High-throughput sequencing reveals the core gut microbiota of the mud crab (Scylla paramamosain) in different coastal regions of southern China
Scylla paramamosain is a commercially important mud crab. The microbiota is a community that inhabits the crab intestine, and is important for physiological functional and host health.
Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria were the dominant phyla of the 36 representative phyla. Eleven genera of the 820 representative genera were considered as core gut microbiota and were distributed in the five dominant phyla. The core genus of the Proteobacteria included Arcobacter, Photobacterium, Vibrio, Shewanella and Desulfovibrio. The other four phyla contained one or two genera. Male and female crab samples had two different core genera, (male samples: Psychrilyobacter & Lactococcus; female samples: Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma).
This is the first time core intestinal microbiota have been identified in crab from nine coastal regions of southern China. This study provides sequencing data related to the gut microbiota of S. paramamosain, and may contribute to probiotic development for S. paramamosain aquaculture industries.
KeywordsScylla paramamosain Core gut microbiota Illumina MiSeq sequencing 16S rRNA
Hele county, Wanning city, Hainan province
Hepu county, Behai city, Guangxi zhuang autonomous region
Ruian county, Wenzhou city, Zhejiang province
- S. paramamosain
Sanmen county, Taizhou city, Zhejiang province
Shantou city, Guangdong province
Taishan county, Zhuhai city, Guangdong province
Xiapu county, Ningde city, Fujian province
Yangjiang city, Guangdong province
Yunxiao county, Zhangzhou city, Fujian province
Scylla paramamosain is a commercially important mud crab distributed along the coasts of southern China and other Indo-Pacific countries [1, 2, 3, 4]. Mud crab production reached 231,467 tons in 2017 in China . Currently, thanks to its richness, rapid growth, and high market value, the species is important in both fisheries and aquaculture in southern China [6, 7, 8].
The microbiota inhabits the intestine which is an important physiological functional organ in S. paramamosain, and is closely related to host health [9, 10]. Much research in humans has shown that the gut microbiota plays basic roles in nutrient absorption and immune function, which is beneficial to host health [11, 12]. Some pathological conditions, such as, inflammatory bowel disease , liver cirrhosis , cancer , obesity , and Type 1 Diabetes  appear to be caused by disruption to its normal balance. Research has shown that the gut microbiota are widely involved in organ development, nutrition, immunity and crustacean diseases [18, 19, 20, 21]. Other gut microbiome research has shown that the health, eating habits and crustacean habitats are key to the formation of a symbiotic gut bacteria model [22, 23]. Although a close relationship between the crab and its gut microbiota is increasingly accepted, limited data are available on the gut microbiota of S. paramamosain from Southern Chinese coasts. As part of aquaculture development, it is crucial to develop better probiotics to facilitate S. paramamosain industries, by unraveling gut microbial composition.
In this study, Illumina MiSeq sequencing of 16S rRNA was used to identify gut microbial composition in S. paramamosain. Samples from southern Chinese coasts were compared to characterize core gut microbiota.
Overview of sequencing data and alpha-diversity of samples from the nine coastal regions of southern China
26,157 ± 1969
215 ± 57
3.48 ± 0.216
0.84 ± 0.030
338 ± 125.87
0.995 ± 0.0016
26,168 ± 2337
755 ± 148
5.44 ± 0.993
0.91 ± 0.021
858 ± 109.83
0.988 ± 0.0008
27,904 ± 860
154 ± 53
3.42 ± 0.495
0.80 ± 0.040
189 ± 52.33
0.997 ± 0.0008
26,118 ± 2418
453 ± 280
4.29 ± 1.123
0.88 ± 0.070
527 ± 317.45
0.992 ± 0.0047
28,219 ± 821
286 ± 176
4.57 ± 2.047
0.89 ± 0.080
318 ± 150.82
0.997 ± 0.0014
27,103 ± 1061
270 ± 113
3.41 ± 0.367
0.79 ± 0.066
353 ± 146.69
0.994 ± 0.0025
27,495 ± 1216
318 ± 193
4.79 ± 1.872
0.90 ± 0.079
385 ± 133.53
0.996 ± 0.0015
25,757 ± 2074
179 ± 43
3.85 ± 0.594
0.86 ± 0.075
213 ± 65.40
0.997 ± 0.0011
26,952 ± 1164
174 ± 66
3.09 ± 1.118
0.75 ± 0.143
248 ± 107.24
0.996 ± 0.0015
27,729 ± 2215
429 ± 30
4.84 ± 1.043
0.92 ± 0.045
596 ± 91.95
0.991 ± 0.0022
22,555 ± 3723
530 ± 242
3.55 ± 0.896
0.76 ± 0.075
617 ± 269.36
0.990 ± 0.0044
26,197 ± 1560
186 ± 97
3.55 ± 0.579
0.84 ± 0.071
266 ± 170.60
0.996 ± 0.0030
28,377 ± 698
87 ± 14
2.47 ± 0.471
0.70 ± 0.068
129 ± 57.67
0.998 ± 0.0006
26,811 ± 1415
92 ± 43
2.43 ± 0.984
0.66 ± 0.217
153 ± 103.13
0.998 ± 0.0015
26,447 ± 657
594 ± 368
5.12 ± 2.241
0.89 ± 0.086
695 ± 394.79
0.991 ± 0.0054
21,993 ± 5211
378 ± 80
5.52 ± 1.623
0.92 ± 0.077
394 ± 70.29
0.998 ± 0.0016
25,662 ± 2052
219 ± 186
4.74 ± 1.923
0.91 ± 0.066
242 ± 167.04
0.998 ± 0.0004
25,138 ± 6631
480 ± 8
6.98 ± 0.149
0.98 ± 0.003
487 ± 15.34
0.998 ± 0.0004
The characterization of bacterial community richness, diversity and sequencing depth was performed using the alpha diversity index (Table 1). The Chao1 indices, which ranged from 129 ± 57.67 to 858 ± 109.83, were used to determine bacterial community richness in S. paramamosain. There were no differences in Shannon and Simpson indices. The Good’s coverage estimator of the samples ranged from 0.990 to 0.998 (Table 1), indicating that sequencing depths covered all species in samples. Meanwhile, the sparse curve reaches the saturation platform (Additional file 1: Figure S1A), manifesting that the sequencing depth is large enough to obtain a stable and unbiased estimate of species richness. In addition, the specaccum accumulation curves tend to gradually, indicating that the sample size is sufficient to reflect the abundance of the community richness, the results reflect the rate of increase in new species observed as the sample size continues to increase during the overall sampling of the sample. The number of OTUs increased rapidly from 1 to 54 and began to level off at the end of our sampling, indicating that bacterial diversity was largely saturated (Additional file 1: Figure S1B).
Composition of microbial communities in S. paramamosain
At the taxonomic level, six different patterns of intestinal microbial composition were distinguished. As shown (Additional file 1: Table S1), the number of taxonomic units detected in each region were present. The pattern of gut microbial composition in male samples was greater than female samples (Additional file 1: Table S1). Additional file 1: Figure S2-S4 show microbial community composition at Class, Order, and Family levels.
At the genus level, sequences from samples represented 820 genera (Additional file 1: Table S1). Genera in male samples numbered 45 more than in female samples (711 genera in female samples and 756 genera in male samples). The top 15 genera are listed in Fig. 1b and Additional file 1: Table S3; Candidatus_Hepatoplasma, Arcobacter, Photobacterium, Vibrio, Carboxylicivirga, Bacteroides, Spirochaeta_2, Psychrilyobacter, Sunxiuqinia, Parabacteroides, Defluviitaleaceae_UCG_011, Lachnoclostridium, Shewanella, Enterococcus, and Clostridium_sensu_stricto_11. These 15 genera accounted for nearly half or more of the total sequences in the samples. Candidatus_Hepatoplasma and Shewanella abundance differed significantly in female samples when compared to male samples (P = 0.0286, P = 0.0291, respectively). No significant differences were found for the other thirteen genera.
Core gut microbiota at the genus level in S. paramamosain
The core genera identified in samples
Relative abundance (%)
We also investigated these intergeneric the co-occurrence patterns of these genera based on Spearman’s rank correlations (Fig. 2c). We observed that the genus Candidatus_Hepatoplasma was inversely associated with almost every other genera (Spearman’s rank correlation coefficients (ρ) ranged from − 0.45 to 0.17) and Bacteroides showed relatively strong negative correlations with Photobacterium (ρ = − 0.53). Vibrio was positively associated with other genera besides Candidatus_Hepatoplasma, Lactobacillus and Bacteroides (Spearman’s rank correlation coefficients (ρ) ranged from − 0.27 to 0.68). Other genera were positively or inversely correlated with each other to different degrees.
The core genera identified in female samples
Relative abundance (%)
The core genera identified in male samples
Relative abundance (%)
Gut microbiota relationships across S. paramamosain, in nine regions
S. paramamosain are usually cultured in brackish, seawater ponds along the coasts of southern China and other Indo-Pacific countries. It is a commercially important mud crab distributed [3, 27]. Breeding of S. paramamosain mainly occurs along the coasts of southern China (Additional file 1: Figure S1), including Zhejiang, Fujian, Guangdong, Guangxi, Hainan province. Although gut microbiota regulates many aspects of digestive function, nutrition, metabolism, fat storage and gut-associated mucosal immunity , little is known about gut bacterial community structures in S. paramamosain. Hence, this study sought to examine gut microbial diversity and core gut microbiota of S. paramamosain from nine coastal regions in southern China. To the best of our knowledge, this study is the first to characterize core gut microbiota from S. paramamosain from southern Chinese coasts using state of the art, Illumina MiSeq sequencing methodologies.
Analysis of gut microbiota composition demonstrated that the dominant bacteria of the fifty-four samples belonged to six phyla, Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria, and the first four phyla were also found in the Eriocheir sinensis gastrointestinal tract . These results were consistent with a previous study on gut bacterial assemblages of Eriocheir sinensis from Lake Tai (286 km from Lake Gucheng in China) . These dominant genera may play major roles in gut function or adapt to the environment by the digestive tract.
The 11 core genera constituted a phylogenetic core of the genus, accounting for 48.81% of total sequences. Among them, Tenericutes from the genus Candidatus_Hepatoplasma, accounted for the greatest average relative abundance. Previous research had discovered that isopods with intestinal tract based Candidatus_Hepatoplasma, had higher survival rates when food was deficient . However, this has not yet been reported in S. paramamosain. In China, artificially cultured crabs are located in ponds, with little phytoplankton or zooplankton. Similarly, breeding densities are higher. In addition, farmers feed crabs at fixed times, therefore, S. paramamosain may be in hungry environments for prolonged periods. So, we speculated that the reason the mud crab could be able to adapt to thehunger environment is the regulation of Candidatus_Hepatoplasma. However, this conjecture must be corroborated by further research.
The core genera; Arcobacter, Photobacterium, Vibrio, Shewanella and Desulfovibrio belong to Proteobacteria. The genus Arcobacter is common in many marine invertebrates, such as crabs , mussels , abalones , and oysters . The genus Photobacterium, which is one of the nine genera in the family Vibrionaceae (order “Vibrionales”, class Gammaproteobacteria), is the largest genera after Vibrio [36, 37]. Some of its species exhibit bioluminescence and pathogenesis mechanisms , with one study reporting that Photobacterium is a potential freshwater fish pathogen . Worldwide, Vibrio is widely distributed in aquatic environments. However, many Vibrio members are considered primary pathogens in causing disease and death in aquaculture animals , and they seriously jeopardize the development of aquaculture. Many studies have shown that Vibrio provides a benefit to the host, for example, Asfie et al.,  isolated multiple strains of Protease-producing bacteria from the gingiva intestinal tract, and showed that some proteases secreted by Vibrio are beneficial to gums, growth and development. Similarly, Hamid et al.,  observed that Vibrio secreted amylases, proteases, lecithinases and chitinases to help digest important nutrients such as fat, proteins and carbohydrates in the host body. Further research also showed that Vibrio was present in both healthy and diseased S. paramamosain . We therefore speculated that Vibrio may digest important nutrients such as fat, protein and carbohydrates in the host body by secreting amylases and proteases to maintain normal activities in healthy crabs. Therefore, it was not surprising that Vibrio was found in samples in this study. Our study has also illustrated the diversity of Vibrio and its beneficial role as a dominant bacteria in the intestine. The separation and identification of beneficial Vibrio species may promote crab aquaculture production.
Firmicutes are often found in the gut of marine invertebrates, such as sea squirt (Ciona intestinalis) , black tiger shrimp (Penaeus monodon)  and the Atlantic blue crab (Callinectes sapidus) . The Lactobacillus genus belongs to the Firmicutes, which are commonly found in the gastrointestinal tracts of humans and other animals. In this study, Lactobacillus was also found in these nine regions, therefore, we speculated it may have potential probiotic properties in S. paramamosain. Studies have shown that due to its relevance in industrial applications in certain species, such as L. lactis, the central metabolic pathway of this genus has been extensively studied. These bacteria can convert large hexose sugar substrates to pyruvate via glycolysis and then to lactate . Lactococcus is the focus of intensive research in carbohydrate catabolism, the industrial fermentation process  and its role in promoting health, such as the prevention and protection of diarrhea and intestinal infections, are important for a well-balanced gut microbiota [47, 48]. So, on one hand, due to it can prevent and protect diarrhea and intestinal infections, it is not surprising that it can be found in all samples from nine regions. On the other hand, it is worthy of further study to isolate and characterize the functional bacteria of this genus from intestinal samples, and it may develop probiotics for the S. paramamosain breeding industries. Moreover, the genus Bacteroides from the Bacteroidetes phyla has been associated with animal protein metabolism, a variety of 354 Yang et al., amino acids and saturated fats . And other core genera of Shewanella, Desulfovibrio, Romboutsia, Carboxylicivirga and Spirochaeta_2 in functional study have not yet been reported.
According to the experience of many farmers, there were differences in development processes between male and female crabs. However, there were 13 identical genera in the mid-gut population of male and female crabs (Fig. 3). This meant that gender had no significant effects on gut composition in S. paramamosain. This was consistent with Jin et al.,  and their study, the intestinal flora of Eriocheir sinensis in Yangcheng West Lake. An interesting discovery from this study was the difference between male and female samples in terms of the core gut microbiota, at the genus level. There were two different core genera in female samples (Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma), and two additional core genera in male samples (Psychrilyobacter and Lactococcus). At present, the functions of these four genera have not been reported. We speculated these genera may be the reason for differences in flavors between male and female crabs. It will be interesting to determine which gut microbiota cause flavor differences. In this study, the 11 core genera were distributed in the five dominant phyla. Among these, five genera (Arcobacter, Photobacterium, Vibrio, Shewanella, Desulfovibrio) belonged to Proteobacteria, while the other four phyla contained one or two genera, such as Bacteroidetes (genus Carboxylicivirga & Bacteroides), Firmicutes (genus Lactobacillus & Romboutsia), Tenericutes (genus Candidatus_Hepatoplasma), and Spirochaetae (genus Spirochaeta_2). We speculated that these phyla and genera present in the S. paramamosain’ gut might have many reasons. Firstly, in terms of internal factors, in China, although the seedlings of these S. paramamosain have broken through the key technologies of large-scale breeding , the artificial breeding technology is not quite ripe yet. So mud crab seedlings are mainly derived from natural sea areas, these crabs in nine regions may be not the same source, they are the same species, S. paramamosain, therefore they are hereditary. We suspect that they have these common core genera that may be genetic factors.
On the other hand, environmental factors have also been reported to affect gut microbial composition . Our analyses found 11 core genera in nine different regions. This observation may indicate that environmental factors do little to affect the structure of the gut microbiota of S. paramamosain or the lack of an effect may have been due to similar natural conditions (Additional file 1: Table S4), such as temperature, salinity, pH, dissolved oxygen (DO) and so on. It may be that these nine regions are all at suitable temperatures and salinities and they live in ponds that are separated by mud with black plastic membranes to prevent the loss of crabs.
In this study, we used Illumina MiSeq sequencing to clarify the gut microbiota of composition from S. paramamosain. This was the first time we identified core intestinal microbiota from nine coastal regions of southern China. We analyzed 472,782 valid tags from nine regions samples, of which 2552 OTUs were identified. Our results showed that Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria were the dominant phyla of the 36 representative phyla. 11 genera of the 820 representative genera were considered as core gut microbiota and were distributed in the five dominant phyla. The core genera distributed in Proteobacteria were the most genera, including Arcobacter, Photobacterium, Vibrio, Shewanella, Desulfovibrio. While other four phyla contain one or two genera. Moreover, there were differences between male and female samples in the core gut microbiota, at the genus level. There were two special genera in female samples (Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma), and two special genera in male samples (Psychrilyobacter and Lactococcus). This study has generated much sequencing data related to the gut microbiota of S. paramamosain and may contribute to probiotic development for mud crab aquaculture industries in the future.
Sample collection in different areas
Mud crab breeding is mainly distributed along the coast of southern China (Additional file 1: Figure S5), including Zhejiang, Fujian, Guangdong, Guangxi, Hainan provinces. Firstly, we purchased crabs from local farmers in nine areas between May and June 2017. These crab collection areas were at Sanmen county, Taizhou city, Zhejiang province (SM, 29°06′16.81″N, 121°23′44.45E), Ruian county, Wenzhou city, Zhejiang province (RA, 27°46′42.17″N, 120°39′18.65″E), Xiapu county, Ningde city, Fujian province (XP, 26°53′6.61″N, 120°0′20.02″E), Yunxiao county, Zhangzhou city, Fujian province (YX, 23°57′29.02″N, 117°20′22.74″E), Shantou city, Guangdong province (ST, 23°21′22.19″N, 116°40′39.89″E), Yangjiang city, Guangdong province (YJ, 21°51′39.78″N, 111°58′39.04″E), Taishan county, Zhuhai city, Guangdong province (TS, 22°12′24.13″N, 113°17′50.43″E), Hepu county, Behai city, Guangxi zhuang autonomous region (HP, 21°39′48.50″N, 109°12′10.98″E), Hele county, Wanning city, Hainan province (HL, 18°54′0.52″N, 110°28′32.39″E). In these locations, we caught mud crab. Each region chose good limbs and good vitality 12 female and male crabs (body weight: 250-450 g), immediately isolated their intestinal tracts and contents and froze samples in liquid nitrogen. Samples were transported to the lab on dry ice. When extracting DNA, every three samples were mixed into one, so each region had six samples, with a total of 54. All samples were stored in sterile containers.
DNA extraction, 16S rRNA gene amplification, and Illumina MiSeq sequencing
Genomic DNA was extracted from each sample using the DNeasy PowerLyzer PowerSoil Kit (QIAGEN, Hilden, Germany). DNA concentration and purity were assessed on 1% agarose gels. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the barcode-fusion forward primer 343F (5′- TACGGRAGGCAGCAG-3′) and the reverse primer 798R (5′- AGGGTATCTAATCCT − 3′). The PCR was performed in a 30 μl volume with 15 μl of 2 × HiFi Hot Start Ready Mix, 1.0 μl of forward and reverse primers (10 μmol/L), and 50 ng of genomic DNA as template. Thermal cycling consisted of initial denaturation at 94 °C for 5 min; followed by 26 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, elongation at 72 °C for 30 s and a final extension at 72 °C for 7 min.
The PCR products were purified using Agencourt Ampure XP beads (Beckman, CA, USA) according to the manufacturer’s instructions. 5 μl purified product were detected by 1% agarose gel electrophoresis and 1 ul purified product for concentration detection using a NanoDrop Lite spectrophotometer (Thermo Scientific). The purified DNA was then used as a template to perform a second PCR amplification using the same primer sequences and the protocol described above, however, 8 cycles were employed. After a further purification with the AMPure XP beads, the amplicon was measured concentration using NanoDrop Lite spectrophotometer. Purifed amplicons were pooled in equimolar and pairedend sequenced (2 × 300) on an Illumina MiSeq platform.
Raw sequencing data came in FASTQ format. Paired-end reads were preprocessed using Trimmomatic software , to detect and remove ambiguous bases (N). The software also removed low quality sequences, with average quality scores below 20, using the sliding window trimming approach. After trimming, paired-end reads were assembled using FLASH software . The parameters for assembly were: 10 bp minimal overlap, 200 bp maximum overlap and a 20% maximum mismatch rate. Sequences were performed further denoising as follows: contigs with ambiguous, homologous sequences or below 200 bp were abandoned. Reads with 75% of bases above Q20 were retained. Contigs with chimeras were detected and removed. These steps were achieved using the QIIME software (version 1.8.0) . Clean reads were subjected to primer sequence removal and clustering to generate operational taxonomic units (OTUs) using UPARSE software, with 97% similarity cutoff . The representative read of each OTU was selected using QIIME software. All representative reads were annotated and blasted against the Silva database Version 123 (or Greengens) (16 s rDNA) using RDP classifier (confidence threshold was 70%) . For alpha-diversity metrics, Observed Species, Chao1 estimator, Shannon Wiener Index, Simpson diversity index, Good’s Coverage with QIIME (Version 1.8.0) and displayed with R software (Version 2.15.3). For beta-diversity metrics, the weighted UniFrac distance matrix  was calculated and visualized using Principal Coordinate Analysis (PCoA) in QIIME. Biomarker discovery analysis of each taxonomic unit was performed using LeFse (version 1.0.7) . All figures were generated with customized R scripts.
All statistical analyses were performed using SPSS (SPSS 21.0). ANOVA was also used for statistical analyses. Correlations between the core genera of gut microbiota were appraised by calculating nonparametric Spearman’s rank correlation coefficients, which were displayed in a correlation matrix. Results were considered significant at P < 0.05.
We thank Oebiotech (Oebiotech, Shanghai, China) for sequencing consultation and support.
CW and HW1 (Huan Wang) conceived and designed the study. HW2 (Hongling Wei), HW1and LT took samples of experimental animals. HW2, HW1, LT, CM, CY and LC performed and analyzed all the other experiments. HW2 wrote the manuscript with support from all authors. All authors read and approved the final manuscript.
This work was supported by the Major Science and Technology Special Project of Zhejiang Province (No. 2016C02055–8); the Ministry of Agriculture of China and China Agriculture Research System (No. CARS-48); the Marine and Fishery Bureau, Sanmen County, Zhejiang Province (HK2017000088); and the K. C. Wong Magna Fund in Ningbo University. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethics approval and consent to participate
The animals used in this study are crabs, which are invertebrates and are exempt from ethical requirements. S. paramamosain is not an endangered or protected species. All animal work was conducted according to the relevant national and international guidelines. No specific permissions are required to work with invertebrates in China. Similarly, no specific permissions are required for the collection of S. paramamosain from sample sites because they were not collected from protected areas.
Consent for publication
The authors declare that they have no competing interests.
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