Analysis of 16S rRNA sequencing results
Six samples were taken from each group, but only 5, 5 and 3 samples from BL, BH and SH groups respectively accorded with the requirements of library construction, because the rest samples of PCR products were no purpose bands or low concentrations. After a series of puriﬁcation and ﬁltration processes on the sequencing results, 13,740 to 28,143 effective sequences were collected from each sample, resulting in a total of 246,700 sequences from the 13 samples. The statistics of the ﬁltered sequencing data of each sample are shown in Table S1 (Additional file 1). The proportion of effective tags among Raw_Tags was in the 60% to 80% range at each sample. The reads sequences in the corresponding length range of each sample after quality control ﬁltration were counted. The eﬀective sequence length distribution is shown in Fig. S1 (Additional file 1). 400 bp to 440 bp were the most eﬀective sequence length distribution region.
The number of sequences in OTU of each sample was obtained within the 97% sequence similarity threshold. Meanwhile, the classiﬁcation information for each species corresponding to each OTU was also obtained by comparing the OTU representative sequences with a microbial reference database. The bacteria that could be detected were classiﬁed into 13 phyla, 21 classes, 41 orders, 77 families, and 120 genera. The composition of each sample community was calculated at phylum, class, order, family, genus and species levels, respectively. Table S2 (Additional file 1) was showed the number of each species at diﬀerent levels, and Table S3 (Additional file 1) was recorded the total number of OTUs covered by each sample in their subordinate levels. The number of OTUs increased with the depth of sequencing, which was confirmed by the dilution curves of the OTUs measured in this study. The ﬁnal curve became stable, which signified that the amount of sequencing data is somewhat reasonable (Fig. 1).
Comparison of core intestinal microﬂora in sturgeons from three experimental groups
Venn diagrams were used to show the core intestinal microﬂora of the sturgeons in three experimental groups. The core microﬂora of sturgeons meant the common bacterial populations in three experimental groups. As shown in Fig. 2, the number of OTUs shared by all sturgeon in three experimental groups was 243, while the number of OTUs was 510 sharing by sturgeon in BL and BH groups, and 278 for sturgeon in BH and SH groups. The main bacterial phyla in the intestines of each group of sturgeon are shown in Fig. 3. Three dominant phyla in BL and BH groups were Proteobacteria, Firmicutes and Cyanobacteria, while SH group was Proteobacteria, Firmicutes and Actinobacteria. The core intestinal microﬂora in fish from three experimental groups were the same, but their relative abundances were different. The top five core intestinal microﬂora at phyla in BL group showed the order from high to low was Proteobacteria, Firmicutes, Cyanobacteria, Bacteroidetes and Actinobacteria (P<0.05), whereas the core intestinal microﬂora in BH groups showed the difference with the order of Cyanobacteria>Proteobacteria>Firmicutes>Actinobacteria>Bacteroidetes. And the order from high to low of core intestinal microﬂora in SH group was Proteobacteria, Firmicutes, Actinobacteria, Cyanobacteria, and Bacteroidetes.
A heatmap (Fig. 4) is a graphical representation that uses a system of colored gradients to represent the size of values in a data matrix, and the cluster data are also expressed in heatmap according to species or the abundance similarity of samples. In order to reﬂect the similarities and diﬀerences between multiple sample communities, high-abundance and low-abundance species are clustered by color gradient and similarity. Based on the species composition and relative abundance of each sample, a heatmap analysis was performed to extract the species at each taxonomic level. Mapping was achieved using R language tools, and a heatmap cluster analysis was performed at the levels of the phylum, class, order, family, genus, and species, respectively. The result was found in Fig. 4 that the vertical clustering between BH and SH groups showed the long branch length, which indicated the richness of intestinal microbiota between two groups was clearly different. Similarly, BL and BH groups have a certain degree of similarity in richness, because the short branch length was found in BL and BH groups at Fig. 4. In addition, the relative abundance of intestinal flora at genus level among BL, BH and SH groups were also showed in Fig. 4. The major intestinal microﬂora at genus level in BL group were Acinetobacter, Pseudomonas, Bacillus, and so on, as same as the BH group, while the SH group included Bacillus, Staphylococcus and Acinetobacter intestinal microﬂora at genus level.
Alpha diversity analysis of microbial communities in sturgeons from three experimental groups
Alpha diversity refers to the richness and diversity of a single sample species. There were several indices for measurement of alpha diversity, such as the Chao1, Observed_species, Shannon, PD_whole_tree, and Good's coverage. The Chao1 index measures the richness of species (i.e., the numberof species), whereas the Shannon index measure the diversity of species. Observed_species shows the number of OTU was observed with the increase of sequencing depth. PD_whole_tree refers to the number of species observed, reflecting the abundance of the colony. The completeness of the sequencing was tested by good's coverage, which was close to 99% in this study, indicating that the majority of the bacterial species present in the sample had been detected.
Table 2 showed the results of alpha diversity for the gut microbiota in sturgeons from three experimental groups. A total of 826 OTUs were obtained at the 97% similarity level. There was a signiﬁcant diﬀerence in the OTUs, Chao1, Good's coverage, Observed species and Shannon indices between the BH and SH groups (P<0.05), but no signiﬁcant diﬀerence was found in the PD whole tree of the two groups (P>0.05). The result showed that the alpha diversity indices were considered to be signiﬁcantly diﬀerent between Siberian sturgeon and Beluga sturgeon when they fed with the same diet (P<0.05).
Meanwhile, although the most alpha diversity indice were no significant difference between BL and BH groups (P>0.05), but the Shannon index was obviously higher in BL group than that in BH group (P<0.05), which pointed that the species diversity of the intestinal microbiota was enhanced when Beluga sturgeon fed with the low fishmeal diet.
Beta diversity analysis of microbial communities in sturgeons from three experimental groups
Non-metric multidimensional scaling (NMDS) (Fig. 5) is a data analysis method that simplifies research objects (samples or variables) in multidimensional space to low-dimensional space for positioning, analysis and classification, while retaining the original relationship between objects. It is applicable to the case that the exact similarity or heterogeneity data between the research objects cannot be obtained, but only the hierarchical relationship data between them can be obtained. Its basic feature is to treat the data of similarity or dissimilarity between objects as a monotone function of the distance between points, and replace the original data with a new data column of the same order for the metric multidimensional scale analysis on the basis of maintaining the original data order relationship. In other words, when the data is not suitable for the direct multidimensional scaling analysis of variable type, the variable transformation is carried out and then the multidimensional scaling analysis of variable type is adopted. For the original data, it is called non-metric multidimensional scaling analysis. Its characteristics are reflected in multi-dimensional space in the form of points according to the species information contained in the sample, and the degree of difference between different samples is reflected by the distance between points, and finally the spatial locus map of the sample is obtained.
The closer the distance between sample points, the higher the similarity. Generally speaking, the samples within the same circle meant the difference between samples was not obvious, while the sample points within circles with no intersection indicated that there was significant difference between the samples. As shown in Fig. 5, the circles were intersect between BL and BH groups, but no intersection was found in BH and SH groups, which indicated the beta diversity between BH and SH groups was significant difference, whereas BH and BH groups was not.
Analysis of the differences in gut microbiota among sturgeons in three experimental groups at the phylum and genus levels
LEfSe (Linear discriminant analysis Eﬀect Size) (Fig. 6) is an algorithm for high-dimensional biomarker discovery and explanation that identifies bacteria of each level of phylum, class, order, family, or genus characterizing the differences among BH, BL and SH three groups (Fig. 6). The cladogram (Fig. 6a) showed diﬀerences in 102 taxa among fish in BL, BH and SH groups. BL fish (enrichment in the Gammaproteobacteria and Proteobacteria) have no similar to BH fish, with a lot of changes occurring in the BH group such as enrichment in Actinobacteria, Corynebacteria and Norynebacteria (Fig. 6b). And the SH group was mainly enriched in Bacillales and Bacillus genus, which was clearly difference with BH group (Fig. 6b).
In addition, Fig. 7 shows the diﬀerences in relative abundances at the phyla level of the top 5 bacterial communities and genera level of the top 8 bacterial communities in the three experimental groups. In the fish from BL group, the relative abundance of Proteobacteria and Bacteroidetes were signiﬁcantly higher than that in the sturgeons from BH groups (P<0.05). In contrast, the relative abundances of the Cyanobacteria phyla in BL group were signiﬁcantly lower than in BH group (P<0.05). Both groups did not show any signiﬁcant diﬀerences in the relative abundances of Firmicutes and Actinobacteria (P> 0.05) (Fig. 7a).
Compared with the sturgeons of BH group, the relative abundance of Proteobacteria and Firmicutes were signiﬁcantly increased in sturgeons from SH group, whereas the relative abundance of Cyanobacteria were clearly decreased (P<0.05). The Bacteroidetes and Actinobacteria relative abundance were no signiﬁcant diﬀerences in sturgeons of BH and SH groups (P> 0.05) (Fig. 7b).
At the genus level, the relative abundances of Pseudomonas and Citrobacter in BL group were signiﬁcantly higher compared with BH group (P<0.05) (Fig. 7c), while the relative abundance of Bacillus, Luteibacter, Staphylococcus, and Oceanobacillus was lower in BH group than in SH group (P<0.05) (Fig. 7d). Three groups did not show any signiﬁcant diﬀerences in the relative abundances of Alpinimonas and Acinetobacter (P>0.05).