Pb accumulation in intestines
Determining the Pb concentration of silver carp intestine by ICP-MS. As shown in Fig. 1, the concentration of Pb in intestines significantly increased to the highest concentration (118.39 mg/kg) at 96 h after Pb exposure.
Histologic observations and analysis of intestines
Observing the morphology of silver carp intestines by a microscope (Nikon, Japan) (Fig. 2A). As shown in Fig. 2B, after Pb exposure for 48 h, the relative intestinal wall thickness was increased significantly and reached the highest level (up to 1.27-fold, p < 0.01). Besides, the relative depth of intestinal crypts decreased and reached the lowest level at 96 h (down to 0.41-fold, p < 0.01) (Fig. 2C), while the goblet cells' number in intestine increased and reached the highest level at 48 h (up to 3.17-fold, p < 0.01) (Fig. 2D).
The activity levels of trypsin and lipase in the intestines
Using the enzyme activity kit to determine the relative activity level of trypsin activity and lipase activity of silver carp intestine after Pb exposure. As shown in Fig. 3A, the relative activity of trypsin in the intestine increased significantly and reached the highest level after 6 h of Pb exposure (up to 6.38-fold, p < 0.01). Similarly, the relative activity level of lipase increased significantly and reached the highest level after 48 h (up to 3.55-fold, p < 0.01) (Fig. 3B).
The expression of immune and structure -related genes in the intestines
The mRNA expressions of immune-related genes (IL-8, IL-10 and TNF-α) and structure-related genes (Claudin-7 and villin-1) in intestine were measured by qRT-PCR. After Pb exposure, as shown in Fig. 4A and B, the expression of TNF-α and IL-10 in intestine significantly increased to the highest level at 48 h (up to 2.36-fold, p < 0.01, and 2.28-fold, p < 0.01, respectively) after Pb exposure and gradually recovered at 96 h. In Fig. 4C, the expression of IL-8 in intestine significantly increased to the highest level at 6 h (up to 2.76-fold, p < 0.01) and gradually recovered later. On the contrary, the expression of Claudin-7 and villin-1 in intestine decreased significantly and reached the lowest level at6 h after Pb exposure (down to 0.31-fold, p < 0.01, and 0.11-fold, p < 0.01, respectively) (Fig. 4D and E).
Characteristics of fish microbiota structures and diversity analysis
Quality and chimera filtration from effective sequences ranging 21,252 to 101,185 (each group, n = 3) per sample by Illumina MiSeq platform, a total of 779,971 quality-filtered sequences were obtained. A 97% similarity cutoff was applied to cluster the high-quality sequences of the microbiota in intestines which divided into 615 operational taxonomic units (OTUs) (excluding monad sequence). Among them, 324 OTUs were shared by all samples (Fig. S1).
Good’s coverage of different samples was more than 99% (Tab. 2), and the statistical estimates of species richness and diversity indexes from each sample were presented in Tab. 2. ACE index ranged from 261.56 to 354.71, while chao1 index ranged from 261.07 to 362.55. The trends of ACE and chao1 after Pb exposure were shown in Fig. 5A and B. The shannon index ranged from 5.12 to 6.22, while the simpson index ranged from 0.90 to 0.97. In all samples, the shannon index and simpson index were the lowest at 48 h after Pb exposure (Fig. 5C and D). The corresponding rarefaction curves tended to reach the saturation plateau (Fig. S2).
Table 2 Diversity and Richness indexes as calculated by MOTHUR software (ver. 1.30.0). Operational taxonomic units (OTUs) are defined at 97% sequence similarity.
Samples
|
Total sequences passed quality check
|
Total OTUs
|
Diversity and Richness indexes1
|
Good’s coverage
|
Ace Index
|
Chao1 Index
|
Simpson Index
|
Shannon Index
|
L0 h
|
221,047
|
884
|
261.56 ± 11.25
|
261.07 ± 8.99
|
0.96 ± 0.01
|
5.85 ± 0.24
|
0.999
|
L6 h
|
231,612
|
958
|
273.66 ± 47.05
|
273.07 ± 46.12
|
0.97 ± 0.03
|
6.22 ± 0.82
|
0.997
|
L48 h
|
122,601
|
905
|
265.86 ± 28.48
|
271.32 ± 29.39
|
0.90 ± 0.04
|
5.12 ± 0.79
|
0.998
|
L96 h
|
204,711
|
844
|
354.71 ± 103.11
|
362.55 ± 102.98
|
0.96 ± 0.02
|
6.02 ± 0.96
|
0.999
|
1: Values represent the average ± S.D (each mean value of 3 determinations).
Change in the bacterial community compositions
Main bacterium of silver carp at the phylum level was shown in Fig. 6, mainly including Proteobacteria, Firmicutes, Bacteroidetes, Cyanobacteria, and Fusobacteria. Before Pb exposure, the most abundant bacterium in intestine was Proteobacteria (29.47%). After Pb exposure, the most abundant bacterium in intestine was changed to Firmicutes (36.05%) at 6 h and then changed to Bacteroidetes at 48 h (39.50%) and Proteobacteria at 96 h (33.37%).
In Fig. 7A, 29 different family of microbiota were confirmed in silver carp’s intestine and six of them were dominant. They were Aeromonadaceae, Weeksellaceae, Burkholderiaceae, Flavobacteriaceae, and Erysipelotrichaceae. After Pb exposure for 6 h, the abundance of Aeromonadaceae in intestines were increased significantly and reached to the highest level (Fig. 7B). The relative abundance of Weeksellaceae and Burkholderiaceae significantly increased to the highest level at 48 h, and then recovered (Fig. 7C and D).
Canonical correlation analysis
CCA was carried out to analyze the relationship between intestinal microbiota, intestinal structure, immune factors, digestive factor, and Pb content. As shown in Fig. 8A, the goblet cell number was positively correlated with the intestinal microbial community in L48 h group, the intestinal crypt was correlation with intestinal microbial community in L0 h and L6 h groups, and the Pb content was positively correlated with the intestinal Pb content in L96 h group. In Fig. 8B, the expression of TNF-α and IL-10 in silver carp were positively correlated with the intestinal microbial community in L48 h group. Meanwhile, the expression of IL-8 correlated with intestinal microbial community in L6 h group. In Fig. 8C, the expressions of trypsin and lipase in intestine were correlated with intestinal microbial community in L6 h and L48 h groups, respectively.
Predictive functional profiling of microbial communities
PICRUST was used to predictive the functions of the microbiota in intestines. In Fig. 9A, 25 functions were identified by Cluster of Orthologous Groups of proteins (COG) analysis in this study. In COG analysis, except for general function prediction only (relative abundance from 11.48 to 11.72%), the highest represented category was transcription (7.04 to 8.07%) and amino acid transport and metabolism (7.87 to 8.20%) function categories. These COG function classification results indicated that the biological functions profiles of all groups were similar with each other.
Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the functions of microbiota in carp intestinal were divided into six categories at level one, namely genetic information processing, cellular processes, human diseases, environmental information processing, metabolism and organismal systems, among which metabolism (47.54-49.26%) was the most abundant (Fig. 9B). At level two, membrane transport (10.94-12.94%) was the most abundant, while immune system and digestive system was the lower abundant but significant changes (Tab. S1). Similarly, cellular antigens had a significant change at level three (Tab. S2). The relative abundance change of membrane transport, immune system, digestive system and cellular antigens were shown in Fig. 9C, D, E and F, respectively. The relative abundance of membrane transport function of intestinal microbiota significantly decreased to the lowest level at 48 h, and the relative abundance of immune system function of microbiota also decreased at 6 h. In contrast, the relative abundance of digestive system and cellular antigens function of intestinal microbiota increased consistently and reached the highest level at 48 h.