Comparative analysis and gut bacterial community assemblages of grass carp and crucian carp in new lineages from the Dongting Lake area

Abstract Gut microbiota are known to play an important role in health and nutrition of the host and have been attracting an increasing attention. Farming of new lineages of grass carp and crucian carp has been developed rapidly as these species were found to outperform indigenous ones in terms of growth rate and susceptibility to diseases. Despite this rapid development, no studies have addressed the characteristics of their gut microbiota as a potential factor responsible for the improved characteristics. To reveal whether microbiomes of the new lineages are different from indigenous ones, and therefore could be responsible for improved growth features, intestinal microbiota from the new lineages were subjected to high‐throughput sequencing. While the phyla Firmicutes, Fusobacteria and Proteobacteria were representing the core bacterial communities that comprised more than 75% in all fish intestinal samples, significant differences were found in the microbial community composition of the new linages versus indigenous fish populations, suggesting the possibility that results in the advantages of enhanced disease resistance and rapid growth for the new fish lineages. Bacterial composition was similar between herbivorous and omnivorous fish. The relative abundance of Bacteroidetes and Actinobacteria was significantly higher in omnivores compared to that of herbivores, whereas Cetobacterium_sp. was abundant in herbivores. We also found that the gut microbiota of freshwater fish in the Dongting lake area was distinct from those of other areas. Network graphs showed the reduced overall connectivity of gut bacteria in indigenous fish, whereas the bacteria of the new fish lineage groups showed hubs with more node degree. A phylogenetic investigation of communities by reconstruction of unobserved states inferred function profile showed several metabolic processes were more active in the new lineages compared to indigenous fish. Our findings suggest that differences in gut bacterial community composition may be an important factor contributing to the rapid growth and high disease resistance of the new fish lineages.

Fish originated over 600 million years ago (Egerton et al., 2018), and over three billion people worldwide depend on fish for at least 20% of their protein intake and approximately 20 kg of fish is con- cian carp (C. auratus) from the Dongting lake area. The research work related to the gut microbiota of fish is dwarfed by that on humans and mammals. The colonization of fish intestinal microorganisms is an extremely complex process because of the influence of fish species, water environments, seasonal changes, and other factors (Ni, Yu, Zhang, & Gao, 2012;Qin et al., 2016;Ye, Amberg, Chapman, Gaikowski, & Liu, 2014). Previous studies focused mainly on pattern of bacterial composition of wild-type fish species, for example, zebrafish Siriyappagouder et al., 2018), common carp (Chang et al., 2019), bighead carp (Li, Zhu, et al., 2018), Atlantic salmon (Klemetsen, Willassen, & Karlsen, 2019), Atlantic cod (Walter, Bagi, & Pampanin, 2019), rainbow trout (Etyemez & Balcázar, 2015), blunt snout bream, and topmouth culter .
Farming of grass carp and crucian carp is an important economic pillar in the Dongting Lake area of Hunan Province (China). The grass carp, an herbivorous freshwater fish, made it be the largest freshwater aquaculture product in China, which the production reached 5.7 million tons in 2015 (Lin et al., 2018). The Pure-line grass carp is a new grass carp lineage that has the advantages of strong disease resistance and rapid growth, which was developed through gynogenesis using ultraviolet-treated heterologous sperm to activate the eggs of grass carp and subsequently cold shock or heat shock treatment on eggs to double the chromosomes (Mao et al., 2019;Zhang et al., 2011). The crucian carp as an omnivorous freshwater fish is one of the most important and popular freshwater aquaculture species in China, in which its total yield is over 3 million tons per year . Crucian carp is rich in nutrients such as the vitamin, protein, unsaturated fatty acids, and inorganic components (Liu, Sha, Wang, Li, & Bureau, 2018 and higher anti-disease ability (~1.5 times) (Chen et al., 2009;Liu et al., 2001;Xiao et al., 2019). Japanese white crucian carp (Carassius auratus curvier) are characterized by strong reproductive ability and rapid growth rates . Both Hefang crucian carp and Xiangyun crucian carp inherit superiority of white crucian carp. All of these freshwater fish are economically very important because of their high nutritional value and are widely distributed in the lakes and reservoirs of China. The present study investigated the intestinal microbial structures of the five groups of fish from Dongting lake area to assess the relationship among genetic differentiation, feeding habits differences, and gut microbiota structure.  Table 1). Experimental fish were euthanized by gut bacterial community composition may be an important factor contributing to the rapid growth and high disease resistance of the new fish lineages.

K E Y W O R D S
connectivity of bacteria, crucian carp, Dongting Lake area, grass carp, gut microbiota, highthroughput sequencing overdose of tricaine methane sulfonate (MS222; 50 mg/L) and were aseptically dissected to obtain midgut samples and their contents (Cordova & Braun, 2007;Song et al., 2016). Samples were stored at −80°C until further processing.

| DNA extraction and high-throughput 16S rRNA amplicon sequencing
Microbial DNA was isolated from the fish gut contents samples using (1 ng/μl), and 2 μl H 2 O . PCR amplification was conducted using the following thermocycles: initial denaturation at 98°C for 1 min; 30 cycles at 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Equal amounts of each sample were combined and gel-purified using a QIAquick Gel Extraction Kit (QIAGEN) before being quantified using PicoGreen (Mardis & McCombie, 2017). All PCR products were then sequenced using on an Illumina HiSeq 2500 platform, and 250 bp paired-end reads were generated (Caporaso et al., 2012) at Novogene Bioinformatic Biotechnology Co., Ltd.
Analysis of similarity statistics (ANOSIM) was estimated using the Bray-Curtis distance matrix to test the significance of differences between the five groups of fish (Douterelo, Sharpe, & Boxall, 2013).
One-way ANOVA and a two-tailed Student's t test were applied to determine differences in intestinal bacterial communities between the five fish groups ; p-value <.05 and p-value <.01 were considered statistically significant and highly significant, respectively.

| Microbial network construction and prediction for the potential function of the OUTs sequences
A network consisting of non-random co-occurrences was constructed according to a Spearman correlation coefficient >0.5 in the R platform without considering the compositional variation . Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt, version 1.0) was used to predict the functional content based on the 16S rRNA sequencing data (Wong et al., 2016).

| Sequencing quality
We obtained the 16S rRNA V4 amplicon sequences of the bacterial communities of 25 intestine samples from five fish species. After the adapter and quality trimming of 1,915,354 raw sequences, a total of 1,789,385 effective sequences were clustered into 4,896 OTUs (Table S1 at Figure A2a). The core species curves were horizontal when the number of samples was greater or equal to 4, thereby allowing us to capture most of the bacterial communities ( Figure A2b). Hence, all sequences may contribute to a complete and even representation of commensal microbiome and are considered to be appropriate for further analysis.

| Alpha-and beta-diversity analysis
The alpha diversity of the microbial community of the five fish groups was expressed using the Shannon and Chao indexes (Figure 1a,b).
The microbial diversity of XYCC group was highest according to

| Microbiota composition analysis
Given that the microbiota community structure of the five fish groups Erysipelotrichaceae genus, classified into Firmicutes, was significantly higher in ICC group (51.30%) as compared to the XYCC group (9.23%, p < .001, one-way ANOVA) and IGC group (16.82%, p < .01, one-way ANOVA), which indicated that norank_f_Erysipelotrichaceae genus played a critical role in Firmicutes due to the higher relative abundance of Firmicutes in the ICC group than that in XYCC and IGC ( Figure A4b).
To explore the variation of the microbial community composition in different fish groups, we performed LEfSe analysis to detect differences in the relative abundance of bacterial taxa (Figure 2d). It was noteworthy that the number of taxa identified in XYCC group (16 taxa Bacterial community richness of HFCC group was significantly higher than that of the XYCC groups. The phylum Actinobacteria in the HFCC group was predominant taxon, whereas the phylum Bacteroidetes in the XYCC group was superior taxon. We further compared the characteristics of intestinal microbiota between herbivorous fish (IGC and PGC) and omnivorous fish (ICC, HFCC, and XYCC). The shared microbiome between the two feeding types comprised of 2,304 OTUs, while there were 745 and 1,847 unique OTUs in herbivore and omnivore, respectively ( Figure 3a).
Bacterial composition at phylum and species level in the two types of feeding habits of fish was similar (Figure 3b,c). However, the relative abundance of Bacteroidetes and Actinobacteria was significantly higher in omnivores compared with herbivores (p < .05, Student's t test) (Figure 3d). Furthermore, Cetobacterium_sp._ZOR0034 was more abundant in herbivores (p < .01, Student's t test) (Figure 3e).

| Association network between bacterial community
The co-occurrence network analyses of top-50 abundant intestinal bacteria in each fish group revealed that their interactive patterns

| Functional prediction
Phylogenetic investigation of communities by reconstruction of unobserved states-predicted COG functional classification showed similar microbial functional features in every fish group, including amino acid transport and metabolism, energy production and conversion, carbohydrate transport and metabolism, nucleotide transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, and signal transduction mechanisms ( Figure 6) 638.v2). Predicted COG profiles also suggested great differences between the microbial processes potentially ongoing in herbivore and omnivore intestinal communities ( Figure A5). The pathways involved in the transport and metabolism of carbohydrates, amino acids, nucleotides, lipids/ fatty acids, secondary metabolites, inorganic ion, F I G U R E 2 Taxonomic composition of the gut microbiota in the five fish groups. (a) Venn diagram showed the shared and unique OTUs in all fish samples. (b) Distribution of microbial community was visualized by Circos for each fish sample at phylum level. (c) Distribution of microbial community was visualized by Circos for each fish sample at genus level. (d) LEfSe was applied to finding the differential abundance using a cutoff of 4 and a significance threshold of p < .05. Y-axis labels are color-coded for different bacterial taxa: Firmicutes-dark green, Proteobacteria-purple, Bacteroidetes-red, Actinobacteria-blue, Fusobacteria-green, Planctomycetes-pink, Spirochaetes-coral, and Chloroflexi-cyan. (e) Statistical comparison of the relative abundance the five fish groups by one-way ANOVA at phylum level. (f) Statistical comparison of the relative abundance the five fish groups by one-way ANOVA at genus level. *p < .05, **p < .01, ***p < .001

F I G U R E 3
Comparative analysis of gut microbiota between herbivore (grass carp) and omnivore (crucian carp). (a) Venn diagram showed shared and unique OTUs between herbivore and omnivore. (b) Distribution of microbial community was visualized by Circos for herbivore and omnivore at phylum level. (c) Relative abundance of top-15 gut microbial was visualized at species level. (d) Student's t test bar plot was employed to reveal statistical differences on phylum level of bacterial communities between herbivore and omnivore. (e) Student's t test bar plot was employed to reveal statistical differences on species level of bacterial communities between herbivore and omnivore. *p < .05, **p < .01 and energy production and conversion were more prominent in the omnivorous fish gut microbiome compared with herbivorous fish.
However, this microbial metabolism, such as coenzyme transport, cell wall/membrane/envelope biogenesis, and signal transduction mechanisms, tended to be more active in herbivorous fish. The results indicated gut microbial community not only played important roles in host metabolism, but also differentiated on the basis of diet and species.

| D ISCUSS I ON
Gut microbiota, which continues to be an intrinsic component of the host, can help host acquire the calories and nutrients present in various complex dietary and has attracted great attention from the scientific community in recent year (Costello, Gordon, Secor, & Knight, 2010;. The establishment of the gut microbiota in fish is a complex process and a specific reflection of microorganisms in rearing water, diet, and the environment. The sort and number of microorganisms presented in fish are lower than that in warmblooded animals and fluctuate greatly with host age, nutrition, and environment (Riiser et al., 2019). In this study, the gut microbiota profiles in the two types of grass carps and three types of crucian carps were characterized and compared, subsequently describing the relationship among genetic differentiation, feeding habits differences, and gut microbiota structure.  Figure 2b). However, the predominant phylum of Firmicutes was found among the core microbiota of fish from Dongting lake area in our study, which are consistent with Li's results . Interestingly, Proteobacteria was the most abundant phylum in many freshwater fish elsewhere (Wu et al., 2012;Yan et al., 2016). This finding might be due to different feeding habits and conditions. The genus Aeromonas, Pseudomonas, and Bacteroides were found to be dominant in majority freshwater fish intestine, followed by Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, and others (Nayak, 2010). The rationality of gut microbiota was aided to enhancement of host's growth (Gong et al., 2019;Xie et al., 2019). It has been suggested that Proteobacteria, Firmicutes, and Bacteroidetes can play important roles in the growth of fishes. To be specific, Proteobacteria was shown to participate in the metabolism and cycling of carbon, nitrogen, and sulfur in fish (Fjellheim, Klinkenberg, Skjermo, Aasen, & Vadstein, 2010;Han et al., 2010); Bacteroidetes was found to be involved in the fermentative process and degradation of oligosaccharides (Li et al., 2015); and Firmicutes was shown to contribute to carbon metabolism (Corrigan, Leeuw,

F I G U R E 4
Network graphs of bacterial relationship in each fish group. Node color corresponds to phylum taxonomic classification. Edge color represents positive (red) and negative (green) correlations F I G U R E 5 Network graphs of bacterial relationship in herbivore (grass carp) and omnivore (crucian carp), respectively. Node color corresponds to phylum taxonomic classification. Edge color represents positive (red) and negative (green) correlations Penaud-Frezet, Dimova, & Murphy, 2015). It was reported that the higher proportion of Firmicutes over Bacteroidetes could be associated with enhanced growth rate of fish (Li et al., 2013 resistance. Cetobacterium as a potential probiotic remained to be further studied on the intestine of fish. Among the Cetobacterium species, the C. somerae could promote the synthesis of vitamin B12 (Zhai et al., 2017). In addition, Aeromonas, even though sometimes pathogenic, have been detected as a necessary community member in the normal intestinal mucosa of several fishes, which could produce hydrolytic enzymes. Thus, they could act as symbionts assisting in the breakdown of dietary components (Wu et al., 2012).
As is well known, the habitat is a key factor for the survival of fish, so fish habitat differences are presumably caused by the differences in the type of food sources, including ingested bacteria, intestinal morphology and digestion, or life habits (Ni et al., 2012;Ye et al., 2014). The dietary habits considerably affect fish intestinal bacterial structure. Remarkable differences between herbivorous fish and omnivorous fish were revealed when the groups were considered separately in pairwise comparisons. Although the strains of Firmicutes dominated the gastrointestinal microbial communities in marine and freshwater fish (Jiang et al., 2019;Riiser et al., 2019;Wu et al., 2012), their abundance exhibited differences between herbivores and omnivore. We analyzed the interaction between bacteria by construction of network graphs and highlighted the significantly abundant and relevant genera in the intestinal microbiota. The network of indigenous fish (IGC and ICC) showed lower overall connectivity, whereas the bacteria of new lineage fish groups (PGC, HFCC, and XYCC) had hubs with more node degree ( Figure 4). Previous studies have shown that the microbiota structure stability declines with an increase in microbial diversity and proportion of cooperative interactions (Coyte, Schluter, & Foster, 2015). We spec- As an inherent lack of power of 16S rRNA amplicon sequencing method to resolve fine-scaled biological complexity, we did not obtain the correlation between the predominant microbes and metabolic functional features. Hence, it would be further determined by metagenomic analysis.
Our results are the first to reveal the gut microbial composition of new lineages-grass carp and crucian carp from Dongting lake area. The observed differences of gut microbiota between new lineages and indigenous may be an important influencing factor for different growth and disease resistance.

ACK N OWLED G M ENTS
This present work was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
None declared. Xia; Funding acquisition-Supporting.

E TH I C S S TATEM ENT
This study was carried out in accordance with the recommendations  (Table S1: Statistics for each sample sequencing;

F I G U R E A 4
One-way ANOVA showed relative abundance differences of (a) Firmicutes and (b) Erysipelotrichaceae in any two groups of fish. *p < .05, **p < .01, ***p < .001