. 1” new variety, created in 2022, has demonstrated improved growth and survival rates compared to wild juveniles [9]. Despite these advancements, the industry still faces a significant shortfall in seedling numbers due to supply limitations and the specific temporal demands of seedling cultivation. This has led to a pronounced reliance on wild seedlings harvesting for aquaculture. In the Zhejiang mud crab aquaculture sector, for instance, a significant portion of wild seedlings is sourced from Fujian. Nonetheless, aquaculture practitioners have noted that seedlings native to the region generally exhibit better adaptability than those sourced externally.

Species with broad distributions are expected to exhibit morphological, physiological, or behavioral differences in response to local conditions, a phenomenon known as local adaptation [10]. Local adaptation is typically driven by genetic variation, which can provide a foundation for molecular breeding [11]. In China, studies have indicated that mud crabs from different coastal regions show a degree of genetic differentiation and local adaptation [12]. However, research on the local adaptation of the mud crabs remains limited, especially in terms of growth performance, morphological indices, and comparative transcriptomics. Most studies have focused on environmental stress and innate immunity [13, 14], with fewer exploring growth-related aspects. A comparative analysis of the claw muscle transcriptome during various fattening stages identified 121 differentially expressed genes (DEGs)[15]. Additionally, four single nucleotide polymorphism (SNP) markers and two candidate genes, multidrug resistance-associated protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR), were associated with growth-related traits in the species [16].

Conventional genetic improvement strategies, which rely on long-term selection, are often labor-intensive and time-consuming [17]. As a result, the development of artificial breeding programs that focus on economically important traits—such as growth rate, disease resistance, and environmental tolerance—is crucial for improving the sustainability of the mud crab farming industry. Molecular breeding techniques, which can overcome the limitations of traditional breeding methods and enhance breeding efficiency, are emerging as a promising alternative [18]. To effectively implement molecular breeding, a thorough investigation of the biological and genetic foundations of key traits in wild mud crabs is essential.

Zhejiang and Fujian, two neighboring provinces in China, are home to mud crab populations that exhibit notable differences in growth rates. This study aims to compare the growth performance, morphological indices, and eyestalk transcriptomes of mud crab native to these regions. The objectives are twofold: (1) to identify the molecular differences that contribute to the observed growth rate variations between the two geographical populations, and (2) to discover candidate genes for potential use in molecular breeding programs. The findings are expected to enhance our understanding of the adaptability of nonlocal varieties, the inherent advantages of local varieties, and growth differences between sexes. This research will provide new insights into advancing the mud crab industry and lay a scientific foundation for future molecular breeding strategies.

2. Materials and Methods

3. Results

3.1. Growth Performance and Morphological Parameters

The daily weight gain and SGR of both female and male crabs were significantly higher (p < 0.01) in the SMC population compared to the ZPC population (Figure 1). There was no significant difference (p > 0.05) in condition factor (CF) between the two populations. The propodus/cheliped length of both female and male crabs was significantly greater (p < 0.01) in the SMC population compared to the ZPC population (Figure 2). However, the carapace width/length and cheliped/carapace length ratios were not significantly different (p > 0.05) between the two populations.

3.4. PCA and Correlation Analysis

In the PCA score plot, the samples were separated into four distinct groups based on population and gender (Figure 3A). PC 1 and PC 2 explained 42.84% of the total variation in the data. Consistent with the PCA results, the correlation analysis of the samples also demonstrated a good intragroup correlation (Figure 3B).

3.5. DEGs

In females, a total of 1048 DEGs were identified, with 425 upregulated genes and 623 downregulated genes in the SMCF group (Figure 4A, Supporting Information 1: Table S1). In males, 1069 DEGs were identified, including 525 upregulated genes and 544 downregulated genes in the SMCM group (Figure 4B, Supporting Information 2: Table S2). Heatmaps illustrated the differences in gene expression patterns between different groups (Figure 5). A total of 44 DEGs were upregulated in both female and male crabs from the SMC population (Figure 6A). Among these, five DEGs, Otop (Otopetrin 1), Smyd (SET and MYND domain containing), AM1159 (Cancer pagurus Cuticle protein AM1159), PPAF1 (PPAR gamma coactivator 1 α), and NARG2 (NMDA receptor regulated 2), might directly regulate the molting and growth (Table 3). Meanwhile, nine DEGs, QDPR (Quinoid Dihydropteridine Reductase), NLRC3 (NLR Family CARD Domain Containing 3), RVT1 (Reverse Transcriptase 1), SMCT (Sodium-Monocarboxylate Cotransporter), NDUFB3 (NADH: Ubiquinone Oxidoreductase Subunit B3), NP4 (Neutrophil peptide 4), FCN (Ficolin), SelM (Selenoprotein M), and FABP3 (Fatty Acid Binding Protein 3), were involved in energy metabolism, nutrient metabolism, and immune response, which could also influence the growth. Eighty-seven DEGs were downregulated in both female and male crabs from the SMC population (Figure 6B). Among them, four DEGs, P5CS (Pyrroline-5-Carboxylate Synthase), PGK (Phosphoglycerate Kinase), FABP6 (Fatty Acid Binding Protein 6), and ATGL (Adipose Triglyceride Lipase), were involved in energy metabolism and fatty acid metabolism, potentially affecting growth.

Table 3. DEGs identified in eyestalk of both female and male.

	Category	Gene	log2FC_F	padj_F	log2FC_M	padj_M
Upregulated genes in sanmen county population	Growth and molting	Otop	3.5349	4.99E-02	3.7036	2.69E-02
		Smyd	3.0439	9.09E-04	2.4411	1.46E-02
		AM1159	2.9388	3.70E-02	5.6633	3.44E-06
		PPAF1	1.3730	2.22E-02	1.7916	1.25E-03
		NARG2	1.0432	2.31E-02	1.2220	1.16E-02
	mTOR pathway	QDPR	5.3993	2.14E-06	2.1761	6.98E-05
		NLRC3	1.8463	3.01E-04	1.2715	1.79E-02
	Nutrient metabolism	SMCT	3.6848	3.27E-10	1.8731	1.69E-05
		RVT1	2.0555	1.44E-07	1.6999	1.13E-06
		FABP3	1.6487	3.41E-02	1.7249	4.33E-02
		NP4	1.1458	2.89E-02	4.1873	2.86E-25
	Energy metabolism	NDUFB3	4.4101	2.35E-03	4.1521	2.74E-03
		SelM	1.4943	1.11E-04	1.6074	1.56E-02
	Immune response	FCN	1.9167	2.44E-02	2.2087	2.38E-02
Downregulated genes in sanmen county population	Fatty acid metabolism	FABP6	−3.9944	3.59E-04	−3.2839	4.55E-03
		ATGL	−2.8915	3.34E-04	−2.9143	4.07E-04
	Energy metabolism	PGK	−1.8925	3.18E-02	−1.8528	2.80E-02
		P5CS	−1.0590	1.02E-05	−1.1852	2.09E-07

• Note: log2FC_F, the log2 fold change of DEG in female; padj_F, the adjusted p value of DEG in female; log2FC_M, the log2 fold change of DEG in male; and padj_M, the adjusted p value of DEG in male.

3.6. GSEA

Four KEGG pathways were upregulated in both female and male crabs from the SMC population (Figure 7A)“Melanogenesis,” “Riboflavin metabolism,” “Choline metabolism,” and “Ribosome biogenesis.” Conversely, three KEGG pathways— “Biotin metabolism,” “Arginine and proline metabolism,” and “Phototransduction”— were downregulated in both female and male crabs from the SMC population. Intrestingly, five KEGG pathways were downregulated in female but upregulated in males: “Various types of N−glycan biosynthesis,” “Cell adhesion molecules,” “cAMP signaling pathway,” “Histidine metabolism,” and “Glycosphingolipid biosynthesis.” Additionally, the KEGG pathway “Ubiquinone and other terpenoid−quinone biosynthesis” was upregulated in females but downregulated in males.

Four GO–BP (biological processes) terms, four were upregulated in both female and male crabs from the SMC population (Figure 7B): “Cell redox homeostasis,” “Translation,” “Protein glycosylation,” and “mRNA splicing, via spliceosome.” Two GO–BP terms—“Actin cytoskeleton organization” and “Signal transduction”— were downregulated in both females and males: Additionally, three GO–MF (molecular functions) terms were upregulated in both sexes: “Structural constituent of ribosome,” “Small GTPase binding,” and “Methyltransferase activity.” Interestingly, two GO–MF terms, “Catalytic activity” and “ligase activity,” were upregulated in females but downregulated in males. Whereas, GO–MF term, “Acetylglucosaminyltransferase activity,” was downregulated in females but upregulated in males.

4. Discussion

Growth rate is critical in aquaculture species, as it directly impacts production efficiency and commercial value. Thus, it is often one of the first traits targeted for genetic improvement [19]. In this study, the growth rates of female and male mud crabs were respectively compared, as these species typically exhibit significant sex-based differences in growth traits [3]. Our results revealed that the SMC population exhibited a faster growth rate than the ZPC population in both sexes, suggesting that it is a suitable model for studying the genetic background of growth trait. Additionally, we observed a morphological difference in the propodus-to-chela ratio between the two populations, which could serve as a key distinguishing characteristic for the SMC population.

The growth of crustaceans occurs through the shedding of the old exoskeleton and the formation of a new one, a special process known as molting, which is primarily regulated by the eyestalk [20, 21]. In the present study, five molting-related DEGs were upregulated in the eyestalk of the SMC population. NARG2, which encodes an NMDA receptor-regulated protein, acts upstream in the methyl farnesoate (MF) signaling pathway [22, 23]. MF is an essential endocrine hormone controlled by the eyestalk and plays an important role in the modulation of molting [24]. The crustacean exoskeleton is constructed from a calcified extracellular matrix called the cuticle. Smyd, encoding lysine methyltransferase, provides a unique epigenetic regulation mechanism for cuticle development and morphogenesis in the Chinese mitten crab (Eriocheir sinensis) [25]. AM1159, which encodes a cuticle protein, interacts with chitin to contribute to cuticle development and structural integrity in crustaceans [26, 27]. Molting also requires the mobilization of CaCO₃ for the exoskeleton mineralization [28, 29]. Otops, an evolutionarily conserved family of proton channels found in various animals, have been reported to be critically involved in the mineralization process, providing an exit route for protons liberated during the formation of CaCO₃ [30, 31]. In addition, PPAF1, which belongs to the serine proteinases family, has been reported to control and regulate molting in the blue crab (Callinectes sapidus) [32]. Taken together, the present study suggests that the faster growth rate of the SMC population may be attributed to enhanced molting regulation, particularly in exoskeleton formation.

Nutrition and energy are essential for growth and molting in crustaceans, and the mTOR signaling pathway acts as a central hub, coordinating nutrient status, energy homeostasis, and growth [33–35]. Previous studies have reported that mTOR signaling could regulate molting in response to environmental conditions by affecting neuropeptide production in the eyestalk of the Dungeness crab Metacarcinus magister [36]. In the present study, two regulators of the mTOR signaling pathway, NLRC3 and QDPR, as well as two energy metabolism-related DEGs, were upregulated in the eyestalk of the SMC population, suggesting that the SMC population may have a better capacity to enhance energy allocation to molting via the mTOR pathway [37, 38]. Additionally, the protein metabolism-related gene RVT1, the fatty acid transport-related genes FABP3 and SMCT, and the mineral metabolism-related gene NP4 were upregulated in the SMC population, implying their important roles in promoting the growth of the mud crab [39–41]. In addition to the DEGs, the upregulation of the “Ribosome biogenesis” KEGG pathway in the SMC population further supports this, as ribosome biogenesis is essential for protein biosynthesis and typically parallels growth rate [42].

Two nutrient metabolism-related pathways, namely “Choline metabolism” and “Riboflavin metabolism,” were identified as being enriched in this study. Choline is an essential precursor for metabolites like the neurotransmitter acetylcholine, the membrane constituent phosphatidylcholine, and the methyl group donor betaine, all crucial for crustacean metabolism [43]. Choline has been shown to enhance growth performance in the shrimp and crab [44, 45]. Our study indicated that mud crabs from the SMC population may utilize choline more efficiently for growth. Riboflavin, an essential component of the cofactors FAD and FMN, is important for energy metabolism and stress response, although its role in invertebrates is less understood [46]. Riboflavin and choline are metabolically interconnected, both participating in one-carbon metabolism, which may influence growth through their interaction.

Light is a critical ecological factor affecting crustacean growth, development, and reproduction. Its effects depend on factors such as wavelength, intensity, and photoperiod [47]. Specific wavelengths, like blue and cyan light, have been shown to promote growth in mud crabs and other shrimp species [48–50]. Light intensity and photoperiod also play key roles, with a 12-h light:12-h dark cycle being particularly beneficial [47, 51]. Shorter photoperiods may enhance growth rates in some species, while longer ones can support regular metamorphosis [52, 53]. The eyestalk, equipped with photoreceptors, is vital for receiving light signals and adapting to environmental conditions. The enrichment of the “Phototransduction” pathway in the eyestalk transcriptome suggests that the SMC population has an enhanced capacity to regulate growth in response to light signals [47].

5. Conclusion

In conclusion, this study demonstrated that the SMC population of mud crabs exhibited a faster growth rate than the ZPC population. Five DEGs related to molting and growth (Otop, Smyd, AM1159, PPAF1, and NARG2) were identified, which may serve as potential candidate genes for improving growth rates through genetic enhancement. The SMC population also appeared to allocate nutrients and energy more effectively to molting and growth via the mTOR signaling pathway, with regulators NLRC3 and QDPR playing key roles. Furthermore, the “Phototransduction” pathway plays a crucial role in the regulation of growth in mud crabs. These findings provide a solid foundation for future research on the molecular mechanisms underlying growth and support advancements in molecular breeding for mud crabs.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Yue Wang: investigation, methodology, data curation, writing–original draft and review, funding acquisition, Xiao-Shan Wang: resource provision, Sheng-Yu Liu: resource provision, Ben-Jian Wang: resource provision, Zhi-Xing Su: investigation, Xiao-Kang Lv: investigation, Jia-Yuan Xu: investigation, Xue-Feng Song: investigation, Bian-Bian Zhang: investigation, Li-Guo Yang: conceptualization, software, supervision, funding acquisition, writing–review and editing, supervision.

Funding

This work was supported by the Bureau of Science and Technology of Zhoushan (2023C41019), and the Scientific Research Foundation for Talents of Zhejiang Ocean University (JX6311101223).

Supporting Information

Additional supporting information can be found online in the Supporting Information section.