Intestinal Microbiota of Grass Carp Fed Faba Beans: A Comparative Study

Many reports of the intestinal microbiota of grass carp have addressed the microbial response to diet or starvation or the effect of microbes on metabolism; however, the intestinal microbiota of crisp grass carp has yet to be elucidated. Moreover, the specific bacteria that play a role in the crispiness of grass carp fed faba beans have not been elucidated. In the present study, 16S sequencing was carried out to compare the intestinal microbiota in the fore-, mid- and hind-intestine segments of grass carp following feeding with either faba beans or formula feed. Our results showed that (1) the hind-intestine presented significant differences in diversity relative to the fore- or midintestine and (2) faba beans significantly increased the diversity of intestinal microbiota, changed the intestinal microbiota structure (Fusobacteria was reduced from 64.26% to 18.24%, while Proteobacteria was significantly increased from 17.75% to 51.99%), and decreased the metabolism of energy, cofactors and vitamins in grass carp. Furthermore, at the genus and species levels, Acinetobacter accounted for 15.09% of the microbiota, and Acinetobacter johnsonii and Acinetobacter radioresistens constituted 3.41% and 2.99%, respectively, which indicated that Acinetobacter of the family Moraxellaceae contributed to changes in the intestinal microbiota structure and could be used as a potential biomarker. These results may provide clues at the intestinal microbiota level to understanding the mechanism underlying the crispiness of grass carp fed faba beans.


Introduction
An accidental discovery was made in the early 1970s in Dongsheng Town, Guangdong Province, China, that feeding crisp grass carp (Ctenopharyngodon idellus C.et V) that had reached a certain level of maturity faba beans led to carp that exhibited more hardness and crispiness in its muscles than ordinary grass carp, and this diet has since become popular. The crisp mechanism of grass carp may be due to a certain factor in faba beans that activates the TGF-β/Smads signaling pathway to regulate type I collagen in muscles, which is the main type of collagen and is closely related to changes in muscle properties [1][2][3]. Furthermore, a proteomic profile demonstrated that muscle fiber hyperplasia was closely related to a protein-protein network of 12 muscle component proteins, and the abundance of amounts, and paired-end sequences (2 × 250) were obtained with an Illumina HiSeq 2500 system (Illumina, California, USA) at Gene Denovo Biological Technology Co. Ltd. (Guangzhou, China).

Quality Control and Read Assembly
Raw reads were filtered to obtain high-quality clean reads, which were merged as raw tags using FLASH [19] (v 1.2.11), with a minimum overlap of 10 bp and mismatch error rates of 2%. Then, raw tags were filtered by the QIIME (V1.9.1) pipeline under specific filtering conditions to obtain high-quality clean tags [20,21]. Clean tags were searched against the reference database to perform reference-based chimera checking. All chimeric tags were removed, and effective tags were finally obtained for further analysis. Raw read data have been submitted to the NCBI Sequence Read Archive (SRA) under the accession number PRJNA563528.

OTU Cluster and Taxonomy Classification
The effective tags were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity using the UPARSE pipeline [22]. The tag sequence with the highest abundance was selected as the representative sequence within each cluster, and a Venn analysis was performed in R to identify unique and common OTUs.
The representative sequences were classified into organisms by a naive Bayesian model using an the Ribosomal Database Project classifier (Version 2.2) based on the SILVA (v128) database [23] (https://www.arb-silva.de/).

Diversity Analysis and Functional Prediction
The Chao1, Simpson and other alpha diversity indexes were calculated in QIIME (V1.9.1) as mentioned above. An OTU rarefaction curve and rank abundance curves were plotted in QIIME. Statistics of the alpha index comparison between groups were calculated by Welch's t-test and the Wilcoxon rank test in R. The alpha index comparison among groups was computed by Tukey's HSD test and the Kruskal-Wallis H test in R (version 3.5.1).
Nonmetric multidimensional scaling (NMDS) of Bray-Curtis dissimilarity index was calculated and plotted in R. An analysis of similarities (ANOSIM) based on the Bray-Curtis dissimilarity index was performed to determine significant differences in the microbial community. R values in ANOSIM were used to detect the overlap in the community as reported by Buttigieg and Ramette (R > 0.75, well separated; 0.50 < R ≤ 0.75, separated but overlapped; 0.25 < R ≤ 0.50, separated but strongly overlapped; and 0.25 ≤ R, barely separated) [24]. The p value was used to indicate significant differences between the two groups (* p < 0.05 and ** p < 0.01).
Differential species analyses between the C and T groups were compared and identified using Welch's t-test in R. Furthermore, to analyze the functions of the intestinal microbiota communities in the C and T groups, the functional prediction of the OTUs was inferred using Tax4Fun (v1.0) [25]. A related analysis was carried out using the online platform of Gene Denovo Biological Technology Co. Ltd. (Guangzhou, China) (http://www.omicsmart.com/).

Statistics of Illumina Sequencing Data and OTUs
A total of approximately 4.4 M million raw reads of bacterial 16S rDNA were obtained by Illumina sequencing. After data quality filter processing, approximately 4.2 M effective reads/tags were obtained. In detail, the total numbers of OTUs were 1290, 1146, 964 for the TF, TM, and TH treatments, respectively, and 572, 618, 482 for the CF, CM, and CH treatments, respectively. A summary of the OTUs and tags of all samples is listed in Table S1.
The differences in OTUs between different samples or groups were illustrated using Venn diagrams, which showed that the total number of OTUs was 1519 for T and 609 for C ( Figure S1C and Table S1). In addition, 246 OTUs overlapped for the FI, MI, and HI OTUs in the treatment and control group ( Figure  S1D). Compared with the C group, the T group had more abundant intestinal microbes, suggesting that more intestinal microbes were present to digest, absorb and manage faba beans. Moreover, the number of OTUs from the hind-intestine was significantly less than that from the fore-and mid-intestine for the T and C groups ( Figure S1A,B and Table S1), thereby indicating that the abundance of the microbial population in the hind-intestine was significantly decreased compared with that of the foreand mid-intestine areas.

Microbiota Structure
To visualize the variation in species abundance of different samples at different taxonomic levels, taxonomy stack distributions were carried out to statistically analyze the species composition of each sample at each level of classification. In this classification, the top 10 species with an abundance of 2% in the sample are shown, and the rest were unified to the other category. The tags that could not be annotated to a specific taxonomy were classified to the unclassified category ( Figure 1 and Table S2). A summary of the profiling for each sample at all taxonomic levels is listed in Table S3. the fore-and mid-intestine for the T and C groups ( Figure S1 A,B and Table S1), thereby indicating that the abundance of the microbial population in the hind-intestine was significantly decreased compared with that of the fore-and mid-intestine areas.

Microbiota Structure
To visualize the variation in species abundance of different samples at different taxonomic levels, taxonomy stack distributions were carried out to statistically analyze the species composition of each sample at each level of classification. In this classification, the top 10 species with an abundance of 2% in the sample are shown, and the rest were unified to the other category. The tags that could not be annotated to a specific taxonomy were classified to the unclassified category ( Figure  1 and Table S2). A summary of the profiling for each sample at all taxonomic levels is listed in Table  S3.
At the family level, the ratios of the other and unclassified categories rapidly increased and even covered two-thirds in the control group. At the species level, the CF, CM and CH of the control group were almost covered by the unclassified category, while the TF, TM and TH of the treatment group showed distributions of approximately 3% for Acinetobacter johnsonii, Acinetobacter radioresistens and the other category.

Diversity Differences and Potential Functions
To analyze the microbial community diversity within the sample, alpha diversity (a single sample diversity analysis) was used to reflect the diversity of microbial communities. The four indexes of alpha diversity analysis, i.e., the Chao1, observed species, Shannon, and Simpson indexes, were calculated for different samples using the Wilcoxon rank sum test. Significant differences are listed in Figure 2. A comparison of the T to C group showed significant differences (p < 0.05) for the above four indexes ( Figure 2A). However, the alpha diversity differences within the group were not significant except for the observed species index for TF/TH, CF/CH and CM/CH (p < 0.05) and Shannon index for TF/TH (p < 0.05) ( Figure 2B). These results indicated that there were significant diversity differences between groups but few differences within groups and that the differences were primarily associated with the differences between the hind-intestine and the fore-or mid-intestine. distributions of approximately 3% for Acinetobacter johnsonii, Acinetobacter radioresistens and the other category.

Diversity Differences and Potential Functions
To analyze the microbial community diversity within the sample, alpha diversity (a single sample diversity analysis) was used to reflect the diversity of microbial communities. The four indexes of alpha diversity analysis, i.e., the Chao1, observed species, Shannon, and Simpson indexes, were calculated for different samples using the Wilcoxon rank sum test. Significant differences are listed in Figure 2. A comparison of the T to C group showed significant differences (p < 0.05) for the above four indexes ( Figure 2A). However, the alpha diversity differences within the group were not significant except for the observed species index for TF/TH, CF/CH and CM/CH (p < 0.05) and Shannon index for TF/TH (p < 0.05) ( Figure 2B). These results indicated that there were significant diversity differences between groups but few differences within groups and that the differences were primarily associated with the differences between the hind-intestine and the fore-or mid-intestine. To compare the diversity between different ecosystems and indicate the response of biological species to the environment, NMDS was carried out to analyze the beta diversity based on the Bray-Curtis dissimilarity index of samples. As shown in Figure 3, the NMDS showed that the T and C groups could be closely clustered together, indicating that the similarity among the groups was higher but the difference between the groups was obvious. Furthermore, to detect significant differences in the community of different groups, a pairwise ANOSIM was performed (Table 1), and the results indicated that the groups were well separated for T/C (ANOSIM−R = 0.87, p = 0.001), TF/CF (ANOSIM−R = 1.00, p = 0.002) and TM/CM (ANOSIM−R = 0.98, p = 0.005), separated but slightly overlapped for TH/CH (ANOSIM-R = 0.62, p = 0.003), and separated but strongly overlapped for CF/CH (ANOSIM−R = 0.30, p = 0.018), CM/CH (ANOSIM−R = 0.28, p = 0.017) and TF/TH (ANOSIM−R = 0.34, p = 0.049). These results indicated that there were significant differences in the community between groups and that the microbial community of the hind-intestine was significantly separated but strongly overlapped compared to the microbial community of the fore-or mid-intestine. To compare the diversity between different ecosystems and indicate the response of biological species to the environment, NMDS was carried out to analyze the beta diversity based on the Bray-Curtis dissimilarity index of samples. As shown in Figure 3, the NMDS showed that the T and C groups could be closely clustered together, indicating that the similarity among the groups was higher but the difference between the groups was obvious. Furthermore, to detect significant differences in the community of different groups, a pairwise ANOSIM was performed (Table 1), and the results indicated that the groups were well separated for T/C (ANOSIM−R = 0.87, p = 0.001), TF/CF (ANOSIM−R = 1.00, p = 0.002) and TM/CM (ANOSIM−R = 0.98, p = 0.005), separated but slightly overlapped for TH/CH (ANOSIM-R = 0.62, p = 0.003), and separated but strongly overlapped for CF/CH (ANOSIM−R = 0.30, p = 0.018), CM/CH (ANOSIM−R = 0.28, p = 0.017) and TF/TH (ANOSIM−R = 0.34, p = 0.049). These results indicated that there were significant differences in the community between groups and that the microbial community of the hind-intestine was significantly separated but strongly overlapped compared to the microbial community of the fore-or mid-intestine. Microorganisms 2019, 7, x FOR PEER REVIEW 6 of 13  Bray-Curtis 0.87 0.001 ** #: * stands for p < 0.05, and ** stands for p < 0.01 Furthermore, the functional profiling of the intestinal microbial communities from all samples was predicted from the 16S rRNA gene amplicon data using Tax4Fun (v1.0). As shown in Figure 4, significantly different results indicated that among the five KEGG pathways (Level 1) of metabolism, environmental information processing, genetic information processing, cellular processes, and human diseases, the main changes in the intestinal microbiota gene functions were focused on metabolism, which contained four of the top six differences in the abundance of KEGG pathways (i.e., carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins) (Level 2). Because higher OTU numbers were observed in the T group, the results showed that "signal transduction", "energy metabolism" and "metabolism of cofactors and vitamins" were significantly decreased in the T group compared to the C group, while "carbohydrate metabolism", "amino acid metabolism" and "membrane transport" were increased, which suggested that metabolic changes occurred in the intestinal microbiota of grass carp caused by the feeding of broad beans.  Furthermore, the functional profiling of the intestinal microbial communities from all samples was predicted from the 16S rRNA gene amplicon data using Tax4Fun (v1.0). As shown in Figure 4, significantly different results indicated that among the five KEGG pathways (Level 1) of metabolism, environmental information processing, genetic information processing, cellular processes, and human diseases, the main changes in the intestinal microbiota gene functions were focused on metabolism, which contained four of the top six differences in the abundance of KEGG pathways (i.e., carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins) (Level 2). Because higher OTU numbers were observed in the T group, the results showed that "signal transduction", "energy metabolism" and "metabolism of cofactors and vitamins" were significantly decreased in the T group compared to the C group, while "carbohydrate metabolism", "amino acid metabolism" and "membrane transport" were increased, which suggested that metabolic changes occurred in the intestinal microbiota of grass carp caused by the feeding of broad beans. Microorganisms 2019, 7, x FOR PEER REVIEW 7 of 13 The following results were obtained based on the various indicators of the alpha and beta diversity analysis and combined functional prediction: (1) the hind-intestinal microbiota was less diverse than the fore-and mid-intestinal microbiota, and a significant diversity difference was observed between the hind-intestine and the fore-or mid-intestine; and (2) the intestinal microbiota in the corresponding parts between the T and C groups were significantly different, which indicates that the faba bean diet likely changed the intestinal microbiota and their corresponding metabolism.

Differences at Different Taxonomic Levels
After categorizing the significant changes in microbiota, the five most abundant terms were obtained at different taxonomic levels. As listed in Figure 5, compared to the C group, the T group showed that at the phylum level, Fusobacteria was reduced significantly from 64.26% to 18.24% and Proteobacteria increased significantly from 17.75% to 51.99%. At the class and order levels, similar results were obtained for Fusobacteria and Proteobacteria (including γ-proteobacteria and αproteobacteria) at the class level and for Fusobacteriales and Pseudomonadales at the order level. Because Fusobacteriales can only be classified as "unclassified" below the order level, levels below family could not be annotated and had no relevant information in this study. At the family level, Moraxellaceae and Caulobacteraceae showed a sharp rise; at the genus level, Acinetobacter and Brevundimonas increased significantly; and at the species level, Acinetobacter johnsonii, Acinetobacter radioresistens and Rhizobium radiobacter significantly increased. Detailed data are listed in Table S4. The following results were obtained based on the various indicators of the alpha and beta diversity analysis and combined functional prediction: (1) the hind-intestinal microbiota was less diverse than the fore-and mid-intestinal microbiota, and a significant diversity difference was observed between the hind-intestine and the fore-or mid-intestine; and (2) the intestinal microbiota in the corresponding parts between the T and C groups were significantly different, which indicates that the faba bean diet likely changed the intestinal microbiota and their corresponding metabolism.

Differences at Different Taxonomic Levels
After categorizing the significant changes in microbiota, the five most abundant terms were obtained at different taxonomic levels. As listed in Figure 5, compared to the C group, the T group showed that at the phylum level, Fusobacteria was reduced significantly from 64.26% to 18.24% and Proteobacteria increased significantly from 17.75% to 51.99%. At the class and order levels, similar results were obtained for Fusobacteria and Proteobacteria (including γ-proteobacteria and α-proteobacteria) at the class level and for Fusobacteriales and Pseudomonadales at the order level. Because Fusobacteriales can only be classified as "unclassified" below the order level, levels below family could not be annotated and had no relevant information in this study. At the family level, Moraxellaceae and Caulobacteraceae showed a sharp rise; at the genus level, Acinetobacter and Brevundimonas increased significantly; and at the species level, Acinetobacter johnsonii, Acinetobacter radioresistens and Rhizobium radiobacter significantly increased. Detailed data are listed in Table S4. Considering that α-proteobacteria accounted for a relatively small proportion (10.67%), γproteobacteria (36.39%) was the main dominant microbiota of the T group. Additionally, according to the taxonomic and expression profiling data of the species, the schematic diagram ( Figure 6) of the Proteobacteria species taxonomic tree was simplified for the main significant genus/species differences in the intestinal microbiota between the T and C groups. The corresponding ratios were as follows: Proteobacteria (17.75%), γ-Proteobacteria (16.75%), Pseudomonas (0.84%), Moraxella (0.76%), Acinetobacter (0.72%), Acinetobacter johnsonii (0.18%) and Acinetobacter radioresistens(0.15%) for the C group; and Proteobacteria (51.99%), γ-Proteobacteria (36.39%), Pseudomonas (17.53%), Moraxellaceae (16.05%), Acinetobacter (15.09%), Acinetobacter johnsonii (3.41%) and Acinetobacter radioresistens (2.99%) for the T group. At the same time, in γ-Proteobacteria, Aeromonas had no significant difference between the T and C groups (14.92% and 11.51%), although Aeromonas was also the main dominant bacterial genus. Additionally, in Proteobacteria, the secondary corresponding ratios of change in the Alphaproteobacteria and below level were as follows: Alphaproteobacteria (0.52%), Caulobacteraceae (0.18%), Brevundimonas (0.18%) for the C group; and Alphaproteobacteria (10.67%), Caulobacteraceae (4.55%), Brevundimonas (4.36%) for the T group. Considering that α-proteobacteria accounted for a relatively small proportion (10.67%), γ-proteobacteria (36.39%) was the main dominant microbiota of the T group. Additionally, according to the taxonomic and expression profiling data of the species, the schematic diagram ( Figure 6) of the Proteobacteria species taxonomic tree was simplified for the main significant genus/species differences in the intestinal microbiota between the T and C groups. The corresponding ratios were as follows: Proteobacteria (17.75%), γ-Proteobacteria (16.75%), Pseudomonas (0.84%), Moraxella (0.76%), Acinetobacter (0.72%), Acinetobacter johnsonii (0.18%) and Acinetobacter radioresistens(0.15%) for the C group; and Proteobacteria (51.99%), γ-Proteobacteria (36.39%), Pseudomonas (17.53%), Moraxellaceae (16.05%), Acinetobacter (15.09%), Acinetobacter johnsonii (3.41%) and Acinetobacter radioresistens (2.99%) for the T group. At the same time, in γ-Proteobacteria, Aeromonas had no significant difference between the T and C groups (14.92% and 11.51%), although Aeromonas was also the main dominant bacterial genus. Additionally, in Proteobacteria, the secondary corresponding ratios of change in the Alphaproteobacteria and below level were as follows: Alphaproteobacteria (0.52%), Caulobacteraceae (0.18%), Brevundimonas (0.18%) for the C group; and Alphaproteobacteria (10.67%), Caulobacteraceae (4.55%), Brevundimonas (4.36%) for the T group.
The results indicated that a faba bean diet changed the intestinal microbiota structure, and the main difference was that Fusobacteria was reduced. Meanwhile, Proteobacteria increased significantly, which led to significant decreases in "energy metabolism" and "metabolism of cofactors and vitamins" in the T group. Furthermore, at the genus level, Acinetobacter of Moraxellaceae mainly played a major role in the changes in the intestinal microbiota structure with the feeding of faba beans to grass carp.

Discussion
The crispiness of grass carp is an extremely complex physiological and biochemical process that eventually leads to excessive crispiness, which directly leads to death due to blood circulation disorders. The degree of crispiness and physical changes are consistent with the toxicological dose (faba bean)-effect response [26]. Studies have shown that faba beans significantly inhibit growth, decrease the ratio of liver to body and gradually lead to excessive accumulation of fat in the hepatopancreas and abdominal cavity of grass carp [27,28]. Additionally, faba beans may increase the abundance of gram-negative and flagellated bacteria and then result in intestinal inflammation in fish, including grass carp, and these changes share similar features with inflammatory bowel disease (IBD) [18].
The composition of fish intestinal microbiota is susceptible to diet, which reflects and plays an important role in the health of the fish intestine [29]. The dominant bacterial species largely determine the function of the intestinal microbiota community of a fish. Thus, understanding the species composition at different taxonomic levels can effectively interpret the formation, changes and ecological impacts of the community structure. Meanwhile, by interacting with the intestinal microbiota, the metabolism in the intestine or host can change and more effectively adapt to changes stemming from new nutrients/environmental conditions. A previous study showed that intestinal microbiota may shape intestinal immune responses in healthy and diseased states [30].
Many studies have described that Proteobacteria blooms in the intestine reflect an unstable microbial community structure or a state of host disease. Moreover, the natural microbial community of the intestines normally contains only a minor proportion of this phylum and is dominated by Firmicutes or Bacteroidetes [31,32]. In contrast, Fusobacteria, a distinct and understudied phylum of bacteria, is divided into two families: Leptotrichiaceae and Fusobacteriaceae. These less-studied bacteria are gram-negative, non-spore-forming, and usually nonmotile anaerobes with unique metabolic capabilities [33]. In this study, a comparison of the C and T groups showed that Fusobacteria was reduced from 64.26% to 18.24% while Proteobacteria increased from 17.75% to 51.99%, thus suggesting that faba beans resulted in an imbalance and changes to the intestinal microbiota in grass carp.
At the family and lower levels, since members of Fusobacteria are unclassified, the dominant species and/or changes were concentrated in the genus Acinetobacter, which belongs to the phylum Proteobacteria, class Gammaproteobacteria, order Pseudomonadales, and family Moraxellaceae. Acinetobacter contains gram-negative, nonfermentative, nonmotile, oxidase-negative, catalase-positive, and strictly aerobic bacteria with a G + C content of 39-47%, and its species are widely distributed in nature, can survive on various surfaces (both moist and dry) and are spread by various means [34][35][36][37].
As significant opportunistic pathogens, they are generally considered to have low virulence and cause a decrease in body resistance and immune function after infection even though they have an astonishing ability to acquire antibiotic resistance genes [38,39]. In addition, these bacteria contribute to the mineralization of aromatic compounds [40,41]. In this study, Acinetobacter accounted for 15.09% and Acinetobacter johnsonii and Acinetobacter radioresistens accounted for 3.41% and 2.99%, respectively. This result showed that Acinetobacter played an important role in the intestinal microbiota by combining with gram-negative and flagellated bacteria introduced by faba beans, which resulted in intestinal inflammation [18,42]. Thus, Acinetobacter might lead to intestinal inflammation and furthermore become a potential cause of crispiness in grass carp. Therefore, Acinetobacter can also be used as a primary indicator/biomarker for detecting crispiness in grass carp. However, as a fish pathogen, Acinetobacter has been rarely reported. Why does Acinetobacter but not other Pseudomonas genera, such as Aeromonas, dominate the intestinal microbiota of grass carp after faba bean feeding? Is it related to gram-negative bacteria that do not ferment sugars and cannot fully utilize and absorb intestinal decomposition products? To answer these questions, further experiments, such as multiomic combinations, are required.
In addition, because the intestine is the main site for nutrient absorption, when the bacteria in the intestine are significantly increased, they often cause gastrointestinal symptoms, such as bloating, abdominal pain and diarrhea, affect the absorption of carbohydrates and fats, especially the absorption of fat-soluble vitamins, and worsen intestinal inflammation [43]. Moreover, a high amount of bacteria leads to the competitive absorption of nutrients from the host [44,45]. This study indicates that faba beans significantly increased the intestinal bacteria and decreased the energy metabolism (energy metabolism) and cofactor and vitamin metabolism (Metabolism of Cofactors and Vitamins) in grass carp, which also aggravated the severity of the symptoms. The distribution of microbiota varies in different intestinal segments, and the results indicated that hind-intestinal microbiota were significantly different from the fore-and mid-intestinal microbiota, which is consistent with previous studies [14,15].
In conclusion, 16S sequencing and comparative analyses using faba beans and formula feed were carried out on the intestinal microbiota in different intestine segments of grass carp. Our analysis showed that there were significant differences in diversity between the hind-intestine and the foreor mid-intestine, and that faba beans shifted the intestinal microbial composition significantly, with the proportion of Fusobacteria decreased, the proportion of Acinetobacter increased, and the overall diversity increased.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/7/10/465/s1, Figure S1: Venn diagram for intestine microbial OTUs of the treatment and control group (T, C). A) Venn diagram for OTUs in the fore-, mid-and hind-intestine in the treatment group (TF, TM, TH). B) Venn diagram for OTUs in the fore-, mid-and hind-intestine in the control group (CF, CM, CH). C) Venn diagram for the total intestine microbial OTUs of the treatment and control group. D) Venn diagram showing the overlap in the fore-, midand hind-intestine OTUs in the treatment and control groups (TF, TM, TH, CF, CM, CH). Table S1: Statistics of OTUs and tags of all samples. Table S3: Summary of profiling for each sample at all taxonomic levels. Table S4: Difference analysis for different taxonomic levels.