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Effects of cadmium exposure on the composition and diversity of the intestinal microbial community of common carp (Cyprinus carpio L.)
Ecotoxicology and Environmental Safety 171 (2019) 92–98
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of cadmium exposure on the composition and diversity of the intestinal microbial community of common carp (Cyprinus carpio L.) Xulu Chang, Hui Li, Junchang Feng, Yongyan Chen, Guoxing Nie, Jianxin Zhang
T
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College of Fisheries, Henan Normal University, Xinxiang 453007, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cadmium Cyprinus carpio Gut microbiota High-throughput sequencing
Cadmium (Cd) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd, the increased accumulation of Cd in fish has become a food safety and public health concern. The present study was conducted to investigate the effects of waterborne Cd exposure on the microbial community composition and diversity in the gut of common carp. Common carp were exposed to three waterborne Cd concentrations (0, 50 and 500 μg Cd L−1) for 4 weeks. Our results indicated that Cd exposure profoundly affected the composition of the gut microbiota in the common carp. At the phylum level, Saccharibacteria were detected in only the 0 μg and 50 μg Cd L−1 exposure groups, and the abundance of Fusobacteria decreased with increasing Cd concentration, while the abundance of Firmicutes increased with increasing Cd concentration. At the genus level, Cetobacterium was the dominant group in the gut of the common carp, and the abundance of Cetobacterium decreased after Cd exposure. Notably, the abundance of Akkermansia muciniphila, a probiotic, was found to decrease after Cd exposure, and the proportions of some Cd-resistant bacteria were found to increase following Cd exposure. Our results also demonstrated that Cd exposure decreased the community diversity of the gut microbiota. These results suggest that Cd exposure may impact the gut homeostasis of common carp and further affect the health of the organism.
1. Introduction Over the past several decades, industrial progress has been accompanied by frequent incidents of metal pollution, leading to significant public health concerns. Cd, a non-essential element, is widely used in industrial activities and can be toxic and carcinogenic to humans and animals (Liu et al., 2014; Zhai et al., 2017a). With rapid industrial development and population growth, increasing contamination of aquatic systems with Cd has been reported around the world (Taweel et al., 2013; Zhai et al., 2017b). As an environmental hazard, Cd enters the aquatic environment from both natural and anthropogenic sources, including agricultural runoff, industrial effluents, and urban and mining sources (Deng et al., 2014; Rajeshkumar et al., 2017). This exposure results in pathological changes in aquatic ecosystems, mostly manifested in fish, which are affected by Cd via the respiratory and digestive systems and through the skin (Kovarova et al., 2009). Cd is associated with a wide range of toxic effects in different experimental animals, including fish (Mu et al., 2018; Zhai et al., 2017a; Zhang et al., 2017). Notably, Cd can be biologically accumulated by fish, which are major protein sources consumed by humans, resulting in ⁎
potential human health risks. Thus, the increased accumulation of Cd in fishes has become a food safety and public health concern. Although the underlying toxic effects of Cd have been well addressed in different fish organs, including liver, kidney, gut and brain (Morgano et al., 2014; Olmedo et al., 2013), the effects of Cd on the gut microbiota of fishes have received little attention. The fish gut microbiota is known to play a significant role in a number of physiological functions, including immune regulation, mineral metabolism maturation and pathogen resistance (Gomez and Balcazar, 2008). Moreover, imbalance of the gut microbiota may mediate the development of numerous diseases in fishes (Perez et al., 2010). The composition of gut microbial communities in fishes is affected by exogenous and endogenous factors, including developmental stage, diet composition, habitat and surrounding environment (e.g., water temperature and salinity), rearing conditions and possibly phylogenetic position (Sullam et al., 2012; Zhang et al., 2016). Cd exposure has been reported to significantly affect the gut microbiota of many organisms (Liu et al., 2014; Mu et al., 2018; Yang et al., 2018; Zhai et al., 2017b). However, the effects of Cd on the intestinal gut of common carp remain unclear. The common carp, Cyprinus carpio L. (C. carpio), is the most widely farmed freshwater fish in China. It was reported that C. carpio is
Corresponding author. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.ecoenv.2018.12.066 Received 14 September 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer's protocol.
resistant to highly polluted habitats and is used as a bio-indicator species to understand the effects of environmental pollution (Yeşilbudak and Erdem, 2014). The aim of this study was to investigate the influence of Cd on the gut microbiota composition and diversity in common carp. To achieve this goal, the gut bacterial composition was investigated using high-throughput sequencing of the V3-V4 segment of the bacterial 16S rRNA gene. We hope that the results will provide insight into the toxic effects of pollution on common carp from a new perspective.
2.4. Sequencing and data processing Purified amplicons were pooled in equimolar amounts and subjected to paired-end sequencing (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Raw reads were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) toolkit (version 1.17). The reads were removed based on the following criteria: reads with average quality scores lower than 25; ambiguous bases; homopolymers of > 7 bases; reads containing primer mismatches; and reads with lengths shorter than 100 bp. For paired-end reads, only sequences that overlapped by more than 10 bp and lacked mismatches were assembled. Reads that could not be assembled were removed. Barcodes and sequencing primers were trimmed from the assembled sequences. The raw reads were deposited into the NCBI short read archive database (accession number: SRP159034).
2. Materials and methods 2.1. Experimental fish Juvenile common carp were obtained from an aquaculture farm in Zhengzhou City, Henan Province, China. After being removed from the water, the animals were randomly placed in tanks (300 L in water volume) supplied with continuous aeration and equipped with temperature-control devices. The juvenile common carp were acclimated for two weeks before being used in experiments. Commercial food (containing 19% casein, 20% fish meal, 20% soybean meal, 5% cottonseed meal, 20% flour, 3% soybean oil, 10% CMC-Na, 0.2% methylcellulose, 0.2% salt, 1.5% CaH2PO3, 0.1% vitamin mixture and 1% mineral mixture) was provided once a day until satiation. No mortality was observed during the acclimatization, and the tank water was partially changed every day.
2.5. Statistical analysis To estimate the bacterial community composition in the intestinal tract of the common carp, we performed an operational taxonomic unit (OTU) analysis. Sets of sequences with at least 97% identity were defined as an OTU, and chimeric sequences were identified and removed using UCHIME (Edgar et al., 2011). The sequence difference cut-off for OTUs was defined at 97%, in accordance with the currently accepted standards for prokaryotic species (Rossello-Mora and Amann, 2001). The number of OTUs in each sample and the number of sequences in each OTU were counted after the OTU output was obtained. Representative OTUs were selected for annotation of taxonomic information by the SILVA ribosomal RNA gene database using a confidence threshold of 70% (Quast et al., 2013). Additionally, rarefaction curves were analysed by plotting the number of identified OTUs against the number of sequences to determine whether the number of reads used in the analysis was sufficient for identifying species/OTUs (Kemp and Aller, 2004). The rarefaction curve was generated by OTUs at a 97% similarity cut-off level. Alpha-diversity analyses, including determination of the ACE community richness index, Shannon community diversity index and a sequencing depth index (Good's coverage), were performed using Mothur software (Schloss et al., 2011). Beta-diversity analysis was performed to examine the diversity among the three exposure groups. Unweighted UniFrac distance metric analysis was performed using OTUs for each sample, and principal coordinates analysis (PCoA) was conducted according to the distance matrix. Hierarchical clustering using Bray-Curtis distances based on the relative abundances of species was performed to cluster the data set. A hierarchical clustering heatmap analysis was performed to identify the species that accounted for the greatest amount of variation among the samples. To determine if statistically significant differences occurred in microbial populations among the 3 groups, Kruskal-Wallis analysis were completed using R software package. Differences between populations were analysed using a one-way ANOVA. P < 0.05 was considered statistically significant.
2.2. Toxicity test A total of 270 uniformly sized juvenile common carp (95 ± 1.66 g) were randomly divided into three groups. Each group was housed in three tanks, with 30 animals per tank. The fish were exposed to three nominal Cd concentrations of 0 μg L−1 (control, without Cd supplementation), 50 μg L−1 and 500 μg L−1. During the experiment, aeration was provided continuously, and one-third of the water in the tank was replaced every day by adding fresh water or water containing CdCl2·5H2O (Sigma-Aldrich, St. Louis, MO, USA) at the same concentration. The Cd levels in the water were measured every other day using atomic absorption techniques. The fish were fed two times each day, at 8:30 and 17:30. The feeding trial lasted for four weeks, as described in previous studies (Zhai et al., 2017b). 2.3. Sample collection and genomic DNA extraction The microflora present in the intestinal tracts of the common carp were determined at the end of the experiment. Six fish were selected at random from each tank and fish were euthanized in tricaine methanesulfonate (10 mg/L). The body surfaces of the sampled fish were washed with 70% ethanol, and the fish were dissected using sterile surgical scissors. Then, the intestines were removed aseptically, and the total contents were collected. Microbial DNA was extracted from 54 intestinal tract content samples using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. Extracts from the same tank were pooled together to minimize the bias in sampling and extraction. The V3-V4 region of the 16S rRNA gene was amplified with the bacteria-specific primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT), where the barcode was an eight-base sequence unique to each sample. Amplification of the 16S rRNA gene sequence was performed using an initial denaturation step of 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s and then a final extension step of 10 min at 72 °C. PCRs were performed in triplicate in a 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase and 10 ng of template DNA. The resulting PCR products were extracted from
3. Results 3.1. Statistical analysis of sequencing data After pyrosequencing, a total of 445,235 raw sequence reads were obtained from the 9 samples (Table S1). All the sequences were delineated into OTUs with 97% sequence similarity values, and the number 93
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suggesting that the diversity of the gut microbial community decreased following Cd exposure. Based on pyrosequencing data, PCoA and clustering analysis were carried out to assess β-diversity. The PCoA score plot (Fig. 3A) indicated that the groups treated with Cd were structurally different from the control group, with the main principal component (PC) scores as follows: PC1 = 51.66%, PC2 = 27.91%. In addition, a hierarchical clustering tree (Fig. 3B) revealed that microbial communities among the three exposure groups were clustered into two groups: (1) Group I included the 0 and 50 μg Cd L−1 exposure groups, and (2) Group II included the 500 μg Cd L−1 exposure group. Taken together, these results indicated that Cd exposure results in the restructuring of microbial communities. 3.3. Microbial community composition All the sequences were classified from phylum to genus based on Mothur software with the default settings, and 15 phyla and 156 genera were identified. At the phylum level, the 0 and 50 μg Cd L−1 exposure groups were dominated by three major phyla: Fusobacteria, Proteobacteria and Bacteroidetes. However, the 3 most prevalent phyla in the 500 μg Cd L−1 exposure group were Proteobacteria, Firmicutes and Bacteroidetes (Fig. 4A). The relative abundances of Cyanobacteria, Chlamydiae, Verrucomicrobia and Saccharibacteria were higher than 1% in at least one sample. The rest of phyla (Others) were also present but at lower abundances (< 1%). The relative abundances of the bacterial community at the genus level are illustrated in Fig. 4B. There were 23 genera with relative abundances higher than 1% in at least one sample, and the remaining genera were grouped as “Others”. The dominant genus in the control group was Cetobacterium, followed by Acinetobacter, Bacteroides, Aeromonas and Pseudomonas. The dominant genera in the 50 μg Cd L−1 exposure group were Cetobacterium, norank_o_Sphingobacteriales, Aeromonas, Flavobacterium and Acinetobacter. However, the 500 μg Cd L−1 exposure group was dominated by Cetobacterium, norank_f_Erysipelotrichaceae, unclassified_f_Comamonadaceae, Pseudomonas and Flavobacterium.
Fig. 1. Rarefaction analysis of the different samples. Rarefaction curves of OTUs clustered at a 97% phylotype similarity level. Horizontal axis: the number of valid sequences; vertical axis: the observed number of operational taxonomic units. Total sample richness estimates were calculated by the richness estimator Sobs. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
of OTUs from all the samples at this similarity cut-off level was 230. The rarefaction curves tended to approach the saturation plateau and indicated a large variation in the total number of OTUs in different samples. The rarefaction curves also indicated that the community richness in the 500 μg Cd L−1 exposure group was the lowest (Fig. 1). Good's coverage estimations revealed that over 99% of the species were identified in the nine samples, which demonstrated that sufficient sampling depth was obtained for all the samples (Table S1). 3.2. Microbial community diversity The α-diversity of the intestinal microbiota was estimated by the community diversity index (Shannon) and richness index (ACE) for the three groups. The ACE (Fig. 2A) index was higher in the 0 and 50 μg Cd L−1 exposure groups than in the 500 μg Cd L−1 exposure group and significantly different between the 50 μg Cd L−1 and the 500 μg Cd L−1 exposure groups (P < 0.01). Together with the rarefaction curves, these results indicated that the community richness in the 500 μg Cd L−1 exposure group was lower than that in the other groups. The Shannon index (Fig. 2B) exhibited a decreasing trend with increasing Cd concentrations and significant differences were observed between the 0 μg Cd L−1 and 500 μg Cd L−1exposure groups (P < 0.05),
3.4. Overall changes in community composition in response to Cd exposure Significant variations in the composition of the gut microbiota of common carp following Cd exposure were observed at the phylum and genus levels using Kruskal-Wallis tests. At the phylum level (Fig. 5A), no significant differences were observed in the relative abundances of Fusobacteria (P = 0.113), Proteobacteria (P = 0.561), Bacteroidetes (P = 0.113), Chlamydiae (P = 0.066), Verrucomicrobia (P = 0.051) and Cyanobacteria (P = 0.061) among the three groups. Notably, the relative abundance of Fusobacteria decreased with increasing Cd Fig. 2. Significant differences between the three groups in terms of alpha-diversity of the intestinal microbial communities in common carp. (A) Bacterial community richness (measured by the ACE index). (B) Bacterial community diversity (measured by the Shannon index). P < 0.05 was considered statistically significant. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
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Fig. 3. (A) Principal coordinates analysis of unweighted UniFrac scores of the microbial communities. Principal components (PCs) 1 and 2 explained 51.66% and 27.91% of the variance, respectively. (B) Hierarchical cluster analysis of Bray-Curtis distances generated from taxa tables summarized at the OTU level. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
were 66 times higher than that in the 0 μg Cd L−1 exposure group and 15 times higher than that in the 50 μg Cd L−1 exposure group. Moreover, the proportions of some Cd-resistant bacteria, such as Methylobacterium and Methylophilus, were found to increase following Cd exposure (Table S3). Therefore, these data demonstrated that the microbial community composition in the common carp changed significantly in response to Cd exposure. In addition, the hierarchical clustering heatmap analysis at the genus level also indicated that microbial community structures were markedly different among the three groups and highlighted the particularly abundant and non-abundant genera among the three groups (Fig. 6).
concentration. The abundance of Firmicutes in the intestinal tracts of common carp exposed to 500 μg Cd L−1 was significantly higher than that of common carp exposed to 0 or 50 μg Cd L−1 (P = 0.038). Moreover, Saccharibacteria (P = 0.024) were detected in only the 0 and 50 μg Cd L−1 exposure groups, not in the 500 μg Cd L−1 exposure group. There were 7 statistically significant differences among the three groups at the genus level (Fig. 5B). The proportions of norank_f_Erysipelotrichaceae, norank_c__Cyanobacteria, Acidovorax, unclassified_f_Comamonadaceae and Candidatus_Odyssella were significantly higher in the 500 μg Cd L−1 exposure group than in the 0 and 50 μg Cd L−1 exposure groups (P = 0.027, P = 0.027, P = 0.027, P = 0.039, P = 0.027). At the genus level, norank_o_Sphingobacteriales (P = 0.033) was detected in only the 0 and 50 μg Cd L−1 exposure groups. Moreover, the proportion of Aeromonas was significantly higher in the 0 and 50 μg Cd L−1 exposure groups than in the 500 μg Cd L−1 exposure group (P = 0.027). In addition, the proportions of some probiotics, such as A. muciniphila, were found to decrease following exposure to Cd in the intestinal tract of common carp (Table S2). The OTU numbers of A. muciniphila in the 0 μg Cd L−1 exposure group were 5 times higher than that in the 50 μg Cd L−1 exposure group, and this bacteria was not detected in the 500 μg Cd L−1 exposure group. In contrast, the proportions of some pathogens, such as Pseudomonas alcaligenes, were found to increase following Cd exposure (Table S2). The OTU numbers of P. alcaligenes in the 500 μg Cd L−1 exposure group
4. Discussion The heavy metal Cd is a hazardous pollutant that has various toxic effects on aquatic animals. Although the maximum allowable concentration of Cd in water is 10 μg L−1 in China (Huang et al., 2015), in many areas of China, Cd levels can reach as high as100–900 μg L−1 (Deng et al., 2014). Thus, Cd contamination and toxicity in aquaculture systems currently pose a significant health hazard to fishes. Although numerous studies have focused on the toxic effects of Cd on various fish organs, the effects of this metal on the gut microflora of fishes have not been thoroughly studied. The gut microbiota, a complex and dynamic community of microbes in the fish gut, is essential for host health. In recent times, the
Fig. 4. Bacterial composition of the different communities at the phylum level (A) and genus level (B). Taxa with abundances < 1% are included in “Others”. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group. 95
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Fig. 5. Kruskal-Wallis test for differences in these microbial taxa among the three exposure groups at the phylum level (A) and genus level (B). Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
freshwater teleosts (Li et al., 2015; Roeselers et al., 2011). Our results indicated that Cd exposure caused significant differences in the composition of the intestinal microbiota at the phylum level. Specifically, Saccharibacteria were not detected in the 500 μg Cd L−1 exposure group, and the abundance of Firmicutes was greatly increased in the 500 μg Cd L−1 exposure group. Moreover, the relative abundance of Fusobacteria decreased with increasing Cd concentration. The phylum Saccharibacteria (former candidate division TM7, hereafter called Saccharibacteria) is a well-described candidate phylum and has been frequently detected in various natural environments, human oral cavities, and activated sludge (Kindaichi et al., 2016; Starr et al., 2018), and these bacteria are also present in the gut of common carp. Much has been learned about the metabolism of Saccharibacteria and the effects of these bacteria on their environment, for instance, the possible immunosuppressive capabilities of the episymbiont Actinomyces odontolyticus in the human mouth (Starr et al., 2018), suggesting that Saccharibacteria may play an important role in host health. Our results revealed that the abundance of Saccharibacteria decreased in the 50 μg Cd L−1 exposure group, and these bacteria were absent in the 500 μg Cd L−1 exposure group. Thus, we speculate that the reduction and disappearance of Saccharibacteria in the common carp gut exposed to Cd may lead to health problems. Firmicutes represent a major component of the intestinal microflora of mice and humans (Ismail et al., 2011). These bacteria have been shown to be involved in energy resorption and associated with the development of diabetes and obesity (Komaroff, 2017), suggesting that the increase in Firmicutes abundance in the gut is associated with metabolic disorder in hosts. We observed that exposure to high levels of Cd increased the abundance of Firmicutes in common carp. This finding is consistent with previous results in which Cd was shown to affect the metabolism of organisms (De Mortel et al., 2008; Xu and Bai, 2007). In the present study, almost all Fusobacteria detected in the fish intestinal samples belonged to the genus Cetobacterium. In addition, Cetobacterium was the dominant group in the gut of common carp among the three groups. This result is consistent with those of previous studies that indicated Cetobacterium to be a predominant genus in the intestines of freshwater fish (Li et al., 2015). Cetobacterium has been shown to produce vitamin B12 with high efficiency and to ferment
relationship between heavy metals and the gut microbiome has been a focus of research aimed at understanding toxicity (Jin et al., 2017; Xia et al., 2018). A number of previous studies have shown that exposure to heavy metals, including Cd, Cu and Pb, can alter the composition of the gut microbiome, leading to disorders of energy metabolism, nutrient absorption, and immune system function or to other toxicity-associated symptoms (Jin et al., 2017). In the present study, our results clearly show that Cd exposure profoundly affects the diversity and composition of the gut microbiota in common carp. In this study, the community diversity index (Shannon) significantly decreased in the 500 μg Cd L−1 exposure group (P < 0.05) (Fig. 2B), suggesting that the diversity of the gut microbial community decreased with increasing in Cd concentration. Zhai et al. (2017b) also found that the gut microflora diversity of Nile tilapia decreased when the fish were exposed to Cd. The same phenomenon was reported by Mu et al. (2018), who found that Cd exposure significantly decreased the gut microflora diversity of Rana chensinensis tadpoles. A previous study demonstrated that the stability of the gut bacterial diversity plays an important role in the maintenance of host health (Jones and Lennon, 2010). Thus, the decrease in the intestinal microbial community diversity induced by Cd may have an adverse effect on common carp. In the present study, PCoA plots and a hierarchical clustering tree revealed that the microbial communities of the three exposure groups were separated into three distinct groups, suggesting that Cd exposure could affect the composition of the gut microbiota in common carp. Previous studies have also confirmed that Cd exposure leads to changes in intestinal microbial composition in other organisms (Mu et al., 2018; Zhai et al., 2017b). Previous research has shown that the occurrence of many diseases is correlated with altered gut microbial composition (Lange et al., 2016). The gut microbiota provides protection against pathogens via the production of inhibitory compounds and via competition for nutrients and space (van Kessel et al., 2011). Therefore, we assume that the changes in intestinal microbial communities caused by Cd exposure might bring health problems of common carp. The gut microbial analysis of the common carp demonstrated that the dominant bacteria at the phylum level in the control group belonged to three phyla: Fusobacteria, Proteobacteria and Bacteroidetes. This finding is consistent with the results of earlier studies on
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Fig. 6. Heatmap of the 50 most abundant genera in the bacterial communities detected from the three groups. Dendrograms of the hierarchical cluster analysis grouping of genera and sample locations are shown at the left and top, respectively. The colour scale represents the relative abundance values normalized by log10. Zero values were included as 1 and transformed by log10. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
expression of tight junction proteins, thus protecting the gut barrier and reducing gut permeability (Belzer and de Vos, 2012; Derrien et al., 2017). In the present study, the abundance of A. muciniphila was found to be decreased after Cd exposure. Previous studies also revealed that toxic metals such as Cd, lead (Pb), copper (Cu) and aluminium (Al) could decrease the abundance of A. muciniphila in mice and common carp (Meng et al., 2018; Zhai et al., 2017a). Taken together, these results suggest that the gut functional barrier had been disrupted following metal exposure. In addition, we found that the abundance of P. alcaligenes, members of which are fish pathogens (Xu et al., 2015), increased following Cd exposure, suggesting that impairment of intestinal function might have occurred and may lead to disease outbreaks in fish exposed to Cd. Metal-resistant bacteria have been reported to reduce metal availability in various environments. However, the function of these bacteria in the gut of fish has received little attention. Previous study has indicated that members of Methylobacterium and Methylophilus are highly
peptides and carbohydrates (Tsuchiya et al., 2008), and these characteristics may make Cetobacterium particularly relevant for fish nutrition. In the present study, we observed that the abundance of Cetobacterium decreased after Cd exposure. This result is consistent with the results of a previous study, which showed that Cd exposure could decrease the abundance of Cetobacterium in the Nile tilapia (Zhai et al., 2017b). Therefore, we assume that the changes in the intestinal microbial communities caused by Cd exposure may lead to changes in the intestinal absorption of nutrients in common carp. Probiotics are useful as both biological control agents against fish disease and activators of nutrient intake by the host and have been widely used in aquaculture. A. muciniphila is an intestinal anaerobe that was initially described as a novel functional microbe with probiotic properties (Gomez-Gallego et al., 2016). This bacterial genus was recently shown to be universally distributed in the intestines of humans and other mammals (Belzer and de Vos, 2012). These microbes might also enhance the repair of intestinal mucosal wounds and induce the
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resistant to As, Cd, Cr, Hg and Pb (Mishra et al., 2018). Metal resistance in these groups of organisms may be result of either increased influx of metal accompanied with transformation or efficient efflux system to remove the toxic concentration of metal out from cellular interior. In the present study, the abundance of Methylobacterium and Methylophilus were found to be increased after Cd exposure. These results revealed that Cd exposure could increase the relative abundances of Cd-resistant microorganisms. However, the role of Cd-resistant bacteria in the gut of common carp exposed to high concentration of Cd remains to be elucidated.
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Ecotoxicol. Environ. Saf. 93, 45–51. Tsuchiya, C., Sakata, T., Sugita, H., 2008. Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium in the intestinal tracts of freshwater fish. Lett. Appl. Microbiol. 46, 43–48. van Kessel, M.A.H.J., Dutilh, B.E., Neveling, K., Kwint, M.P., Veltman, J.A., Flik, G., Jetten, M.S.M., Klaren, P.H.M., Op den Camp, H.J.M., 2011. Pyrosequencing of 16S rRNA gene amplicons to study the microbiota in the gastrointestinal tract of carp (Cyprinus carpio L.). AMB Express. https://doi.org/10.1186/2191-0855-1-41. Xia, J.Z., Jin, C.Y., Pan, Z.H., Sun, L.W., Fu, Z.W., Jin, Y.X., 2018. Chronic exposure to low concentrations of lead induces metabolic disorder and dysbiosis of the gut microbiota in mice. Sci. Total Environ. 631–632, 439–448. Xu, J., Zeng, X.H., Jiang, N., Zhou, Y., Zeng, L.B., 2015. Pseudomonas alcaligenes infection and mortality in cultured Chinese sturgeon, Acipenser sinensis. Aquaculture 446, 37–41. Xu, Z.R., Bai, S.J., 2007. Effects of waterborne Cd exposure on glutathione metabolism in Nile tilapia (Oreochromis niloticus) liver. Ecotoxicol. Environ. Saf. 67, 89–94. Yang, H.L., Wang, J., Lv, Z.Y., Tian, J.X., Peng, Y.D., Peng, X.J., Xu, X., Song, Q.S., Lv, B., Chen, Z.Y., Sun, Z.Y., Wang, Z., 2018. Metatranscriptome analysis of the intestinal microorganisms in Pardosa pseudoannulata in response to cadmium stress. Ecotoxicol. Environ. Saf. 159, 1–9. Yeşilbudak, B., Erdem, C., 2014. Cadmium accumulation in gill, liver, kidney and muscle tissues of common carp, Cyprinus carpio, and nile tilapia, Oreochromis niloticus. Bull. Environ. Contam. Toxicol. 92, 546. Zhai, Q.X., Li, T.Q., Yu, L.L., Xiao, Y., Feng, S.S., Wu, J.P., Zhao, J.X., Zhang, H., Chen, W., 2017a. Effects of subchronic oral toxic metal exposure on the intestinal microbiota of mice. Sci. Bull. 62, 831–840. Zhai, Q.X., Yu, L.L., Li, T.Q., Zhu, J.M., Zhang, C.C., Zhao, J.X., Zhang, H., Chen, W., 2017b. Effect of dietary probiotic supplementation on intestinal microbiota and physiological conditions of Nile tilapia (Oreochromis niloticus) under waterborne cadmium exposure. Anton. Leeuw. Int. J. G. 110, 501–513. Zhang, M.L., Sun, Y.H., Liu, Y.K., Qiao, F., Chen, L.Q., Liu, W.T., Du, Z.Y., Li, E.C., 2016. Response of gut microbiota to salinity change in two euryhaline aquatic animals with reverse salinity preference. Aquaculture 454, 72–80. Zhang, Z., Zheng, Z., Cai, J., Liu, Q., Yang, J., Gong, Y., Wu, M., Shen, Q., Xu, S., 2017. Effect of cadmium on oxidative stress and immune function of common carp (Cyprinus carpio L.) by transcriptome analysis. Aquat. Toxicol. 192, 171–177.
5. Conclusion In conclusion, our results indicate that Cd exposure could affect gut microbial diversity and composition in common carp. However, the mechanisms that underlie the changes in the microbiota and the consequences of these changes need to be investigated in future studies. The cataloguing of 16S rRNA gene sequences is essential but not sufficient to reliably determine the functions of the identified microbial populations in response to Cd. Future investigations of microbial function that analyse the total genes in the carp gut may provide insights into the origin and functional significance of these populations under Cd expose. Acknowledgements This work was supported by the Doctoral Foundation of Henan Normal University (qd16157), Cultivation fund of Henan Normal University (2017PL16) and the National Natural Science Foundation of China (No. 31700446). Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.12.066. References Belzer, C., de Vos, W.M., 2012. Microbes inside-from diversity to function: the case of Akkermansia. ISME J. 6, 1449–1458. De Mortel, J.E.V., Schat, H., Moerland, P.D., Van Themaat, E.V.L., Van der Ent, S., Blankestijn, H., Ghandilyan, A., Tsiatsiani, S., Aarts, M.G.M., 2008. Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd-hyperaccumulator Thlaspi caerulescens. Plant Cell Environ. 31, 301–324. Deng, G., Li, M., Li, H., Yin, L.Y., Li, W., 2014. Exposure to cadmium causes declines in growth and photosynthesis in the endangered aquatic fern (Ceratopteris pteridoides). Aquat. Bot. 112, 23–32. Derrien, M., Belzer, C., de Vos, W.M., 2017. Akkermansia muciniphila and its role in regulating host functions. Microb. Pathog. 106, 171–181. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Gomez-Gallego, C., Pohl, S., Salminen, S., De Vos, W.M., Kneifel, W., 2016. Akkermansia muciniphila: a novel functional microbe with probiotic properties. Benef. Microbes 7, 571–584. Gomez, G.D., Balcazar, J.L., 2008. A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol. Med. Microbiol. 52, 145–154. Huang, M.Y., Duan, R.Y., Ji, X., 2015. The influence of long-term cadmium exposure on phonotaxis in male Pelophylax nigromaculata. Chemosphere 119, 763–768. Ismail, N.A., Ragab, S.H., Abd ElBaky, A., Shoeib, A.R.S., Alhosary, Y., Fekry, D., 2011. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal weight Egyptian children and adults. Arch. Med. Sci. 7, 501–507. Jin, Y.X., Wu, S.S., Zeng, Z.Y., Fu, Z.W., 2017. Effects of environmental pollutants on gut microbiota. Environ. Pollut. 222, 1–9. Jones, S.E., Lennon, J.T., 2010. Dormancy contributes to the maintenance of microbial diversity. Proc. Natl. Acad. Sci. USA 107, 5881–5886. Kemp, P.F., Aller, J.Y., 2004. Bacterial diversity in aquatic and other environments: what 16S rDNA libraries can tell us. FEMS Microbiol. Ecol. 47, 161–177. Kindaichi, T., Yamaoka, S., Uehara, R., Ozaki, N., Ohashi, A., Albertsen, M., Nielsen, P.H., Nielsen, J.L., 2016. Phylogenetic diversity and ecophysiology of candidate phylum Saccharibacteria in activated sludge. FEMS Microbiol. Ecol. 92.
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Effects of cadmium exposure on the composition and diversity of the intestinal microbial community of common carp (Cyprinus carpio L.) Xulu Chang, Hui Li, Junchang Feng, Yongyan Chen, Guoxing Nie, Jianxin Zhang
T
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College of Fisheries, Henan Normal University, Xinxiang 453007, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Cadmium Cyprinus carpio Gut microbiota High-throughput sequencing
Cadmium (Cd) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd, the increased accumulation of Cd in fish has become a food safety and public health concern. The present study was conducted to investigate the effects of waterborne Cd exposure on the microbial community composition and diversity in the gut of common carp. Common carp were exposed to three waterborne Cd concentrations (0, 50 and 500 μg Cd L−1) for 4 weeks. Our results indicated that Cd exposure profoundly affected the composition of the gut microbiota in the common carp. At the phylum level, Saccharibacteria were detected in only the 0 μg and 50 μg Cd L−1 exposure groups, and the abundance of Fusobacteria decreased with increasing Cd concentration, while the abundance of Firmicutes increased with increasing Cd concentration. At the genus level, Cetobacterium was the dominant group in the gut of the common carp, and the abundance of Cetobacterium decreased after Cd exposure. Notably, the abundance of Akkermansia muciniphila, a probiotic, was found to decrease after Cd exposure, and the proportions of some Cd-resistant bacteria were found to increase following Cd exposure. Our results also demonstrated that Cd exposure decreased the community diversity of the gut microbiota. These results suggest that Cd exposure may impact the gut homeostasis of common carp and further affect the health of the organism.
1. Introduction Over the past several decades, industrial progress has been accompanied by frequent incidents of metal pollution, leading to significant public health concerns. Cd, a non-essential element, is widely used in industrial activities and can be toxic and carcinogenic to humans and animals (Liu et al., 2014; Zhai et al., 2017a). With rapid industrial development and population growth, increasing contamination of aquatic systems with Cd has been reported around the world (Taweel et al., 2013; Zhai et al., 2017b). As an environmental hazard, Cd enters the aquatic environment from both natural and anthropogenic sources, including agricultural runoff, industrial effluents, and urban and mining sources (Deng et al., 2014; Rajeshkumar et al., 2017). This exposure results in pathological changes in aquatic ecosystems, mostly manifested in fish, which are affected by Cd via the respiratory and digestive systems and through the skin (Kovarova et al., 2009). Cd is associated with a wide range of toxic effects in different experimental animals, including fish (Mu et al., 2018; Zhai et al., 2017a; Zhang et al., 2017). Notably, Cd can be biologically accumulated by fish, which are major protein sources consumed by humans, resulting in ⁎
potential human health risks. Thus, the increased accumulation of Cd in fishes has become a food safety and public health concern. Although the underlying toxic effects of Cd have been well addressed in different fish organs, including liver, kidney, gut and brain (Morgano et al., 2014; Olmedo et al., 2013), the effects of Cd on the gut microbiota of fishes have received little attention. The fish gut microbiota is known to play a significant role in a number of physiological functions, including immune regulation, mineral metabolism maturation and pathogen resistance (Gomez and Balcazar, 2008). Moreover, imbalance of the gut microbiota may mediate the development of numerous diseases in fishes (Perez et al., 2010). The composition of gut microbial communities in fishes is affected by exogenous and endogenous factors, including developmental stage, diet composition, habitat and surrounding environment (e.g., water temperature and salinity), rearing conditions and possibly phylogenetic position (Sullam et al., 2012; Zhang et al., 2016). Cd exposure has been reported to significantly affect the gut microbiota of many organisms (Liu et al., 2014; Mu et al., 2018; Yang et al., 2018; Zhai et al., 2017b). However, the effects of Cd on the intestinal gut of common carp remain unclear. The common carp, Cyprinus carpio L. (C. carpio), is the most widely farmed freshwater fish in China. It was reported that C. carpio is
Corresponding author. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.ecoenv.2018.12.066 Received 14 September 2018; Received in revised form 19 December 2018; Accepted 21 December 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer's protocol.
resistant to highly polluted habitats and is used as a bio-indicator species to understand the effects of environmental pollution (Yeşilbudak and Erdem, 2014). The aim of this study was to investigate the influence of Cd on the gut microbiota composition and diversity in common carp. To achieve this goal, the gut bacterial composition was investigated using high-throughput sequencing of the V3-V4 segment of the bacterial 16S rRNA gene. We hope that the results will provide insight into the toxic effects of pollution on common carp from a new perspective.
2.4. Sequencing and data processing Purified amplicons were pooled in equimolar amounts and subjected to paired-end sequencing (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to standard protocols by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Raw reads were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) toolkit (version 1.17). The reads were removed based on the following criteria: reads with average quality scores lower than 25; ambiguous bases; homopolymers of > 7 bases; reads containing primer mismatches; and reads with lengths shorter than 100 bp. For paired-end reads, only sequences that overlapped by more than 10 bp and lacked mismatches were assembled. Reads that could not be assembled were removed. Barcodes and sequencing primers were trimmed from the assembled sequences. The raw reads were deposited into the NCBI short read archive database (accession number: SRP159034).
2. Materials and methods 2.1. Experimental fish Juvenile common carp were obtained from an aquaculture farm in Zhengzhou City, Henan Province, China. After being removed from the water, the animals were randomly placed in tanks (300 L in water volume) supplied with continuous aeration and equipped with temperature-control devices. The juvenile common carp were acclimated for two weeks before being used in experiments. Commercial food (containing 19% casein, 20% fish meal, 20% soybean meal, 5% cottonseed meal, 20% flour, 3% soybean oil, 10% CMC-Na, 0.2% methylcellulose, 0.2% salt, 1.5% CaH2PO3, 0.1% vitamin mixture and 1% mineral mixture) was provided once a day until satiation. No mortality was observed during the acclimatization, and the tank water was partially changed every day.
2.5. Statistical analysis To estimate the bacterial community composition in the intestinal tract of the common carp, we performed an operational taxonomic unit (OTU) analysis. Sets of sequences with at least 97% identity were defined as an OTU, and chimeric sequences were identified and removed using UCHIME (Edgar et al., 2011). The sequence difference cut-off for OTUs was defined at 97%, in accordance with the currently accepted standards for prokaryotic species (Rossello-Mora and Amann, 2001). The number of OTUs in each sample and the number of sequences in each OTU were counted after the OTU output was obtained. Representative OTUs were selected for annotation of taxonomic information by the SILVA ribosomal RNA gene database using a confidence threshold of 70% (Quast et al., 2013). Additionally, rarefaction curves were analysed by plotting the number of identified OTUs against the number of sequences to determine whether the number of reads used in the analysis was sufficient for identifying species/OTUs (Kemp and Aller, 2004). The rarefaction curve was generated by OTUs at a 97% similarity cut-off level. Alpha-diversity analyses, including determination of the ACE community richness index, Shannon community diversity index and a sequencing depth index (Good's coverage), were performed using Mothur software (Schloss et al., 2011). Beta-diversity analysis was performed to examine the diversity among the three exposure groups. Unweighted UniFrac distance metric analysis was performed using OTUs for each sample, and principal coordinates analysis (PCoA) was conducted according to the distance matrix. Hierarchical clustering using Bray-Curtis distances based on the relative abundances of species was performed to cluster the data set. A hierarchical clustering heatmap analysis was performed to identify the species that accounted for the greatest amount of variation among the samples. To determine if statistically significant differences occurred in microbial populations among the 3 groups, Kruskal-Wallis analysis were completed using R software package. Differences between populations were analysed using a one-way ANOVA. P < 0.05 was considered statistically significant.
2.2. Toxicity test A total of 270 uniformly sized juvenile common carp (95 ± 1.66 g) were randomly divided into three groups. Each group was housed in three tanks, with 30 animals per tank. The fish were exposed to three nominal Cd concentrations of 0 μg L−1 (control, without Cd supplementation), 50 μg L−1 and 500 μg L−1. During the experiment, aeration was provided continuously, and one-third of the water in the tank was replaced every day by adding fresh water or water containing CdCl2·5H2O (Sigma-Aldrich, St. Louis, MO, USA) at the same concentration. The Cd levels in the water were measured every other day using atomic absorption techniques. The fish were fed two times each day, at 8:30 and 17:30. The feeding trial lasted for four weeks, as described in previous studies (Zhai et al., 2017b). 2.3. Sample collection and genomic DNA extraction The microflora present in the intestinal tracts of the common carp were determined at the end of the experiment. Six fish were selected at random from each tank and fish were euthanized in tricaine methanesulfonate (10 mg/L). The body surfaces of the sampled fish were washed with 70% ethanol, and the fish were dissected using sterile surgical scissors. Then, the intestines were removed aseptically, and the total contents were collected. Microbial DNA was extracted from 54 intestinal tract content samples using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions. Extracts from the same tank were pooled together to minimize the bias in sampling and extraction. The V3-V4 region of the 16S rRNA gene was amplified with the bacteria-specific primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT), where the barcode was an eight-base sequence unique to each sample. Amplification of the 16S rRNA gene sequence was performed using an initial denaturation step of 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s and then a final extension step of 10 min at 72 °C. PCRs were performed in triplicate in a 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase and 10 ng of template DNA. The resulting PCR products were extracted from
3. Results 3.1. Statistical analysis of sequencing data After pyrosequencing, a total of 445,235 raw sequence reads were obtained from the 9 samples (Table S1). All the sequences were delineated into OTUs with 97% sequence similarity values, and the number 93
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suggesting that the diversity of the gut microbial community decreased following Cd exposure. Based on pyrosequencing data, PCoA and clustering analysis were carried out to assess β-diversity. The PCoA score plot (Fig. 3A) indicated that the groups treated with Cd were structurally different from the control group, with the main principal component (PC) scores as follows: PC1 = 51.66%, PC2 = 27.91%. In addition, a hierarchical clustering tree (Fig. 3B) revealed that microbial communities among the three exposure groups were clustered into two groups: (1) Group I included the 0 and 50 μg Cd L−1 exposure groups, and (2) Group II included the 500 μg Cd L−1 exposure group. Taken together, these results indicated that Cd exposure results in the restructuring of microbial communities. 3.3. Microbial community composition All the sequences were classified from phylum to genus based on Mothur software with the default settings, and 15 phyla and 156 genera were identified. At the phylum level, the 0 and 50 μg Cd L−1 exposure groups were dominated by three major phyla: Fusobacteria, Proteobacteria and Bacteroidetes. However, the 3 most prevalent phyla in the 500 μg Cd L−1 exposure group were Proteobacteria, Firmicutes and Bacteroidetes (Fig. 4A). The relative abundances of Cyanobacteria, Chlamydiae, Verrucomicrobia and Saccharibacteria were higher than 1% in at least one sample. The rest of phyla (Others) were also present but at lower abundances (< 1%). The relative abundances of the bacterial community at the genus level are illustrated in Fig. 4B. There were 23 genera with relative abundances higher than 1% in at least one sample, and the remaining genera were grouped as “Others”. The dominant genus in the control group was Cetobacterium, followed by Acinetobacter, Bacteroides, Aeromonas and Pseudomonas. The dominant genera in the 50 μg Cd L−1 exposure group were Cetobacterium, norank_o_Sphingobacteriales, Aeromonas, Flavobacterium and Acinetobacter. However, the 500 μg Cd L−1 exposure group was dominated by Cetobacterium, norank_f_Erysipelotrichaceae, unclassified_f_Comamonadaceae, Pseudomonas and Flavobacterium.
Fig. 1. Rarefaction analysis of the different samples. Rarefaction curves of OTUs clustered at a 97% phylotype similarity level. Horizontal axis: the number of valid sequences; vertical axis: the observed number of operational taxonomic units. Total sample richness estimates were calculated by the richness estimator Sobs. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
of OTUs from all the samples at this similarity cut-off level was 230. The rarefaction curves tended to approach the saturation plateau and indicated a large variation in the total number of OTUs in different samples. The rarefaction curves also indicated that the community richness in the 500 μg Cd L−1 exposure group was the lowest (Fig. 1). Good's coverage estimations revealed that over 99% of the species were identified in the nine samples, which demonstrated that sufficient sampling depth was obtained for all the samples (Table S1). 3.2. Microbial community diversity The α-diversity of the intestinal microbiota was estimated by the community diversity index (Shannon) and richness index (ACE) for the three groups. The ACE (Fig. 2A) index was higher in the 0 and 50 μg Cd L−1 exposure groups than in the 500 μg Cd L−1 exposure group and significantly different between the 50 μg Cd L−1 and the 500 μg Cd L−1 exposure groups (P < 0.01). Together with the rarefaction curves, these results indicated that the community richness in the 500 μg Cd L−1 exposure group was lower than that in the other groups. The Shannon index (Fig. 2B) exhibited a decreasing trend with increasing Cd concentrations and significant differences were observed between the 0 μg Cd L−1 and 500 μg Cd L−1exposure groups (P < 0.05),
3.4. Overall changes in community composition in response to Cd exposure Significant variations in the composition of the gut microbiota of common carp following Cd exposure were observed at the phylum and genus levels using Kruskal-Wallis tests. At the phylum level (Fig. 5A), no significant differences were observed in the relative abundances of Fusobacteria (P = 0.113), Proteobacteria (P = 0.561), Bacteroidetes (P = 0.113), Chlamydiae (P = 0.066), Verrucomicrobia (P = 0.051) and Cyanobacteria (P = 0.061) among the three groups. Notably, the relative abundance of Fusobacteria decreased with increasing Cd Fig. 2. Significant differences between the three groups in terms of alpha-diversity of the intestinal microbial communities in common carp. (A) Bacterial community richness (measured by the ACE index). (B) Bacterial community diversity (measured by the Shannon index). P < 0.05 was considered statistically significant. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
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Fig. 3. (A) Principal coordinates analysis of unweighted UniFrac scores of the microbial communities. Principal components (PCs) 1 and 2 explained 51.66% and 27.91% of the variance, respectively. (B) Hierarchical cluster analysis of Bray-Curtis distances generated from taxa tables summarized at the OTU level. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
were 66 times higher than that in the 0 μg Cd L−1 exposure group and 15 times higher than that in the 50 μg Cd L−1 exposure group. Moreover, the proportions of some Cd-resistant bacteria, such as Methylobacterium and Methylophilus, were found to increase following Cd exposure (Table S3). Therefore, these data demonstrated that the microbial community composition in the common carp changed significantly in response to Cd exposure. In addition, the hierarchical clustering heatmap analysis at the genus level also indicated that microbial community structures were markedly different among the three groups and highlighted the particularly abundant and non-abundant genera among the three groups (Fig. 6).
concentration. The abundance of Firmicutes in the intestinal tracts of common carp exposed to 500 μg Cd L−1 was significantly higher than that of common carp exposed to 0 or 50 μg Cd L−1 (P = 0.038). Moreover, Saccharibacteria (P = 0.024) were detected in only the 0 and 50 μg Cd L−1 exposure groups, not in the 500 μg Cd L−1 exposure group. There were 7 statistically significant differences among the three groups at the genus level (Fig. 5B). The proportions of norank_f_Erysipelotrichaceae, norank_c__Cyanobacteria, Acidovorax, unclassified_f_Comamonadaceae and Candidatus_Odyssella were significantly higher in the 500 μg Cd L−1 exposure group than in the 0 and 50 μg Cd L−1 exposure groups (P = 0.027, P = 0.027, P = 0.027, P = 0.039, P = 0.027). At the genus level, norank_o_Sphingobacteriales (P = 0.033) was detected in only the 0 and 50 μg Cd L−1 exposure groups. Moreover, the proportion of Aeromonas was significantly higher in the 0 and 50 μg Cd L−1 exposure groups than in the 500 μg Cd L−1 exposure group (P = 0.027). In addition, the proportions of some probiotics, such as A. muciniphila, were found to decrease following exposure to Cd in the intestinal tract of common carp (Table S2). The OTU numbers of A. muciniphila in the 0 μg Cd L−1 exposure group were 5 times higher than that in the 50 μg Cd L−1 exposure group, and this bacteria was not detected in the 500 μg Cd L−1 exposure group. In contrast, the proportions of some pathogens, such as Pseudomonas alcaligenes, were found to increase following Cd exposure (Table S2). The OTU numbers of P. alcaligenes in the 500 μg Cd L−1 exposure group
4. Discussion The heavy metal Cd is a hazardous pollutant that has various toxic effects on aquatic animals. Although the maximum allowable concentration of Cd in water is 10 μg L−1 in China (Huang et al., 2015), in many areas of China, Cd levels can reach as high as100–900 μg L−1 (Deng et al., 2014). Thus, Cd contamination and toxicity in aquaculture systems currently pose a significant health hazard to fishes. Although numerous studies have focused on the toxic effects of Cd on various fish organs, the effects of this metal on the gut microflora of fishes have not been thoroughly studied. The gut microbiota, a complex and dynamic community of microbes in the fish gut, is essential for host health. In recent times, the
Fig. 4. Bacterial composition of the different communities at the phylum level (A) and genus level (B). Taxa with abundances < 1% are included in “Others”. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group. 95
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Fig. 5. Kruskal-Wallis test for differences in these microbial taxa among the three exposure groups at the phylum level (A) and genus level (B). Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
freshwater teleosts (Li et al., 2015; Roeselers et al., 2011). Our results indicated that Cd exposure caused significant differences in the composition of the intestinal microbiota at the phylum level. Specifically, Saccharibacteria were not detected in the 500 μg Cd L−1 exposure group, and the abundance of Firmicutes was greatly increased in the 500 μg Cd L−1 exposure group. Moreover, the relative abundance of Fusobacteria decreased with increasing Cd concentration. The phylum Saccharibacteria (former candidate division TM7, hereafter called Saccharibacteria) is a well-described candidate phylum and has been frequently detected in various natural environments, human oral cavities, and activated sludge (Kindaichi et al., 2016; Starr et al., 2018), and these bacteria are also present in the gut of common carp. Much has been learned about the metabolism of Saccharibacteria and the effects of these bacteria on their environment, for instance, the possible immunosuppressive capabilities of the episymbiont Actinomyces odontolyticus in the human mouth (Starr et al., 2018), suggesting that Saccharibacteria may play an important role in host health. Our results revealed that the abundance of Saccharibacteria decreased in the 50 μg Cd L−1 exposure group, and these bacteria were absent in the 500 μg Cd L−1 exposure group. Thus, we speculate that the reduction and disappearance of Saccharibacteria in the common carp gut exposed to Cd may lead to health problems. Firmicutes represent a major component of the intestinal microflora of mice and humans (Ismail et al., 2011). These bacteria have been shown to be involved in energy resorption and associated with the development of diabetes and obesity (Komaroff, 2017), suggesting that the increase in Firmicutes abundance in the gut is associated with metabolic disorder in hosts. We observed that exposure to high levels of Cd increased the abundance of Firmicutes in common carp. This finding is consistent with previous results in which Cd was shown to affect the metabolism of organisms (De Mortel et al., 2008; Xu and Bai, 2007). In the present study, almost all Fusobacteria detected in the fish intestinal samples belonged to the genus Cetobacterium. In addition, Cetobacterium was the dominant group in the gut of common carp among the three groups. This result is consistent with those of previous studies that indicated Cetobacterium to be a predominant genus in the intestines of freshwater fish (Li et al., 2015). Cetobacterium has been shown to produce vitamin B12 with high efficiency and to ferment
relationship between heavy metals and the gut microbiome has been a focus of research aimed at understanding toxicity (Jin et al., 2017; Xia et al., 2018). A number of previous studies have shown that exposure to heavy metals, including Cd, Cu and Pb, can alter the composition of the gut microbiome, leading to disorders of energy metabolism, nutrient absorption, and immune system function or to other toxicity-associated symptoms (Jin et al., 2017). In the present study, our results clearly show that Cd exposure profoundly affects the diversity and composition of the gut microbiota in common carp. In this study, the community diversity index (Shannon) significantly decreased in the 500 μg Cd L−1 exposure group (P < 0.05) (Fig. 2B), suggesting that the diversity of the gut microbial community decreased with increasing in Cd concentration. Zhai et al. (2017b) also found that the gut microflora diversity of Nile tilapia decreased when the fish were exposed to Cd. The same phenomenon was reported by Mu et al. (2018), who found that Cd exposure significantly decreased the gut microflora diversity of Rana chensinensis tadpoles. A previous study demonstrated that the stability of the gut bacterial diversity plays an important role in the maintenance of host health (Jones and Lennon, 2010). Thus, the decrease in the intestinal microbial community diversity induced by Cd may have an adverse effect on common carp. In the present study, PCoA plots and a hierarchical clustering tree revealed that the microbial communities of the three exposure groups were separated into three distinct groups, suggesting that Cd exposure could affect the composition of the gut microbiota in common carp. Previous studies have also confirmed that Cd exposure leads to changes in intestinal microbial composition in other organisms (Mu et al., 2018; Zhai et al., 2017b). Previous research has shown that the occurrence of many diseases is correlated with altered gut microbial composition (Lange et al., 2016). The gut microbiota provides protection against pathogens via the production of inhibitory compounds and via competition for nutrients and space (van Kessel et al., 2011). Therefore, we assume that the changes in intestinal microbial communities caused by Cd exposure might bring health problems of common carp. The gut microbial analysis of the common carp demonstrated that the dominant bacteria at the phylum level in the control group belonged to three phyla: Fusobacteria, Proteobacteria and Bacteroidetes. This finding is consistent with the results of earlier studies on
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Fig. 6. Heatmap of the 50 most abundant genera in the bacterial communities detected from the three groups. Dendrograms of the hierarchical cluster analysis grouping of genera and sample locations are shown at the left and top, respectively. The colour scale represents the relative abundance values normalized by log10. Zero values were included as 1 and transformed by log10. Control: 0 μg Cd L−1 exposure group; S2: 50 μg Cd L−1 exposure group; S3: 500 μg Cd L−1 exposure group.
expression of tight junction proteins, thus protecting the gut barrier and reducing gut permeability (Belzer and de Vos, 2012; Derrien et al., 2017). In the present study, the abundance of A. muciniphila was found to be decreased after Cd exposure. Previous studies also revealed that toxic metals such as Cd, lead (Pb), copper (Cu) and aluminium (Al) could decrease the abundance of A. muciniphila in mice and common carp (Meng et al., 2018; Zhai et al., 2017a). Taken together, these results suggest that the gut functional barrier had been disrupted following metal exposure. In addition, we found that the abundance of P. alcaligenes, members of which are fish pathogens (Xu et al., 2015), increased following Cd exposure, suggesting that impairment of intestinal function might have occurred and may lead to disease outbreaks in fish exposed to Cd. Metal-resistant bacteria have been reported to reduce metal availability in various environments. However, the function of these bacteria in the gut of fish has received little attention. Previous study has indicated that members of Methylobacterium and Methylophilus are highly
peptides and carbohydrates (Tsuchiya et al., 2008), and these characteristics may make Cetobacterium particularly relevant for fish nutrition. In the present study, we observed that the abundance of Cetobacterium decreased after Cd exposure. This result is consistent with the results of a previous study, which showed that Cd exposure could decrease the abundance of Cetobacterium in the Nile tilapia (Zhai et al., 2017b). Therefore, we assume that the changes in the intestinal microbial communities caused by Cd exposure may lead to changes in the intestinal absorption of nutrients in common carp. Probiotics are useful as both biological control agents against fish disease and activators of nutrient intake by the host and have been widely used in aquaculture. A. muciniphila is an intestinal anaerobe that was initially described as a novel functional microbe with probiotic properties (Gomez-Gallego et al., 2016). This bacterial genus was recently shown to be universally distributed in the intestines of humans and other mammals (Belzer and de Vos, 2012). These microbes might also enhance the repair of intestinal mucosal wounds and induce the
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resistant to As, Cd, Cr, Hg and Pb (Mishra et al., 2018). Metal resistance in these groups of organisms may be result of either increased influx of metal accompanied with transformation or efficient efflux system to remove the toxic concentration of metal out from cellular interior. In the present study, the abundance of Methylobacterium and Methylophilus were found to be increased after Cd exposure. These results revealed that Cd exposure could increase the relative abundances of Cd-resistant microorganisms. However, the role of Cd-resistant bacteria in the gut of common carp exposed to high concentration of Cd remains to be elucidated.
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5. Conclusion In conclusion, our results indicate that Cd exposure could affect gut microbial diversity and composition in common carp. However, the mechanisms that underlie the changes in the microbiota and the consequences of these changes need to be investigated in future studies. The cataloguing of 16S rRNA gene sequences is essential but not sufficient to reliably determine the functions of the identified microbial populations in response to Cd. Future investigations of microbial function that analyse the total genes in the carp gut may provide insights into the origin and functional significance of these populations under Cd expose. Acknowledgements This work was supported by the Doctoral Foundation of Henan Normal University (qd16157), Cultivation fund of Henan Normal University (2017PL16) and the National Natural Science Foundation of China (No. 31700446). Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.12.066. References Belzer, C., de Vos, W.M., 2012. Microbes inside-from diversity to function: the case of Akkermansia. ISME J. 6, 1449–1458. De Mortel, J.E.V., Schat, H., Moerland, P.D., Van Themaat, E.V.L., Van der Ent, S., Blankestijn, H., Ghandilyan, A., Tsiatsiani, S., Aarts, M.G.M., 2008. Expression differences for genes involved in lignin, glutathione and sulphate metabolism in response to cadmium in Arabidopsis thaliana and the related Zn/Cd-hyperaccumulator Thlaspi caerulescens. Plant Cell Environ. 31, 301–324. Deng, G., Li, M., Li, H., Yin, L.Y., Li, W., 2014. Exposure to cadmium causes declines in growth and photosynthesis in the endangered aquatic fern (Ceratopteris pteridoides). Aquat. Bot. 112, 23–32. Derrien, M., Belzer, C., de Vos, W.M., 2017. Akkermansia muciniphila and its role in regulating host functions. Microb. Pathog. 106, 171–181. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Gomez-Gallego, C., Pohl, S., Salminen, S., De Vos, W.M., Kneifel, W., 2016. Akkermansia muciniphila: a novel functional microbe with probiotic properties. Benef. Microbes 7, 571–584. Gomez, G.D., Balcazar, J.L., 2008. A review on the interactions between gut microbiota and innate immunity of fish. FEMS Immunol. Med. Microbiol. 52, 145–154. Huang, M.Y., Duan, R.Y., Ji, X., 2015. The influence of long-term cadmium exposure on phonotaxis in male Pelophylax nigromaculata. Chemosphere 119, 763–768. Ismail, N.A., Ragab, S.H., Abd ElBaky, A., Shoeib, A.R.S., Alhosary, Y., Fekry, D., 2011. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal weight Egyptian children and adults. Arch. Med. Sci. 7, 501–507. Jin, Y.X., Wu, S.S., Zeng, Z.Y., Fu, Z.W., 2017. Effects of environmental pollutants on gut microbiota. Environ. Pollut. 222, 1–9. Jones, S.E., Lennon, J.T., 2010. Dormancy contributes to the maintenance of microbial diversity. Proc. Natl. Acad. Sci. USA 107, 5881–5886. Kemp, P.F., Aller, J.Y., 2004. Bacterial diversity in aquatic and other environments: what 16S rDNA libraries can tell us. FEMS Microbiol. Ecol. 47, 161–177. Kindaichi, T., Yamaoka, S., Uehara, R., Ozaki, N., Ohashi, A., Albertsen, M., Nielsen, P.H., Nielsen, J.L., 2016. Phylogenetic diversity and ecophysiology of candidate phylum Saccharibacteria in activated sludge. FEMS Microbiol. Ecol. 92.
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