Alpha diversity
When the silkworms grew to the fifth instar larvae by feeding the antibiotics-treated mulberry leaves, PCR amplification and agarose electrophoresis detection results showed that the negative amplification rate of 18S rRNA gene ITS1-ITS2 region of silk-worm feces was 85% (17/20) in the mulberry leaves treated with amphotericin. Six fecal samples with negative 18S rRNA gene ITS1-ITS2 PCR amplification were selected for V3-V4 region amplification of 16S rRNA, and gut bacteria were profiled by high-throughput sequencing. As regarding to the silkworms feeding by penicillin-streptomycin treated mulberry leaves, the negative rate of V3-V4 region of 16S rRNA gene in silkworm feces was 90% (18/20), and six fecal samples with negative 16S rRNA PCR amplification were selected for 18S rRNA gene ITS1-ITS2 region amplification and gut fungal high-throughput sequencing. At the same time, 6 mulberry leaves and 6 fecal samples of the control group were selected for both 16S rRNA gene V3-V4 region and 18S rRNA gene ITS1-ITS2 region amplification simultaneously. Ultimately, average 37830 ± 6702 16S rRNA gene sequences and 44136 ± 9361 18S ITS1-ITS2 gene sequences were obtained from each sample, and 607 bacterial OTUs and 533 fungal OTUs were obtained according to 97% similarity.
Alpha diversity analysis of the flora was conducted by the original OTUs distribution (Table.1). For bacteria, compared with the mulberry leaf group, the richness index (Ace, Chao, Observed OTUs) and diversity index (Shannon) of silkworm intestinal bacteria in the control group were significantly increased (P < 0.05), but the intestinal bacterial richness and diversity index of silkworm which fed mulberry leaves treated with 10 µg mL− 1 amphotericin were observably decreased, indicating that the bacterial richness and diversity of silkworm were markedly reduced by the loss of intestinal fungi. For fungi, compared with the mulberry leaf group, the richness and diversity index of silkworm intestinal fungi in the control group were signally decreased (P < 0.05), and there was no conspicuous change in the richness index of silkworm in-testinal fungi after feeding mulberry leaves treated with 400 U mL− 1 penicillin and 400 µg mL− 1 streptomycin (P < 0.05), but the fungal diversity index was remarkably increased (P < 0.05), indicating that antibiotic treatment could improve the diversity of intestinal fungi of silkworm.
Table.1 Alpha diversity analysis of intestinal flora of Bombyx mori
| Ace | Chao | Shannon | Simpson | Observed OTUs |
Bacteria | Control (n = 6) | 178.2 ± 139.9 | 179.8 ± 139.5 | 2.533 ± 1.429 | 0.194 ± 0.101 | 168.2 ± 144.6 |
Amphotericin (n = 6) | 115.5 ± 10.9* | 115.5 ± 12.4 | 1.845 ± 0.125* | 0.256 ± 0.031* | 105.4 ± 10.4* |
Mulberry Leaf (n = 6) | 123.3 ± 18.8* | 124.1 ± 21.0 | 1.740 ± 0.079* | 0.277 ± 0.030* | 105.4 ± 20.2* |
H(P) | 1.434(0.488) | 1.647(0.439) | 6.313(0.043) | 6.918(0.031) | 0.261(0.878) |
Fungi | Control (n = 6) | 95.5 ± 18.9 | 83.5 ± 30.3 | 2.598 ± 0.950 | 0.211 ± 0.262 | 75.0 ± 30.5 |
Ampicillin + Streptomycin (n = 6) | 100.3 ± 57.8 | 97.5 ± 61.4 | 3.173 ± 0.629△ | 0.089 ± 0.050△ | 90.3 ± 56.3 |
Mulberry Leaf(n = 6) | 253.2 ± 53.6△ | 251.6 ± 55.1△ | 3.497 ± 0.254△ | 0.073 ± 0.031△ | 212.8 ± 39.9△ |
H(P) | 9.018(0.011) | 9.029(0.011) | 3.71(0.156) | 2.946(0.229) | 8.404(0.015) |
* For bacteria, compared with control group, P < 0.05. △ For fungi, compared with control group, P < 0.05. |
Beta diversity analysis
Principal component analysis (PCA) is to simplify the original complex data by dimensionality reduction, so as to facilitate analysis. The similarity of sample composition was reflected as the distance between each point on the two-dimensional coordinate diagram (Deng and Zhang 2018). The results showed that the bacterial and fungal community structure of mulberry leaf were significantly different from silkworm gut. After feeding the mulberry leaves treated with amphotericin or antibiotics, the intestinal bacterial or fungal community structure of silkworm changed notably (Fig. 1). In terms of bacteria, the distance between the control group and the mulberry leaf group was far, suggesting that the similarity of bacterial community between mulberry leaf and silkworm gut was low. After feeding amphotericin-treated mulberry leaves, the bacterial community structure of silkworm gut was obviously changed, indicating that the loss of intestinal fungi observably affected the bacterial community structure (Fig. 1A). On the other hand, the similarity of fungal community between mulberry leaf and silkworm gut was not high also, and the intestinal fungal community structure of silkworm changed markedly after feeding mulberry leaves treated with antibiotics. Antibiotic group was close to the control group and located in the same quadrant, indicating that the fungal community structure of them was similar, and the loss of bacteria mainly resulted in the difference in PC1 direction between the two groups (Fig. 1B).
Analysis of flora structure
The community structures of the bacteria and fungi were analyzed based on the homogenized OTUs distribution. A total of 20 known bacterial phyla were detected, among which the relative abundances of three dominant bacterial phyla (Firmicutes, Cyanobacteria, and Proteobacteria) accounted for 90–96%. The relative abundances of four known fungi phyla (Ascomycota, Basidiomycota, Chytridiomycota, and Mucoromycota) ranked from 81–99%.
At genus level, 8 dominant bacterial genera were found: Bacillus, Chloroplast-norank, Lactococcus, Carnobacterium, Mitochondria-norank, Streptococcus, Exiguobacterium, and Enterococcus. Compared with the mulberry leaf group, the relative abundance of silkworm gut bacteria in the control group was apparently different. After feeding the mulberry leaves treated with 10 µg mL− 1 amphotericin, the relative abundance of silkworm gut bacteria had no evident change (Fig. 2A), indicating that the loss of fungi had little effect on it. The dominant fungal genera were Cladosporium, Nigrospora, Tausonia, Aureobasidium, Penicillium, et al. Compared with the mulberry leaf group, the relative abundance of silkworm gut fungi in the control group was significantly different too. After feeding the mulberry leaves treated with 400 U mL− 1 penicillin and 400 µg mL− 1streptomycin, the relative abundance of each fungal genus obviously changed (Fig. 2B), indicating that the antibiotic treatment had a great effect on the relative abundance of intestinal fungi genera of silkworm.
Among the 154 bacterial genera, Venn analysis results presented 56 shared bacterial genera (Fig. 2C), such as Bacillus (15.92%), chloroplast-norank (15.70%), Carnobacterium (15.53%) and Lactococcus (15.26%). Among the 221 fungal genera, here observed 38 shared fungal genera (Fig. 2D), including Trichosporon, Trichomerium, Didymella and Ceratocystis.
LDA discriminant analysis of flora
LDA was used to compare two or more groups and to identify the potential markers (Ma et al. 2019). The histogram below showed the LDA scores of prominent genera in each group at the OTU level (Fig. 3). Seven bacterial genera, a bacterial taxonomic group and 18 fungal genera with significantly high abundance were found in the mulberry leaf group; 12 bacterial genera and 4 fungal genera were found in the control group, which have extremely high abundance. Multiple bacterial genera were also enriched in the amphotericin group, such as Aerococcus, belonging to the order Lactobacillales, phylum Firmicutes. In the antibiotic group, markedly abundant fungal genera included Aspergillus, belonging to the order Eurotiales, phylum Ascomycota. To sum up, we think amphotericin or antibiotic treatment can affect the composition of bacterial or fungal genera in the intestine of silkworm.
Analysis of symbiotic relationship of flora
Among the shared 56 bacterial genera (Fig. 2C) and 38 fungal genera (Fig. 2D), bacteria and fungi genera with average relative abundance greater than 0.1% were considered as the core flora, and 37 bacterial genera and 21 fungal genera were arranged to conduct symbiotic network. In general, the changing tendency of the bacteria was homologous between the mulberry leaf (Fig. 4A) and the control group (Fig. 4B). After the silkworms were exposed to amphotericin B, the tendency of the bacteria in silkworm intestine did not change significantly, while the tendency of the fungi was notably changed after the exposure to penicillin and streptomycin (Fig. 4C). Based on the flora of mulberry leaf, the status of the dominant bacteria in the silkworm gut was more closed related to the mulberry leaf bacteria than that of the dominant fungi, however, the bacterial intercept became lower (Fig. 4D) while the fungal intercept became higher after the exposure of antibiotics (Fig. 4E). The results suggested that intestinal bacteria and fungi may come from mulberry leaf, the deletion of gut fungi weaken the correlation between gut bacteria and diet-deriving bacteria, but the deletion of bacteria strengthen the correlation between gut bacteria and diet-deriving fungi. In a word, exposure of dietary antibiotics exerted different influence on the host’s gut flora.
Finally, based on core genera, the symbiotic networks were constructed (Fig. 5). In the mulberry leaves, multiple bacteria with higher abundance showed significantly positive or negative correlation in aggregation, such as the first 8 bacteria genera with higher relative abundance (Bacillus, Lactococcus, Carnobacterium, Streptococcus, Exiguobacterium, Enterococcus, Pseudomonas, and Paenibacillus). These 8 bacterial genera were negatively correlated with 5 fungal genera (Cladosporium, Penicillium, Alternaria, Aspergillus, and Moesziomyces) which have higher relative abundance (Fig. 5A). In the gut of silkworm, the above 8 bacterial genera were positively correlated with 9 genera such as Geobacillus, and the correlation coefficient was slightly reduced (Fig. 5B). The results indicated that the bacterial symbiosis changed signally in different habitats (mulberry leaf and silkworm gut), but the fungal symbiosis had no obvious rule. In the gut of silkworms which fed mulberry leaves treating with amphotericin B, the bacteria with high abundance showed apparently positive correlation, such as the population of positively correlated bacteria expanded dramatically, including 17 genera (Bacillus, Lactococcus, Carnobacterium, Streptococcus, Exiguobacterium, Enterococcus, Pseudomonas, Paenibacillus, Geobacillus, Acinetobacter, Brochothrix, Leuconostoc, Fusobacterium, Cronobacter, Enhydrobacter, Enterobacter, and Lysinibacillus), and the total relative abundance reached more than 47% (Fig. 5C). In the gut of silkworms which fed mulberry leaves treating with antibiotics (penicillin and streptomycin), the number of pairs of fungi with significantly positive correlation increased from 2 to 7 (such as Golubevia and Periconia), while the number of pairs of fungi with dramatically negative correlation increased from 3 to 5, suggesting that the loss of bacteria in the gut of silkworm resulted in the abnormal proliferation of fungi (Fig. 5D).