The mud crab, Scylla paramamosain, holds significant economic importance and has become a major contributor to the global seafood market (Tang et al., 2020; Perea and Festijo, 2022). Renowned for its rapid growth, large size, high meat yield, and delectable flavor, the mud crab is widely favored in the coastal regions of southern China. In 2022, China’s cultivation area for mud crabs reached an extensive 24,170 hectares, with an annual production of 154,661 tons. This production accounts for over half of the total marine aquaculture output in the country (Wang et al., 2019; Xie et al., 2021; China Fishery Statistical Yearbook, 2023).

Situated in the central region of Zhejiang Province, Sanmen Bay (29°02′53″N, 121°42′38″E) is a semi-enclosed bay that faces the East China Sea and falls under the jurisdiction of Sanmen County. With an approximate area of 775 square kilometers and an average water depth of 5–10 meters, the bay is renowned for its fishing and seawater aquaculture (Liu et al., 2018). Due to the favorable water quality in Sanmen Bay, which is ideal for mud crab cultivation, as well as its long-established history and efficient cultivation techniques, Sanmen Bay has emerged as one of the primary production areas for high-quality mud crab in China. The mud crabs from Sanmen have long enjoyed a reputation for their superior quality and are acknowledged as the finest specimens in the field. Sanmen County, often referred to as the “Hometown of Chinese mud crab” encompasses Huaqiao Town, Jiantiao Town, Pubagang Town, Shaliu Town and Shepan Town, where mud crabs are artificially bred in seawater ponds utilizing the bay’s resources. These regions serve as the primary breeding grounds for mud crabs in Sanmen Bay.

Research has demonstrated the influence of gut microbes on host growth and development, immunity, metabolism and nutrition (Rolfe, 1984; Sánchez et al., 2017). Gut microbes play a vital role in maintaining gut health by producing vitamins and enzymes that aid in nutrient digestion and absorption, replenishing vitamins and inhibiting the growth of harmful bacteria (Tremaroli and Bäckhed, 2012; Wei et al., 2019). The relationship between gut microbes and their hosts is characterized by intricate interdependencies (Linares et al., 2016). Additionally, gut microbes can generate various beneficial small-molecule metabolites that contribute to the host’s physiological activities (Krishnan et al., 2015). These metabolites participate in the host’s energy metabolism, modulate immune function and maintain the balance of the microbial community (Canfora et al., 2019; Garrett, 2020). Simultaneously, the host provides a stable and nourishing environment for the growth of gut microbes.

Although the term “core microbiome” is commonly used, there is still confusion and disagreement regarding its relevance in ecological and biological research. Frequently, the core microbiome pertains to the collection of microbial species or taxa that consistently inhabit a specific environment, sample, or group of organisms. While the overall composition of the microbiome may fluctuate among individual organisms due to factors like diet, environment, and genetics, the core microbiome constitutes a steady and enduring subset of microorganisms that play a role in the holistic functioning of the host organism (Turnbaugh et al., 2007; Tschöp et al., 2009; Kokou et al., 2019). The core microbiome plays a critical role in maintaining individual and gut health, significantly impacting the microbial community (Perlman et al., 2022). Microbial communities exhibit highly complex taxonomic diversity and there is a positive correlation between biodiversity and stability. In terms of maintaining microbial community stability, the impact of specific species outweighs the overall number of species in the community. Consequently, the core microbiome plays a crucial role in ensuring the functional stability of soil microbial communities (Xun et al., 2021; Jiao et al., 2022).

To date, limited studies have explored the core gut microbiome of mud crab. Understanding the core microbiome is essential for maintaining the stability and resilience of the host’s gut microbial community, as it is closely associated with beneficial functions for the host. Therefore, a comprehensive investigation of the gut microbiota of mud crab is necessary to identify its core gut microbes and fully comprehend the richness and significance of these microbes in the host’s health and ecosystem (Zhou et al., 2009; Sehnal et al., 2021; Sun and Xu, 2021; Zhang and Sun, 2022).

In this study, we utilized Illumina MiSeq’s 16S rRNA sequencing technology to examine the gut microbial composition, diversity and function of mud crab from multiple regions of Sanmen Bay. We observed variations in gut microbes based on sex. Moreover, we discovered the existence of a core mud crab gut microbiome with remarkably similar populations. Identifying these core microbiomes can assist in recognizing the assembly and drivers of microbial communities, thereby significantly enhancing our ability to manipulate the microbiome and address microbial perspectives to overcome challenges encountered in mud crab production activities.

The present study, we observed no significant differences in the diversity of the intestinal microbial community among the five sampling sites in Sanmen Bay’s mud crab. However, variations in community richness were evident across different sampling sites (Figure 1). At the phylum level, the dominant bacterial phyla in the gut microbial community of mud crabs were Firmicutes, Proteobacteria, Bacteroidota and Campilobacterota (Supplementary Figure S5). These phyla are commonly found in the gut microbiota of aquatic invertebrates, including mud crabs, which share similar gut microbial community compositions (Wei et al., 2019; Chen et al., 2021; Ma et al., 2021). Campilobacterota is widely present in the oral and intestinal microbiota of both vertebrates and some invertebrates, highlighting its importance in various animal gut microbial communities (Ma et al., 2020; Farag et al., 2022). Firmicutes and Bacteroidota play crucial roles in the growth, digestion, metabolism and immune function of aquatic animals (Foysal et al., 2021; Yang et al., 2023). At the genus level, we identified 19 core genera (Table 1) primarily engaged in symbiotic interactions (Figure 3C). These core genera accounted for 65.45% of the total sequences and were distributed across six phyla, including the dominant phyla mentioned earlier, as well as Fusobacteriota and Spirochaetota. Comparing the core genera identified in this study with the research by Wei et al. (2019), we found that Candidatus_Hepatoplasma, Vibrio, Bacteroides, Photobacterium and Carboxylicivirga were also identified as core genera in the gut microbiota of mud crab from different coastal regions in southern China. These core gut microbiota members may have established stable mutualistic symbiotic relationships with the mud crab (Oku et al., 2008; Leclercq et al., 2014; de Souza Valente and Wan, 2021). Furthermore, the core genera in mud crabs from Sanmen Bay exhibited quantitative and compositional differences compared to those in other coastal regions in southern China, suggesting potential geographical variations in the core gut microbiota of mud crab. This finding implies that the gut microbial community structure of mud crab may be influenced by geographical factors.

Significant differences exist in the average relative abundance of core genera within the intestinal microbiota of mud crabs in Sanmen Bay. Among these genera, Photobacterium exhibits the highest average relative abundance (Figures 3A,B). Photobacterium is a marine bacterium belonging to the Vibrionaceae family, commonly found in the intestines of fish and other marine organisms (Moi et al., 2017). It possesses the capability to produce various beneficial substances, including polyunsaturated fatty acids, cold-adapted lipase, asparaginase, esterases and antimicrobial compounds (Le Doujet et al., 2019). Previous studies have demonstrated that certain strains of Photobacterium exhibit probiotic activity in animal models. They promote the growth of beneficial bacterial communities, inhibit the growth of pathogenic bacteria, enhance the host’s immune response and maintain intestinal health (Oku et al., 2008; Urbanczyk et al., 2011). Consequently, it is possible that Photobacterium may play a similar role in promoting the growth of beneficial bacterial communities in the mud crab intestine, inhibiting the growth of pathogens, enhancing the host’s immune response and maintaining intestinal microbiota diversity. However, it is important to note that these functions have not been specifically studied or validated in the context of mud crabs and therefore require further investigation.

The core microbial taxa have a significant influence on the formation and stability of the intestinal microbial community (Lemanceau et al., 2017; Jiao et al., 2022). In this study, we observed significant differences in the stability of the intestinal microbial community among different sampling sites in Sanmen Bay (SP and SL, PBG) (Figure 4A). This suggests that the stability of the gut microbiota is influenced by geographical location, possibly due to variations in environmental conditions among different regions (Supplementary Table S3). Moreover, we found a significant positive correlation between core microbial taxa and the stability of the gut microbiota (Figure 4), indicating the crucial role of these core taxa in maintaining gut microbiota stability. Further analysis identified OTU5, OTU3550 and OTU4402 as the core taxa most significantly positively correlated with gut microbiota stability (p < 0.05; Figure 4C). OTU5 belongs to Firmicutes, although its genus level has not been identified; OTU3550 and OTU4402 belong to Proteobacteria and Photobacterium, respectively, at the phylum and genus levels. Bacteria belonging to the Firmicutes are known to produce a wide range of digestive enzymes, facilitating host digestion and nutrient absorption (Hudson and Egan, 2022). Proteobacteria dominate the gut microbiota of aquatic crustaceans (Ding et al., 2017; Apine et al., 2021) and have the ability to synthesize essential micronutrients like vitamin K2. They also regulate gut immunity by enhancing immune cell production and producing beneficial short-chain fatty acids, which contribute to energy metabolism and intestinal health (Saqib et al., 2023). Bacteria within the Photobacterium genus possess various potential functionalities, including the production of antimicrobial compounds and the promotion of beneficial bacterial growth, exhibiting probiotic activities (Cheung et al., 2015; Zhang et al., 2020). Therefore, the core intestinal microbiota may have the potential to regulate the intestinal microbial community in mud crab, enhancing the immune response and adaptability of the crabs. These findings reveal the significant role of core microorganisms in maintaining the stability of the intestinal microbial community, providing valuable insights into understanding the ecological functions of the intestinal microbiota and improving gut health.

Furthermore, we observed differences in the composition of the gut microbiota and the relative abundance of specific species between different sexes of the mud crab, as reflected in the core genera (Supplementary Figures S7, S8). While the influence of sex on the gut microbiota of aquatic animals is relatively small compared to environmental and genetic factors, it should not be ignored. Studies have shown that the gut microbiota structure and composition can vary between male and female fish, possibly due to metabolic differences between the sexes (Li et al., 2016). Similarly, the diversity of the gut microbiota in male wild Chilean octopus (Octopus mimus Gould, 1852) is higher than that in females, which may be related to their different feeding habits (Iehata et al., 2015). Gender has also been found to play a dominant role in shaping the functional aspects of the fish gut microbiota, as observed in Betta splendens (Gruneck et al., 2022). In our study, we observed that similar sex differences existed in the gut microbiota of mud crab, which are similar to previous findings in Eriocheir sinensis (Ma et al., 2021). The differences in gut microbiota between male and female crabs may be related to developmental disparities, which can impact the structure of the gut microbiota and subsequently influence the development of mud crabs. This intriguing observation warrants further investigation to elucidate the underlying mechanisms and potential implications for mud crab aquaculture. Overall, while sex may have a relatively minor influence on the gut microbiota compared to environmental and genetic factors, our study highlights the importance of considering sex as a factor that can contribute to variations in the gut microbiota composition in mud crabs. Further research is needed to fully understand the specific mechanisms driving these sex-related differences and their implications for the health and development of mud crabs in aquaculture settings.

The functional prediction of the core and non-core microbiota in the gut of mud crab in Sanmen Bay was conducted using PICRUSt2, revealing valuable insights into the potential functions and roles of the gut microbiota in this species. The functional pathways primarily involved Global and overview maps, Carbohydrate metabolism, Amino acid metabolism, Energy metabolism and Membrane Transport. These findings suggest that the gut microbiota plays a crucial role in nutrient utilization, metabolism, growth rate enhancement and environmental adaptability of the mud crabs. Furthermore, significant differences in the enrichment levels of functional pathways were observed between males and females in both the core and non-core microbiota. This indicates that gender influences the functional profiles of the microbial community, aligning with previous studies on gut microbiota and gender in other organisms (Mueller et al., 2006; Gomez et al., 2012; Markle et al., 2013; Abeles et al., 2014). However, limited research has explored the effects of gender on gut microbiota in crustaceans, highlighting the need for further investigations in this area. The composition and functional analysis of the gut microbiota in mud crab provide valuable insights into the impact of the microbiota on the health and growth of this species. In summary, this study successfully identified the core gut microbiota shared among mud crab in Sanmen Bay. These core microbial communities have been found to play a crucial role in the growth, immune function, and environmental adaptation of the crabs. Importantly, these findings hold promise for regulating the gut microbiota and enhancing the adaptability of mud crab in aquaculture settings. The discovery of these core microbial communities has significant biological implications, underscoring the importance of understanding their role in the gut of mud crab. By further elucidating the interactions between the host and its core gut microbiota, we can unlock new avenues for improving the health and performance of these commercially important crustaceans.

We conducted a comprehensive analysis of the structure, diversity, functionality, and stability of the gut microbiota in S. paramamosain from Sanmen Bay, China. We identified the core microbiome in this context. The clustering analysis yielded a total of 5,773 Operational Taxonomic Units (OTUs), with 34 of them classified as core OTUs. Within these 34 core OTUs, we identified 19 core genera. These 19 core genera account for 65.45% of the total sequences and span across 6 phyla. Among the core genera, Photobacterium had the highest average relative abundance and possessed probiotic activity. This suggests that it is highly adaptable to its host and has the potential to enhance immunity and maintain gut health in S. paramamosain. Moreover, our investigation revealed gender-specific differences in the composition and relative abundance of specific taxa, as well as the functionality of the gut microbiota in S. paramamosain. These differences may be attributed to variations in developmental processes and metabolic activities that occur between males and females. Furthermore, it is noteworthy that the core microbial group plays a vital role in maintaining the stability of the gut microbial community.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The requirement of ethical approval was waived by the committee on the Ethics of Animal Experiments of Ningbo University (no. SYXK20190005) for the studies involving animals, because the animal subjects used in the present study are crabs, which are invertebrates and are exempt from this requirement. The studies were conducted in accordance with the local legislation and institutional requirements.

HW and XJ conceived and designed the study and wrote the manuscript with support from all authors. XJ, MN, KQ, YH, YL, CC, CW, and CM performed all other experiments and analyzed data. All authors contributed to the article and approved the submitted version.

This research was supported by the National Natural Science Foundation of China (42276106), the Major Science and Technology Innovation Tackling Project of Wenzhou (ZF2022008), the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties (2021C02069-6), the earmarked fund for CARS (CARS-48), ZheJiang Agricultural Science and Technology Cooperation Project (2021SNLF029), and K. C. Wong Magna Fund in Ningbo University.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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