Lead Exposure Induces Structural Damage, Digestive Stress, Immune Response and Microbiota Dysbiosis in Intestine of Silver Carp (Hypophthalmichthys Molitrix)

Lead (Pb) is one of the most common toxic heavy metals in water, and it can cause harm to aquatic animals and humans when released into the environment. In the present study, the effects of Pb exposure on the morphology, digestive enzyme activity, immune function and microbiota structure of silver carp (Hypophthalmichthys molitrix) intestines within 96 h were detected. Moreover, the correlation between them was analyzed. The results showed that Pb exposure could severely damage the intestinal morphology on the one hand, including signicantly shortening the intestinal villi’s length, increasing the goblet cells’ number, causing the intestinal leukocyte inltration, and thickening the intestinal wall abnormally, and on the other hand, increasing the activity of intestinal digestive enzyme (trypsin and lipase). In addition, the mRNA expressions of structure-related genes (Claudin-7 and villin-1) were down-regulated, and the immune factors (IL-8, IL-10 and TNF-α) were up-regulated after Pb exposure. Furthermore, data of the MiSeq sequencing showed that the abundance of membrane transport, immune system function and digestive system of silver carp intestinal microbiota was decreased, and the cellular antigens was increased. Finally, the canonical correlation analysis (CCA) found that there were correlations between silver carp’s intestinal microbiota and intestinal morphology and immune factors. In conclusion, it is speculated that Pb may damage the intestinal barrier of silver carp, leading the microbiota dysbiosis, which further affects the intestinal immune and digestive function.


Introduction
In recent years, industrialization and related human activities have led to a signi cant increase in heavy metal content in freshwater environment (Sin et Tao et al. 2012). Lead (Pb), as one of the most common pollutants, is carcinogenic, teratogenic and to a certain extent mutagenic to animals like most other heavy metals (Eisler 1998;Bonacker et al. 2005).
Several studies have reported that Pb pollution may affect the structure and function of sh intestines, cause sh intestinal morphology disturbance, and affect the activity of intestinal enzymes activity and immune function (Crespo et al. 2006; Lei et al. 2020). In addition, it is reported that the stability of intestinal structure and function is closely related to the intestinal microbiota structure (Ni et al. 2009), and the studies have shown that water pollutants can destroy the structure of sh intestinal microbiota and cause harm to the physiological functions of sh intestines (Qiao et al. 2019a).
Intestinal microbiota is an important ecosystem in organism, which has a lot to do with metabolism and There are many studies on the in uence of foreign substances on the structure of sh intestinal microbiota, such as immunosuppression changed the intestinal microbiota structure of the silver carp, which in turn increased the susceptibility to pathogens (Qi et al. 2019). Although previous studies have suggested that heavy metal has a toxic effect on aquatic organisms, the relevant in uence mechanism among the intestinal microbiota, intestinal structure, and intestinal function of sh under Pb exposure is still unclear.
Fish is one of the most important aquatic organisms in the aquatic environment and plays a key-role in human diet and provides an important source of high-quality protein (Milenkovic et al. 2019). Over the last twenty years, world consumption of sh has increased with increasing concerns about its therapeutic and nutritional bene ts (Rajeshkumar and Li 2018). Among them, silver carp is one of the most common freshwater sh species in the environment, and is well-known as a food sh in many countries (Buchtova and Frantisek 2011;Islam et al. 2019). Meanwhile, silver carp as a lter-feeding sh, can reduce phytoplankton biomass, control algae bloom, and promote nutrient regeneration in water environment ). Therefore, silver carp plays an important role in maintaining the balance of the water environment. In this study, we simulated the basic environment of the pond, and added Pb pollution. We found that Pb was accumulated in the intestine, and the intestinal structure, digestive enzyme activity, and the expression of immune factors was changed after Pb pollution. Then, the dynamics of microbiota in silver carp intestine were detected and analyzed.

Fish maintenance
Purchased silver carp from an aquaculture company in Qingyuan (Guangdong, China), transported to the experimental base in oxygenated polythene bag, and then cultured in an earthen pond The average body length of silver carp was 10.72 ± 0.35 cm and the average weight was 12.31 ± 0.54 g. Before sampling, sh were fed twice a day with a commercial sh food for two weeks as describe before  Eighty silver carp were randomly selected from the pond and cultured in a four cubic meter of aquarium (to simulate sh original living environment, the culture water is taken directly from the local ponds where the sh originally lived). Before Pb exposure, twelve silver carp were randomly collected and sampled (L0 h group). Then, sh were exposed to a SC (3.84 mg/g) of Pb(NO 3 ) 2 until 96 h, and twelve silver carp were randomly collected at 6 h, 48 h and 96 h (L6 h, L48 h and L96 h group), respectively. The Pb content in water was keep around 3.84 mg/L during the experiment. Before sampling, sh were anesthetized in 0.05% MS-222 (Aladdin, China) to euthanize, posterior intestines from each group were sampled to detect the activities of digestive enzyme, Pb content, and RNA expression of gene (n = 3). The samples were immediately stored frozen liquid nitrogen and stored at −80°C until analysis. For histological analyses, posterior intestines were collected from the sh euthanized and xed in 4% paraformaldehyde solution (n = 3). For DNA Extraction and MiSeq sequencing, posterior intestines from the sh euthanized were collected and xed in 95% alcohol (n = 3).

Pb accumulation
Posterior intestines from each group were sampled to detect Pb concentration. According to the method published by previous study, the Pb concentrations were measured . Brie y, the samples were collected and homogenized, using the 4 mL HNO 3 to digest for 1 h, and then put into microwave digestion apparatus (QiYao, China). Microwave digestion procedure: hold at 120°C for 10 min, hold at 150°C for 15 min, and then hold at 190°C for 25 min. After that, the Pb concentration of the sample was determined by ICP-MS ).

Histological analysis
For histological analysis, using 4% paraformaldehyde solution (Biosharp, China) to x the posterior intestine (1 cm segment before the anus) of all samples (each group, n = 3) for 48 h, and then processed according to the method described before (Liu et al. 2021). Using the light microscope (Nikon, Japan) to observe the number of goblet cells, intestinal villi, in ltration of leucocyte and wall thickness.

Digestive enzyme activities analysis
For digestive enzymes activities analysis, obtain intestinal homogenate: mix the intestine with saline solution in a ratio of 1 : 9, mechanical homogenate, and ice bath for 3-5 min to prepare tissue homogenate. Then, the intestinal homogenate was centrifuged at 4,000 × g for 10 min at 4℃, and the supernatant was collected. Finally, use the total protein assay kit (Jiancheng, China), trypsin assay kit (Jiancheng) and lipase assay kit (Jiancheng) to determine the trypsin and lipase enzymes activities of silver carp intestine according to the manufacturer's instructions.
Quantitative real-time PCR for analysis intestinal structure and immune-related genes Using Trizol reagent (Vazyme, China) to extract the total RNA of sample according to the method published by previous study. Brie y, tissues (100 mg / samples) were collected and ground under liquid nitrogen conditions, lysed with 1 mL Trizol, and RNA was extracted with chloroform and isopropanol.
Dissolved RNA in DEPC-treated water. Dissolved the RNA in DEPC-treated water and then determined the RNA concentration and quality according to the previous method (Tan et al., 2018). Using SYBR® qPCR Master Mix (Vazyme, Nanjing, China) and the Bio-Rad CFX Connect PCR (Bio-Rad, USA) to performe the quantitative real-time PCR (qRT-PCR). The β-actin of the silver carp was used as a reference gene (GenBank accession NO. JX274220.1). The primers of gene shown in Tab. 1. Detected the primer's e ciency and speci city before the qRT-PCR analysis and processed the qRT-PCR as the method of Fu et al (Fu et al. 2019). Table 1 The primer sequences used in this study for qRT-PCR.

Primers
Nucleotide The posterior intestinal samples of all groups (each group, n = 3) were sent to Guangzhou JiRui Gene The results were analysed by SPSS 19.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) and R. Using image pro plus program to analyze intestinal structure indicators. One-way ANOVA was used to check for the signi cance of difference between the means of each group. Data were expressed as (Mean ± S.D.). Difference was considered signi cant at 0.01 < p < 0.05(*). Extremely signi cantly difference was considered signi cant at p < 0.01(**).

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 signi cantly 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 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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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 sh microbiota structures and diversity analysis Quality and chimera ltration 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-ltered 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).

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 con rmed 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 signi cantly and reached to the highest level (Fig. 7B). The relative abundance of Weeksellaceae and Burkholderiaceae signi cantly 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 pro ling of microbial communities PICRUST was used to predictive the functions of the microbiota in intestines. In Fig. 9A, 25 functions were identi ed 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 classi cation results indicated that the biological functions pro les 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 signi cant changes (Tab. S1). Similarly, cellular antigens had a signi cant 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 signi cantly 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.

Discussion
Nowadays, the water environment is generally polluted by heavy metals, which seriously affects the survival of sh in the water (Luczynska et al. 2018). Pb may cause damage to human nerves, hematopoietic system and kidneys (Sivaperumal et al. 2007;Hasschon et al. 2008). It has been reported that the accumulation of Pb in sh may adversely affect the structure and function of sh intestines. In this study, we found that Pb was enriched in the intestine of silver carp and the changes in the morphological structure, digestive enzyme's activity, and immune factors of the sh intestine. In addition, we also used high-throughput sequencing technology to detect changes of microbiota in silver carp's intestine after Pb exposure. CCA analysis showed that there is a signi cant correlation between the silver carp's intestinal microbial community and intestinal structural indicators, digestive enzyme activity levels and immune factors. The function prediction analysis further indicated that the changes of silver carp intestinal structure and function may be related to the intestinal microbiota. Therefore, Pb may destroy the structure and function of carp intestine by destroying the composition of silver carp intestinal microbiota.
Previous studies reported that heavy metals exposure could in uence the intestinal function such as active transport, immune response, and so on (Iturri and Peña 1986; Wang and Chen 2015). In this study, the concentration of Pb in the silver carp intestine was signi cantly increased after Pb exposure ( Fig. 1). Moreover, the intestinal morphology changed after Pb exposure, including in ltrated leukocytes, increased goblet cells' number, shallowed the depth of crypts, and increased intestinal wall thickness (Fig. 2). Crypts and intestinal wall thickness changes are related to in ammation, and the goblet cells' number increased and leukocyte in ltration will further promote in ammation (Lofgren et al. 2002;Qiao et al. 2019b;). These results were consistent with the mRNA expression of structure-related genes (Claudin-7 and villin-1) and immune-related genes (TNF-α, IL-8 and IL-10) (Fig. 4). Meanwhile, the activity levels of intestinal digestive enzyme (trypsin and lipase) also increased signi cantly after Pb exposure (Fig. 3), which may be due to the compensation mechanism triggered by the environmental stress (Zare et al. 1996;Cao et al. 2010;Babaei et al. 2020). The data at the genetic, protein and structural levels indicated that Pb exposure can cause physiological damage to the intestinal barrier, which may be related to intestinal digestive function and immune response. After Pb exposure, Firmicutes and Bacteroidetes became the most abundant bacterium at the phylum level in intestine (Fig. 6). Firmicutes, a common phylum in the intestines of sh (Burgos et al. 2018;Meng et al. 2020). The increase of Firmicutes in relative abundance in this study may be due to its higher tolerance to heavy metals (Guo et al. 2019). Bacteroidetes, a Gram-negative bacterium, which closely relate to the occurrence of intestinal in ammation (Marchesi et al. 2016). At the family level, both Aeromonadaceae, Burkholderiaceae and Weeksellaceae in silver carp intestines were increased after Pb exposure (Fig. 5). Aeromonadaceae, belonging to Gammaproteobacteria, is a common pathogenic bacterium in shes, which destroys the intestinal lining, causes intestinal cell damage, promotes in ammation and alter intestinal morphology (Ring et  ). As a lter-feeding sh, the structure and function of silver carp intestines maybe more closely related to intestinal microbiota (Dong and Li 1994). The results in our study indicated that changes in intestinal microbiota could affect the structure and immune function of the intestine.

Conclusion
In this study, the effects of Pb exposure on silver carp intestinal structure, digestive enzyme, and immune function within 96 h were detected. Through CCA analysis, we speculated that these effects may be related to the changes of intestinal microbial community. In addition, the function prediction results were also consistent with the correlation analysis. Overall, the results of this present study provide new information about the toxic effects of Pb on silver carp and gain a better understanding of the relationship between intestinal microbes and intestinal structure and function.

Con ict of Interest
The authors declare no con ict of interests  Accumulation of Pb in silver carp within 96 h.

Figure 2
Representative micrographs of intestines from silver carp (A). intestinal wall relative thickness (B), intestine crypt relative depth (C), and goblet cells relative number (D) were analyzed. Leukocyte in ltration occurs in the area indicated by the arrow. The asterisk represents a statistically signi cant difference (*0.01 < p < 0.05 and **p < 0.01).

Figure 3
Relative enzyme activity of trypsin (A) and lipase (B) in silver carp intestines.

Figure 5
The effects of Pb on the richness and diversity of microbiota in the silver carp intestines in each sample.