Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets

Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets

Journal Pre-proof Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets Yunfei Sun, Wenfeng H...

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Journal Pre-proof Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets

Yunfei Sun, Wenfeng Han, Jian Liu, Feng Liu, Yongxu Cheng PII:

S0044-8486(19)31759-4

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734518

Reference:

AQUA 734518

To appear in:

aquaculture

Received date:

11 July 2019

Revised date:

6 September 2019

Accepted date:

13 September 2019

Please cite this article as: Y. Sun, W. Han, J. Liu, et al., Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734518

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© 2019 Published by Elsevier.

Journal Pre-proof Microbiota comparison in the intestine of juvenile Chinese mitten crab Eriocheir sinensis fed different diets Yunfei Sun1,2,3 , Wenfeng Han1,2,3 , Jian Liu1,2,3 , Feng Liu4 and Yongxu Cheng1,2,3,* 1

Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai

Ocean University, Shanghai, China 2

Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University,

Shanghai, China

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National Demonstration Centre for Experimental Fisheries Science Education, Shanghai

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Ocean University, Shanghai, China

Shandong Freshwater Fisheries Research Institute, Jinan, China

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*Correspondence:

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Professor Yongxu Cheng, College of Fisheries and Life Science, Shanghai Ocean University, 999# Huchenghuan Road, Pudong district, Shanghai,

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201306, P.R. China. Email: [email protected]

Abstract: Intestinal microflora, as a component of the host bacteria ecosystem, can influence the healthy growth of crab. The aim of this study was to determine the intestinal microflora of 1

Journal Pre-proof the juvenile Chinese mitten crab Eriocheir sinensis with different feeding modes. We investigated the bacterial communities of crab intestine resulting from three different feeding modes using high- throughput sequencing technology, including traditional, formulated, and mixture (combined traditional and formulated) diet feeding modes. Intestinal microbiota biodiversity and richness were not affected by the different feeding modes. The most dominant community members at the phylum level were Tenericutes, Bacteroidetes, Firmicutes, and Proteobacteria. Significant differences were only observed in the relative

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abundance of Tenericutes (P = 0.0390) and Proteobacteria (P = 0.0273) in the female crab intestines among different feeding modes. The seven most abundant genera in the intestine of

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male and female crabs included Candidatus Bacilloplasma, Candidatus Hepatoplasma,

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Prolixibacter, Vibrio, Dysgonomonas, Coprobacillus, and Lactovum. Moreover, high average

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similarity of the microbial community was observed in the intestine of male and female crabs among the three different feeding modes and also between male and female crabs within

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these feeding modes. The most abundant genera in the latter (>10%) were Candidatus

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Bacilloplasma, Candidatus Hepatoplasma, and Prolixibacter. In addition, unique microbial

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genera were found in male or female crab intestine among different feeding modes. Differences were also observed in the relative enrichment of the dominant bacteria in male and female crab intestine among different feeding modes. Nevertheless, these findings suggested that the microbial communities in the intestines of crabs showed overall high average similarity among the three feeding modes or between the sexes as reflected by the biodiversity, richness of intestinal microbiota, and the most dominant community members at the phylum and genera levels. The results of this study provide a theoretical reference regarding the diversity of intestinal bacteria in juvenile E. sinensis.

Keywords:intestine, Eriocheir sinensis, 16s rRNA, bacterial community, feeding mode, sex 2

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1. Introduction The Chinese mitten crab Eriocheir sinensis is of considerable commercial significance and is bred extensively in Eastern and Northern China (Liu et al., 2013). Aquaculture constitutes a complex ecosystem wherein microorganisms in the water, sediment, and gastrointestinal tracts interact with each other to affect the health of aquatic animals (Cheng et al., 2009). The term “microbiota” refers to the community of microorganisms harbored in a

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specific ecosystem (Zhang et al., 2016). It is believed that the commensal microbes are evolutionarily stable, positively or negatively influence host health in the intestine or other

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organs (Goffredi et al., 2014; Lai et al., 2009), and contribute to the development and

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metabolism of the host (Semova et al., 2012; Shin et al., 2011). However, with the rapid

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growth of intensive cultivation of E. sinensis, various diseases have appeared that markedly affect the sustainable development of the crab industry (Chaiyapechara et al., 2012). In

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later stage of these crabs.

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particular, the health condition of the juvenile E. sinensis intestine is very important for the

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As the intestinal bacterial microbiota of farmed aquatic animals can play a role in nutrition, immunity, and disease resistance of animals (Cardona et al., 2016), probiotics, constituting live microbial feed supplements, have been used to enhance aquaculture and beneficially affect the host animal by improving its intestinal microbial balance (Fuller, 2010). However, before the intestinal microbial balance in E. sinensis can be manipulated using probiotics, the diversity of their intestinal bacteria and the bacterial community differences in crab intestine among different feeding modes must first be understood. Three primary feeding modes are used for juvenile E. sinensis including traditional, formulated, and combined formulated and traditional diets. Nevertheless, few studies have investigated the intestinal microbiota among different feeding modes. 3

Journal Pre-proof Recently, an increasing number of studies have focused on the microbial community compositions in aquaculture water (Cheng et al., 2009; Zhang et al., 2016), sediment (Liu et al., 2013), and crab guts, gills (Zhang et al., 2016), and intestines (Li et al., 2007), investigating the differences of bacterial communities in the crab intestine and surrounding environments. These results showed that the dominant bacteria in the crab pond sediment were Proteobacteria and Bacteroidetes (Li et al., 2007). Proteobacteria also has been indicated to be dominant in crab pond water (Cheng et al., 2009), and Proteobacteria and

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Bacteroidetes are predominant in the gut of E. sinensis (Li et al., 2007). These results also implied that the microbes in sediment may influence the microbial community structure in

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the gut of E. sinensis. In addition, the relationship between environmental factors and the

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microbial community structure in surrounding environments of aquaculture ponds has also

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been investigated, revealing that salinity was a major contributor to the structure and function of the microbial communities in shrimp cultural enclosure ecosystems of China (Hou et al.,

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2017). Comparison of the microbial community and the expression of immunity genes in the

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foregut, midgut, and hindgut of E. sinensis indicated that the expression of antimicrobial

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peptides were significantly elevated in the hindgut and the gene expression of EsRelish was higher than that of Toll signaling pathway genes (Dong et al., 2018). Some researchers reported differences in the intestinal bacteria between pond-raised and wild crabs, and indicated that the abundance of Bacteroides and the total bacterial load were approximately 4 to 10 times higher in pond-raised crabs than those in wild crabs (Li et al., 2007). However, to our knowledge no study has examined the potential differences of the microbial communities in the intestine between juvenile female and male crabs. To address these issues, in this study we characterized the microbial communities of female and male crab intestine samples obtained from three different crab culture systems (representing the three feeding modes) and evaluated the effect of sex and feeding mode on 4

Journal Pre-proof the bacterial community composition of the crab intestine. The results presented in this study provide a theoretical reference regarding the diversity of intestinal bacteria in juvenile E. sinensis that will inform efforts for their manipulation using probiotics.

2. Materials and methods 2.1 Sample collection Juvenile crab intestine samples were collected from a crab farm in Dongtan, Shanghai

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City, China (31.62°N, 121.40°E) including three types of feeding modes. Each feeding mode encompassed three ponds (A1, A2, A3, B1, B2, B3, C1, C2, C3). Water and sediment were

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collected from three sites in each pond. Water samples were collected at 50 cm below the

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water surface and the sediments were collected at the surface. Water samples (250 ml) taken

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from the three sites of the pond were mixed as a single sample, then filtered using a 0.22-μM filter for DNA extraction. Sediment samples (50 g) were mixed and then placed into a 15- ml

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centrifuge tube. A total of 10 female and 10 male crab samples were taken from each pond

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and then the respective intestinal samples were combined as one sample per sex. Thus, three

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intestine samples were obtained for each mode (A1FI, A1MI, A2FI, A2MI, A3FI, and A3MI for the traditional diet, B1FI, B1MI, B2FI, B2MI, B3FI, and B3MI for the formulated diet, and C1FI, C1MI, C2FI, C2MI, C3FI, and C3MI for the combined diet, where FI and MI represent the female and male crab intestine, respectively). The crab carapace width was approximately 25 mm. The surfaces of crabs were sterilized with 70% ethanol, then the intestines were aseptically dissected from the musculature and placed into a 2- ml sterile centrifuge tube. All samples were stored at −80°C until DNA extraction.

2.2 DNA extraction, amplification, and sequencing DNA was extracted from all samples using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, 5

Journal Pre-proof Norcross, GA, U.S.) base on the manufacturer’s instructions. The V4 region of the bacteria 16S ribosomal RNA gene was amplified by PCR (95°C for 2 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min) using primers

515F:

5-barcode-GTGCCAGCMGCCGCGG-3

and

907R:

5-CCGTCAATTCMTTTRAGTTT-3, where barcode represents an eight-base sequence unique to each sample. PCR was performed in triplicate 20-μl mixtures containing 4 μl of 5 ×

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FastPfu Buffer, 2 μl of 2.5 mM dNTPs, 0.8 μl of each primer (5 μM), 0.4 μl of FastPfu

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Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City,

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CA, U.S.) according to the manufacturer’s instructions and quantified using QuantiFluor™

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-ST (Promega, Madison, WI, U.S.). Purified PCR products were quantified using Qubit®3.0

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(Life Technologies, Invitrogen, Carlsbad, CA, U.S.) and every 24 amplicons whose barcodes were different were mixed equally. The pooled DNA product was used to construct Illumina

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Pair- End libraries following the Illumina genomic DNA library preparation procedure. The n,

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the amplicon library was paired-end sequenced (2 × 250) using an Illumina MiSeq platform

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(Shanghai BIOZERON Co., Ltd., China) according to standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA550313).

2.3 Bioinformatics analyses Sequencing reads of the 16S rRNA gene were processed and analyzed using UPARSE (Edgar, 2013) and QIIME (Caporaso et al., 2010), as described previously by Ramírez and Romero (2017), except that the sequences were trimmed to 270 bp. Raw fastq files were demultiplexed and quality- filtered using Quantitative Insights Into Microbial Ecology (QIIME, version 1.17) software with the following criteria: (i) the 250 bp reads were 6

Journal Pre-proof truncated at any site receiving an average quality score <20 over a 10-bp sliding window, discarding the truncated reads that were shorter than 50 bp; (ii) exact barcode matching: 2 nucleotide mismatches in primer matching and reads containing ambiguous characters were removed; and (iii) only sequences that overlapped longer than 10 bp were assembled according to their overlap sequence. Reads that could not be assembled were discarded. The sequences were subsequently classified into operational taxonomic units (OTUs) at the 97% sequence similarity level using UPARSE (version 7.1) and chimeric sequences were

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identified and removed using UCHIME (Edgar, 2010). The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed using the Ribosomal Database Project (RDP)

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Classifier (http://rdp.cme.msu.edu/) against the Silva (SSU123)16S rRNA database using a

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confidence threshold of 70%.

2.4 Statistical analysis

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The Simpson and Chao diversity parameters were calculated using MOTHUR ver. 1.35.1

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software and comparatively analyzed by Welch’s t test using SPSS ver. 22.0 software (IBM

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Corp., Armonk, NY, U.S.). P < 0.05 was considered significant. A heatmap was drawn in R environment according to the cluster result. The Kruskal–Wallis H test was applied on the four most abundant phyla in male and female crab intestine among different feeding modes. The Wilcoxon rank-sum test was used on the four most abundant phyla between male and female crab intestine. Venn diagrams were produced using the free online platform of I-Sanger (www.i-sanger.com). A non- metric multidimensional scaling (NMDS) ordination to illustrate bacterial community composition variation of juvenile crab intestines was conducted using I-Sanger based on the Bray-Curtis distance of OTUs. Furthermore, linear discriminant analysis (LDA) effect size (LEfSe) was performed to present the enrichment of intestinal microbial communities within different treatments using I-Sanger. 7

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3. Results 3.1 Microbial diversity in juvenile male and female crab intestine among different feeding

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modes

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Figure 1 Analysis of rarefaction curves and alpha diversity comparisons of the microbial communities of crab intestine in different feeding modes. (A) Shannon-Wiener curve using Shannon as the metric for crabs fed various diets. (B) Rarefaction curve. (C) Simpson diversity indices for A, B, and C intestinal samples. Error bars indicate SD (n=3). (D) Chao species richness indices for A, B, and C intestinal samples. Error bars indicate SD (n=3).

The Shannon-Wiener curve indicated that the dataset from the diversity analysis was sufficiently large to reflect the bacterial diversity information of the samples (Figure 1A). The rarefaction curve showed that the sequencing was relatively comprehensive in covering the bacterial diversity, as the rarefaction curves tended to approach saturation (Figure 1B). The 8

Journal Pre-proof Simpson index varied between 0.21 ± 0.07 in the male crab intestine of treatment A, 0.31 ± 0.05 for treatment B, and 0.30 ± 0.05 for treatment C; no significant differences was detected between any two treatments (A and B, P = 0.11; A and C, P = 0.15; B and C, P = 0.75; Welch’s t test; Figure 1C). The Simpson index in the female crab intestine of treatment A, B, and C was 0.26 ± 0.08, 0.30 ± 0.09, and 0.26 ± 0.02, respectively; no significant difference was observed in any two treatments (A and B, P = 0.60; A and C, P = 0.99; B and C, P = 0.54; Welch’s t test). Moreover, Welch’s t test indicated that no significant differences existed in the

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Simpson index of intestinal bacterial communities between male and female crabs in treatment A (P = 0.49), B (P = 0.85), and C (P = 0.30). No significant difference was

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observed in the Chao1 index of male or female crab intestine among treatment pairs (A and B,

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P = 0.33, 0.45; A and C, P = 0.38, 0.37; B and C, P = 0.50, 0.92, respectively; Welch’s t test;

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Figure 1D). No significant differences were detected in the Chao1 index between male and

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female crab intestine in treatment A (P = 0.61), B (P = 0.20), and C (P = 0.12).

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3.2 Microbial community composition at the phylum level in the juvenile crab intestine of the

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three different feeding modes

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Figure 2 Relative abundances (%) of dominant phyla and comparison of bacterial

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abundances in the intestine at the phylum level. (A) Relative abundances (%) of dominant phyla from all samples based on 16S rRNA gene amplicon sequencing data. The phylum

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level distribution presented is based on 70% similarity cluster OTUs. Unclassified phyla with

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relative abundances lower than 1% were assigned as “others.” The four major dominant phyla

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included Proteobacteria, Firmicutes, Bacteroidetes, and Tenericutes. (B) Kruskal–Wallis H test on the four most abundant phyla in male and female crab intestine among different feeding modes. The vertical axis represents the microbial community name at the phylum level, the column length corresponding to the species represents the average relative abundance of the species in each group, and A, B, and C represent the traditional, formulated, and combined formulated and traditional diet feeding mode, respectively. *0.01 < P < 0.05. (C) Wilcoxon rank-sum test on the four most abundant phyla between male and female crab intestine in the three different feeding modes.

Of the classifiable sequences, 12 phyla were identified. The most dominant community 10

Journal Pre-proof members, Tenericutes, Bacteroidetes, Firmicutes, and Proteobacteria accounted for 66.41, 14.95, 9.89, and 8.40% of the total microbial community, respectively (Figure 2A). For female crabs, no significant differences were observed in the relative abundance of Bacteroidetes (P = 0.1133) and Firmicutes (P = 0.4911) among the three feeding modes (Figure 2B). However, significant differences were observed in the relative abundance of Tenericutes (P = 0.0390) and Proteobacteria (P = 0.0273) among these different feeding modes, with the relative abundance of Tenericutes (from high to low) being group C

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(69.65%), B (64.60%), and A (57.02%) and that of Proteobacteria (from high to low level) being group A (12.30%), C (9.67%), and B (4.43%). For male crabs, no significant

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differences were observed in the relative abundance of Tenericutes (P = 0.1931),

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Bacteroidetes (P = 0.1133), Firmicutes (P = 0.4911) and Proteobacteria (P = 0.0509) among

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the three feeding modes. No significant differences were observed for any feeding mode in the relative abundance of Tenericutes (P = 0.6625, 0.6625, 0.3827), Bacteroidetes (P =

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0.6625, 0.6625, 0.3827), Firmicutes (P = 0.0809, 1.0000, 0.1904), and Proteobacteria (P =

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0.0809, 1.0000, 0.3827) between female and male crabs, for modes A, B, and C, respectively

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(Figure 2C).

3.3 Microbial community composition at the genus level in the juvenile crab intestine of the three feeding modes

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Figure 3 Relative abundance of the top 50 most abundant genera and comparison of bacterial

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abundances in the intestine at the genus level. (A) Heatmap showing the relative abundance

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of the top 50 most abundant genera in bacterial communities in the eighteen samples. Dendrograms for hierarchical cluster analysis grouping genera are shown at the left. The

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color scale represents the normalized values of relative abundances by log10. (B) Mean

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relative richness of seven high abundance genera and five high variance genera in male and female crab intestine selected for multiple comparisons among different feeding modes. *0.01 < P ≤ 0.05, **0.001 < P ≤ 0.01, ***P ≤ 0.001. (C) Mean relative richness of seven genera of high abundance genera and five genera of high variance selected for comparisons between male and female crab intestine in different feeding modes. Positive differences in mean relative abundance indicate genera overrepresented in the female crab intestine, whereas negative differences indicate greater abundance in the male crab. *0.01 < P ≤ 0.05, **0.001 < P ≤ 0.01, ***P ≤ 0.001.

A heat map was used to graphically illustrate the male and female crab intestinal bacterial 12

Journal Pre-proof community at the genus level in the three feeding modes (Figure 3A). The heat map revealed that there were no differences in the abundance of the male crab and female crab intestinal bacterial community at the genus level among the three different feeding modes (Figure 3B and 3C). The seven high abundance genera were selected for multiple comparisons, including Candidatus Bacilloplasma, Candidatus Hepatoplasma, Prolixibacter, Vibrio, Dysgonomonas, Coprobacillus, and Lactovum. The Kruskal–Wallis H test showed that the most abundant seven genera did not significantly differ among different feeding modes in the male and

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female intestine. The five high variance genera including Bacteroides, Albimonas, Aeromonas, Paracoccus, and Shewanella were also selected for multiple comparisons. The Kruskal–

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Wallis H test showed that no significant differences existed in the five genera between the

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male and female intestine.

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The heat map showed that no differences existed in the abundance of the bacterial community between the male and female crab intestine at the genus level for the different

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feeding modes (Figure 3D-F). The seven high abundance and five high variance genera were

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selected for multiple comparisons. The Student’s t-test showed that the seven genera did not

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significantly differ between the male and female crab intestine in A and C feeding modes. The Kruskal–Wallis H test revealed that the five genera did not significantly differ between the male and female crab intestine for the A and C feeding modes. In comparison, in the B feeding mode, only the relative abundance of Dysgonomonas was significantly higher in the female crab intestine than that in the male (P = 0.0328). The Student’s t-test showed that no significant differences existed for the other six high abundance genera between female and male crabs. The Kruskal–Wallis H test further indicated that the five high variance genera did not significantly vary between the male and female crab intestine.

3.4 Unique and shared bacterial taxa 13

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Figure 4 Venn diagram analysis of microbial communities in the crab intestine at the genus level between different samples. The Venn diagram illustrates overlapping and unique

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bacterial genera in the juvenile crab intestine among the three feeding modes (A and B) and

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between female and male juvenile crabs for the three feeding modes (C, D, and E). Numbers

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in the overlapping regions indicate the number of shared microbial genera between different treatments. The number of unique microbial genera indicate these genera were identified in at least one of the treatments.

Bacterial genera in the crab intestine for the three feeding modes were investigated as shown in Figure 3A and 3B. The analyses showed that 75 and 70 genera were shared in female and male crab intestines among the various feeding modes, which corresponded to 88.2% and 81.4% of the total genera, respectively. For the female crab, the most abundant genera in the shared genera (>10%) were Candidatus Bacilloplasma (50.51%), Prolixibacter (12.06%), and Candidatus Hepatoplasma (10.67%). The unique microbial genus in the crab 14

Journal Pre-proof intestine of feeding mode B was Mesorhizobium, and those of A were Flavobacterium and Chitinophaga; however, no unique genus was identified for feeding mode C. For the male crab, the most abundant genera in the shared genera (>10%) were Candidatus Bacilloplasma (44.88%), Candidatus Hepatoplasma (22.34%), and Prolixibacter (8.98%). The unique microbial genus in the crab intestine for feeding mode A was Hyphomicrobium and that for C was Collinsella; however, no unique genus was identified in the crab intestine for feeding mode B.

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In feeding mode A, 82 shared microbial genera were identified between the female and male crab intestines, and three unique genera (Planctomyces, Mycobacterium, and

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Mesorhizobium) were detected in the male crab intestine whereas no unique genus was found

male crab

intestines,

with

five (Chryseobacterium,

Mycobacterium,

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female and

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in the female crab intestine. In feeding mode B, 74 shared genera were observed between

Rhodothermaceae, g__norank_f__LD29, and Variovorax) and one (Taibaiella) unique genera

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in the female and male crab intestine, respectively. In feeding mode C, 76 shared genera were

Taibaiella,

Hyphomicrobium)

and

four

(Marinifilum,

Collinsella,

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Bacteroidetes,

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identified between female and male crab intestines, with five (Fusobacterium, Sphingopyxis,

Flavobacterium, Chitinophaga) unique genera found in the female and male crab intestine, respectively.

3.5 Effect of feeding mode on the bacterial community composition of the juvenile female and male crab intestine

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Figure 5 Non-metric multidimensional scaling (NMDS) of intestinal bacterial communities based on Bray–Curtis similarities among different feeding modes and between female and

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male crabs. Each symbol represents a sample from one crab aquaculture pond. Symbol

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shapes denote the different feeding modes (circles = traditional diet, rhombus = formulated

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diet, square = combined traditional and formulated diet). Symbol colors denote the gender of

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juvenile crabs (red, green, and purple = female; blue, orange, and pink = male).

Bacterial community compositions of juvenile crab intestines from NMDS analysis based on the Bray-Curtis similarity distance are shown in Figure 5. Stress value was used to test the results of NMDS analysis. It is generally believed that stress value < 0.2 can be represented by a two-dimensional point graph of NMDS, which has certain explanatory significance; stress value < 0.1 can be considered as a good sort; stress value < 0.05 indicates good representativeness. Thus, the observed 0.11 stress value indicated that the NMDS ordination plot is a good representation of the difference in the data. For the female juvenile crab, the bacterial community composition in the intestine of mode A differed from those of modes B and C. For the male crab, crab intestines from mode A exhibited bacterial communities 16

Journal Pre-proof distinct from those in mode C. This indicated that the feeding status could change the microbiota community structure of the crab intestine. Nevertheless, the results of NMDS analysis showed that the overall bacterial community compositions of intestines between female and male crabs were similar in the three feeding modes.

3.6 Microbial communities with statistically significant differences in the juvenile crab

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intestines from different feeding modes

Figure 6 Cladogram showing the phylogenetic distribution of the bacterial lineages

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associated with the male and female crab intestine from the three feeding modes. (A).

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Taxonomic representation of statistically and biologically consistent differences in the male

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crab intestine from the three feeding modes. Differences are represented by the color of the most abundant class (red indicates traditional diet feeding mode; blue, formulated diet

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feeding mode; green, combined traditional and formulated diet feeding mode; yellow,

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non-significant) (B). Taxonomic representation of statistically and biologically consistent

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differences in the female crab intestine from the three feeding modes. (C-E) Indicator bacteria with linear discriminant analysis (LDA) scores of ≥ 2 in bacterial communities associated with the male and female crab intestine for the traditional, formulated, and combined traditional and formulated diet feeding modes, respectively. (a-e) Histogram of the LDA scores for differentially abundant genera. Circles indicate phylogenetic levels from domain to genus. The diameter of each circle is proportional to the abundance of the group.

Apart from determining α- and β-diversities, another primary goal of comparing microbial communities is to identify specialized communities in samples. LEfSe allows the analysis of microbial community data at any clade; however, as analysis of the large number 17

Journal Pre-proof of OTUs detected in the present study would be excessively computationally complex, statistical analysis was performed only from the domain to the genus level. Groups are shown in cladograms and LDA scores of ≥ 2 were confirmed by LEfSe (Figure 6A and 6B). We compared taxa not only on the basis of statistical significance but also taking into account biological consistency of the results and effect relevance. Significantly different bacteria were found in the male or female intestine among different feeding modes as well as between the male and female crab intestine for the three feeding modes. These results confirmed that four

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groups of bacteria were significantly enriched in the male intestine of mode A; i.e., Caulobacterales (from order to genus), Planococcaceae (from family to genus),

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Family-XII-O-Bacillales (from family to genus), and Pseudomonadales (at the order level).

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In addition, one group of bacteria was significantly enriched in the male intestine of mode B;

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i.e., Fusobacteria (from phylum to genus). No group of bacteria was significantly enriched in the male intestine of mode C. We also found that Proteobacteria (from phylum to genus) and

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Anaerorhabdus-furcosa- group (at the genus level) were more abundant in the female intestine

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of mode A compared with the other two modes, and that Tenericutes (from phylum to class)

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was more abundant in the female intestine of mode C. However, no uniquely abundant bacteria were identified in the intestine of female crabs feeding on the formulated diet. Male crabs of mode A (Figure 6C) exhibited significant enrichment in the intestine for Firmicutes and Proteobacteria (at the phylum level), unclassified- f-Rhodospirillaceae (at the genus level), Caulobacterales, Rhodobacterales, Bacillales, and Propionibacteriales (from order to genus), Methylocystaceae and Planococcaceae (from family to genus), and Cyanobacteria (from phylum to genus), whereas female crabs showed no significant enrichment for bacteria. In mode B (Figure 6D), the intestine of female crabs exhibited significant enrichment for c-unclassified-p-Firmicutes, Flavobacteriia (from class to genus), Pseudomonadales,

Xanthomonadales

(from 18

order

to

genus),

Methylocystaceae,

Journal Pre-proof Erythrobacteraceae,

Porphyromonadaceae

(from

family

to

genus),

and

g-Anaerorhabdus- furcosa-group and g-norank-f-Erythrobacteraceae (at the genus level), whereas male crabs showed no significant enrichment for bacteria. In mode C (Figure 5E), the intestine of female crabs showed significant enrichment for Chintinophagaceae (from phylum

to

genus),

Clostridia,

o-Unclassified-p-Bacteroidetes, Enterobacteriales,

Betaproteobacteria,

Sphingobacteriia

Xanthomonadales

(from

(from class to

order

to

genus),

Deltaproteobacteria, genus),

Bacillales,

Staphylococcaceae,

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Erythrobacteraceae (from family to genus), and g- unclassified- f-Flavobacteriaceae (at the genus level), whereas the male crab intestine was significantly enriched for Rickettsiales

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(from order to genus).

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4. Discussion

E. sinensis

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4.1 Effect of feeding mode on diversity and composition of intestinal bacterial communities in

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This study analyzed the bacterial communities in the intestine of male and female

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Chinese mitten crabs with different feeding status (traditional, formulated, and mixture diet); the results indicated that feeding status did not change the bacterial diversity, dominant bacterial communities, or bacterial community similarities. No significant differences were observed in the bacterial diversities among the intestines of juvenile crabs fed on traditional, formulated, and combined diets. This differs from the results obtained in other organisms; for example, Tanaka et al. found that the bacterial diversity in the intestines of abalone Haliotis discus hannai fed with artificial food was higher than that in individuals fed with sea algae Laminaria (Tanaka et al., 2004). In the intestines of puffer fish Takifugu obscurus, researchers also found that the bacterial communities differed between T. obscurus fed a natural diet and those fed with an artificial diet (Yang et al., 2007), concluding that the 19

Journal Pre-proof intestinal bacteria in aquatic animals were correlated with the feed. Diets also appeared to influence the composition of the intestinal microbiota in fish such as Arctic char Salvelinus alpinus L. (Ringø and Olsen, 1999) and Atlantic cod Gadus morhua (Ringø et al., 2006). However, our results showed that crab feed did not substantially affect intestinal microbial communities. Alternatively, Bolnick revealed that there was a negative relationship between diet diversity and microbial diversity in stickleback Gasterosteus aculeatus and perch Perca fluviatilis, with individuals with more generalized diets having less diverse microbiota than

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dietary specialists (Bolnick et al., 2014). In particular, they postulated that the negative relationship was driven by complex interactions between sex, size, and diet (Bolnick et al.,

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2014).

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In the traditional diet treatment (A), of which the main components were wheat, corn,

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soybean, and bran, the predominant bacterial communities at the phylum level were Tenericutes, Bacteroidetes, Firmicutes, and Proteobacteria, and Candidatus Baciloplasma,

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Candidatus Hepatoplasma, Prolixibacter, Vibrio, Dysgonomonas, Coprobacillus, and

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Lactovum at the genus level. Similar results were obtained for the formulated (B) and mixture

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diets (C). This indicated that the feeding mode did not change the predominant bacterial communities in the intestine of juvenile E. sinensis. Feng et al obtained a similar result by analyzing the bacterial community in yellow grouper Epinephelus awoora, showing that the different diets did not change the predominant phyla and genera in the intestine of E. awoora, with the dominant bacterial species identified in intestinal- non-adherent bacteria in both groups of fish feeding on natural and complete diets including Pseudomonas, Shwanella, and Aeromonas punctata, belonging to only two phyla, γ-Proteobacteria and Verrucomicrobia (Feng et al., 2010). In the present study, the nutritional composition of the traditional diet after digestion was similar with that of the formulated diet. When the digested food moved to the crab intestine, the nutritionally diverse intestinal environments were similar among the 20

Journal Pre-proof three treatments, which could sustain a few similar competitively dominant microbes at high abundance. The high similarities (> 91%) in intestinal- non-adherent bacteria between E. awoora feeding on natural and complete diets indicated that stable microbial communities existed in these specific ecological niches in these fish (Feng et al., 2010). In the present study, our results showed that 88.2% and 81.4% of total genera were shared in female and male crab intestines among the three feeding modes, which indicated high similarities of the bacterial

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communities in the female and male crab intestines with different feeding status and suggested that stable microbial communities existed in the intestinal digesta in E. sinensis.

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Alternatively, previous research revealed that the intestinal microbiota were not only affected

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by the gastrointestinal tract and host condition, but were also readily affected by the culture

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water environment (Cahill, 1990; Hansen and Olafsen, 1999). We speculated that these microbial communities may depend in a large part on the aquaculture environment including

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water body, sediment, and animal species. As the culture freshwater bodies of the traditional,

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formulated, and mixture diet groups in the present study were from the same river and the

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ponds were located in the same general site, the microbial communities in the intestinal digesta among our three groups of crab were highly similar. In contrast, previous studies regarding the microbial communities in the intestines of crickets Acheta domesticus (Kaufman et al. 2000), gypsy moths (Broderick et al. 2004), cockroaches (Kane and Breznak 1991), and wasps (Reeson et al. 2003) showed that these microbes are essentially extrinsic and the intestinal microflora changed when the diet changed. A similar conclusion was also reached for the larval and adult midgut of the colydiid beetle Dastarcus helophoroides (Wang et al., 2014). Considerable differences were also observed between two intestinal-adherent bacteria (IAB) samples; i.e., compared with the effects of natural diet fed to E. awoora, higher bacterial species richness and less abundance 21

Journal Pre-proof appeared to exist in IAB in fish fed with complete diets, probably indicating that the community structures in IAB were readily and significantly affected by diet (Feng et al., 2010). Nevertheless, the present study represented that the diet does not constitute the only factor that could affect the intestinal microbiota; rather, the microbes of the host diet and water environment, along with host physiology or intestinal nutrition likely contribute to reported dietary effects on microbial composition.

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4.2 Effect of gender on diversity, composition, and community structure of intestinal bacterial communities

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Previous research indicated that significant difference was observed in the intestinal

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microbial composition and structure between males and females, which demonstrated that

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human intestinal microbial composition was related to sex (Li et al., 2008). In addition, a recent study revealed that the intestinal microbial communities of wild largemouth bronze

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gudgeon (Coreius guichenoti) significantly differed between male and female fish (Li et al.,

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2016). Shannon’s and inverse Simpson’s diversity indices also significant differed between

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male and female C. guichenoti (Li et al., 2016). However, some researchers found the opposite results in two fish species. The quadratic relationship between dietary and microbial diversity was independent of sex in threespine stickleback G. aculeatus, whereas in Eurasian perch P. fluviatilis the quadratic effect was sex-dependent (Bolnick et al., 2014). In the present study, we found that there were no significant differences in intestinal microbacterial diversity, composition at the phylum and genus level, or structure between male and female E. sinensis. Specifically, the dominant community members at the phylum level included Tenericutes, Bacteroidetes, Firmicutes, and Proteobacteria in the intestine of male and female crabs, with no difference being detected. At the genus level, the most abundant community members were similar between male and female crabs, including 22

Journal Pre-proof Candidatus Bacilloplasma, Candidatus Hepatoplasma, Prolixibacter, Vibrio, Dysgonomonas, Coprobacillus, and Lactovum. This indicated that sex had no effect on the dominant intestinal microbiota of E. sinensis. However, a previous study revealed that phylum Proteobacteria was dominant in the intestine of male in contrast to female C. guichenoti, in which the five phyla Tenericutes, Proteobacteria, Firmicutes, Fusobacteria, and Spirochaetes were dominant. The genus profile revealed that genera Shewanella and Unclassified bacteria were dominant in male C.

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guichenoti, whereas genus Mycoplasma was dominant in females (Li et al., 2016). The different dominant intestinal microbiota were considered to occur because hormones

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associated with each sex might affect the composition of the microbiota, with different feed

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preference also potentially underlying the variations in the intestinal microbiota (Li et al.,

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2016). Notably, although the dominant genus (Dysgonomonas) in the present study was the same between male and female crab intestine, its relative abundance was higher in the female

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crab intestine than that in males in the formulated diet feeding mode. The genus

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Dysgonomonas is abundant in the gut of cockroaches and termites (Mikaelyan et al., 2015)

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and play important roles in assisting termites to digest lignocellulose and in their immunity and reproduction (Brune, 2014; Fraune and Bosch, 2010; Scharf, 2015; Su et al., 2016; Warnecke et al., 2007; Werren et al., 2008). Although E. sinensis belongs to the crustacean class and the arthropod phylum, as do termites, whether these bacteria are related to intestinal nutrient absorption or the level of immunity in crab, and whether they function as beneficial bacteria, require further exploration. Moreover, in the traditional diet feeding mode, Paracoccus were more highly enriched in the male crab intestine than in the female crab intestine in this study. Some species of Paracoccus are known to be beneficial to the host, which has positive effects on nutrient digestibility and absorption, immune responses, and the growth of aquatic animals. 23

Journal Pre-proof Caulobacter were also found to be much more enriched in the intestine of male crabs. Caulobacter cells are typically found in aquatic ecosystems, in which the most common limiting nutrient is phosphorus (Paerl, 1982). Phosphorus is an essential element and is preferentially imported in the form of inorganic phosphate. When starved for phosphate, bacteria increase their ability to take up inorganic phosphate and to utilize organic phosphate sources (Rao and Torriani, 2010). This suggested that phosphorus was more highly enriched in the intestine of male crabs than in female crabs, with the different intestinal environment

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and physiological condition potentially leading to the different enrichment of microbiota between male and female crabs.

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In turn, in the formulated diet feeding mode, Methylocystis showed more enrichment in

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the female crab intestine than that in the male crab intestine in this study, as determined by

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LEfSe analysis. The genus Methylocystis is one of the five genera that were included in the first taxonomic framework of methanotrophic bacteria created by Whittenbury and Dalton

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(1981). This genus belongs to the class Alphaproteobacteria, family Methylocystaceae, and it

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encompasses strictly aerobic obligate utilizers o f C1 compounds with type II intracytoplasmic

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membranes and the serine pathway of carbon assimilation (Bowman, 2006). Acinetobacter represents another genus found to be more highly enriched in the female crab intestine. The genus Acinetobacter belongs to the family Moraxellaceae; members of this genus are gram- negative, aerobic, oxidase-negative, catalase-positive, and immotile bacteria (Kim et al., 2014). We speculated that the difference of enrichment of these microbiotas between crab sexes occurred because of the different microenvironments of the crab intestine. In mixture diet feeding mode, the female crab intestine also showed significant enrichment for Staphylococcus, whereas the male crab intestine had significant enrichment for Candidatus hepatincola. This suggested that the enriched microbiota in the intestine between male and female crab are different. 24

Journal Pre-proof In summary, the present study showed that the different diets and sex did not influence the overall bacterial diversity, dominant bacterial communities, or bacterial community similarities of E. sinensis intestine. However, feeding status could change the microbiota community structure of the crab intestine. Significantly different bacteria were found in the male or female intestine among different feeding modes in addition to between the male and female crab intestine for the three feeding modes. Understanding the associations between the intestinal bacterial community and diet is important for the healthy aquaculture of Chinese

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mitten crab. Different additives may improve aquatic animal growth and provide reinforcement of the mucus and epithelium of the intestine. The specific relationship between

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diet and the crab intestine and the potential of these diets to affect the immune response of

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crab (e.g., antimicrobial peptide production, change in gene expression) should be further

5. Conclusion

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studied.

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This study investigated the bacterial communities of crab intestine from three different

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feeding modes via high-throughput sequencing technology, including traditional, formulated, and mixture (combined traditional and formulated) diet feeding modes. The results showed that the biodiversity and richness of intestinal microbiota were not affected by the feeding mode or animal sex. The most dominant community members at the phylum and genera levels in the intestine of male and female crabs were not changed by the feeding mode or sex. The microbial communities in the intestines of male and female crabs showed high average similarity among the three feeding modes. Nevertheless, differences were observed in the relative enrichment of the dominant bacteria in male and female crab intestine among different feeding modes. A better understanding of how the feeding modes affect the intestinal microbiota will help improve the microbial management in crab rearing systems. 25

Journal Pre-proof Future research should focus on the relationship between the different diets and intestinal bacteria of the Chinese mitten crab.

Acknowledgments This study was funded by the China Agriculture Research System (No. CARS-48), the National Natural Science Foundation of China (No. 31802320), the China Postdoctoral

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of Integrated Rice and Fishery Breeding (SD2019YY002).

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Science Foundation (2018M641985) and Research and Demonstration on key Technologies

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Highlights

1. Feeding modes and sex didn’t change the intestinal microbial communities of juvenile E. sinensis. 2. Biodiversity and richness weren’t affected by the different feeding modes and gender. 3. The most dominant community members at phylum level were Tenericutes, Bacteroidetes, Firmicutes, and Proteobacteria. 4. This study provided a theoretical guidance for the healthy aquaculture of juvenile crab. 30