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Sep 17, 2015 - ABSTRACT: Goldfish (Carassius auratus) were exposed to 0−100 μg/L pentachlorophenol (PCP) for 28 days to investigate the correlation...
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Correlations of Gut Microbial Community Shift with Hepatic Damage and Growth Inhibition of Carassius auratus Induced by Pentachlorophenol Exposure Haifeng Kan, Fuzheng Zhao, Xu-Xiang Zhang,* Hongqiang Ren, and Shixiang Gao* State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China S Supporting Information *

ABSTRACT: Goldfish (Carassius auratus) were exposed to 0−100 μg/L pentachlorophenol (PCP) for 28 days to investigate the correlations of fish gut microbial community shift with the induced toxicological effects. PCP exposure caused accumulation of PCP in the fish intestinal tract in a time- and dose-dependent manner, while hepatic PCP reached the maximal level after a 21 day exposure. Under the relatively higher PCP stress, the fish body weight and liver weight were reduced and hepatic CAT and SOD activities were inhibited, demonstrating negative correlations with the PCP levels in liver and gut content (R < −0.5 and P < 0.05 each). Pyrosequencing of the 16S rRNA gene indicated that PCP exposure increased the abundance of Bacteroidetes in the fish gut. Within the Bacteroidetes phylum, the Bacteroides genus had the highest abundance, which was significantly correlated with PCP exposure dosage and duration (R > 0.5 and P < 0.05 each). Bioinformatic analysis revealed that Bacteroides showed quantitatively negative correlations with Chryseobacterium, Microbacterium, Arthrobacter, and Legionella in the fish gut, and the Bacteroidetes abundance, Bacteroides abundance, and Firmicutes/Bacteroidetes ratio played crucial roles in the reduction of body weight and liver weight under PCP stress. The results may extend our knowledge regarding the roles of gut microbiota in ecotoxicology.

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INTRODUCTION Pentachlorophenol (PCP) has been widely used as a herbicide in the world for fighting against schistosomiasis.1 Approximately 5.5 million kg of PCP has been sprayed over central China as a molluscicide since the 1960s, resulting in heavy PCP contamination in water environments, including the Yangtze River (0.7−22 ng/L) and Dongting Lake (0.005−71.3 mg/L).2 Previous studies have shown that PCP at 1%) abundance, physical indices (body length, body weight, liver weight, hepatic CAT, SOD, and protein concentration), and enrichment of PCP in liver tissue and intestine content samples. Principal component

explore the correlations of gut microbial community with liver and intestinal tract PCP levels, and with the induced hepatic damage and growth inhibition. The results may help us to understand the underlying relationship between the intestinal flora change and aquatic toxicities.

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MATERIALS AND METHODS Animal Treatment. Juvenile goldfish (C. auratus) used in this study were obtained from the Fisheries Research Institute of Nanjing (Nanjing, China). A total of 150 fish were acclimated in three l50 L tanks (50 fish each) containing dechlorinated and aerated water (pH 7.8 ± 0.5) at 25 ± 1 °C (12:12 h light/dark photoperiod; dissolved oxygen, 6.4 ± 0.4 mg L−1) for at least 7 days before being exposed to PCP. We started the exposure experiments when the total mortality of the fish was 0.5 or 1% in any

analysis (PCA) was conducted according to the matrix of distance using the PAST software. The community-wide species co-occurrence patterns were tested with the checker11896

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Environmental Science & Technology sample) occurred once but on different sites.32 Details of the community-wide species co-occurrence patterns analysis methods are shown in the Methods of the Supporting Information.

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RESULTS PCP Bioaccumulation. Chemical analysis indicated that the concentrations of residual PCP in the fish liver showed increasing trends after PCP exposure and reached the maximal level (6.52, 14.30, and 19.34 μg/g in 10, 50, and 100 μg/L PCP treatment groups, respectively) after a 21 day exposure (Figure 1). The concentrations of PCP in the fish intestinal tract demonstrated temporally continuous enhancement in each treatment group, reaching 38.02 μg/g in the 100 μg/L PCP treatment group at 28 days (Figure 1). However, PCP was not detectable in the liver and intestinal tract of the control group at any time. Fish Performance, Oxidative Stress, and Histopathological Damage. After a 28 day exposure to PCP at different doses, the fish showed no death and no significant changes in body length (Table S1). However, after exposure for >14 days, the body weight and liver weight in all treatment groups were significantly reduced compared to those of the control group (P < 0.05) (Table S1). Short-term exposure to PCP at smaller doses seemed to increase CAT activity in fish livers, but the enzyme activity was strongly inhibited when the exposure dose reached 100 μg/L or the exposure time lasted for >14 days (Figure 1). Similarly, hepatic SOD activity was inhibited when the fish were exposed to >50 μg/L PCP for >14 days (Figure 1). Histopathological observation showed that a 14 day exposure to PCP at 10 μg/L induced multifocal inflammation, cell swelling, and cytoplasmic vacuolization in livers. Focal necrosis, a very severe and irreversible type of damage, appeared in high frequency in the 100 μg/L exposure group (Figure S1). PCP exposure was also found to induce aneurisms, edema, and epithelial lifting in gill (Figure S1). Effect of PCP on the Fish Gut Microbial Community. Pyrosequencing of the 16S rRNA gene showed that PCP exposure obviously induced the gut microbial community shift. Gut bacterial diversity demonstrated great variation among the treatment groups at different exposure time points (Table S2), which is supported by the rarefaction curves (Figure S2). Compared with the control, the fish exposed to PCP had a higher level of Bacteroidetes phylum (P < 0.05) and a lower level of Firmicutes phylum (P > 0.05) in their intestinal tracts (Figures S3 and S5). Within the Bacteroidetes phylum, the Bacteroides genus had the highest abundance, demonstrating increasing trends along with the enhancement of PCP exposure dosage and duration (Figure S4). A PCA score plot revealed that both exposure dosage and duration posed obvious effects on the gut microbial community structure of the fish (Figure 2 and Table S3), which is also supported by Venn diagrams demonstrating that the four groups shared a small proportion of common genera after PCP exposure (Figure S6). The occupancy−abundance plot revealed 12 genera of the core gut microbiaotia, and PCP exposure posed no obvious effects on the occurrence of the core microbiaotia but greatly affected their abundance (Figure 3). Correlation of Liver and Intestinal Tract PCP with Growth Performance, Enzyme Activities, and Gut Microbiota. Statistical analysis showed that liver and intestinal tract PCP levels were negatively correlated with liver weight, body weight, and hepatic SOD and CAT levels (R < −0.5 and P

Figure 3. Occupancy−abundance plot of each genus in the 4 CK (xaxis) and 12 PCP (y-axis) intestinal content samples of goldfish (C. auratus), which occurred at >1% in any sample (listed on the right). Each bacterial genus represented by an average relative abundance is illustrated in the occupancy−abundance plot to split the set of genera into one arbitrarily defined ecological category: generalists (12 genera) widely distributed in both >60% PCP samples (8 samples) and >60% control samples (3 samples). The size of circles was proportional to the average relative abundance of the corresponding genus, with those overlapped inner circles shown. CK indicates the control check (nonPCP treatment). The fish were exposed to 0, 10, 50, and 100 μg/L PCP for 7, 14, 21, and 28 days.

< 0.05 each) (Figure 5). The body weight and liver weight were negatively correlated with the relative abundance of Bacteroidetes phylum and Bacteroides genus (R < −0.5 and P < 0.05 each) but were positively correlated with the Firmicutes/ Bacteroidetes (F/B) ratio (R > 0.5 and P < 0.01) (Figures 4 and 5a). The whole gut bacterial network analysis revealed a total of 78 pairs of significant and robust correlations identified from 23 bacterial genera (Figure 6), with more strong positive correlations observed than negative ones (Figure 5c). The abundance of Chryseobacterium, Microbacterium, Arthrobacter, and Legionella was negatively correlated with Bacteroides abundance in the fish gut (Figure 6b). The three major modules observed in the interaction network (Figure 6c) revealed that Microbacterium, Arthrobacter, and Legionella belonged to module III, and Chryseobacterium, a genus within the Bacteroidetes phylum, is a crucial node between module I and module III. Each genus within modules I and III showed positive correlation with at least one of the four genera negatively correlated with Bacteroides (Figure 6b).

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DISCUSSION This study showed that a 28 day PCP exposure at 10−100 μg/ L caused no fish death but reduced the body weight and liver weight and induced tissue damage of liver and gill. Previous studies have indicated that the lethal concentrations of PCP were up to 100 μg/g in goldfish bodies.21 However, this study indicated that the highest concentrations of PCP were ∼10 μg/ g in liver after a 28 day exposure to 100 μg/L PCP, which may account for the observation of no fish death. A number of studies have reported the direct toxicities of low-dose PCP 11897

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Figure 4. Relative abundance of Bacteroidetes and Bacteroides, Firmicutes/Bacteroidetes (F/B), and body weight in the fish intestinal content after exposure for different periods (a) and correlations of body weight with relative abundance of Bacteroidetes, Bacteroides, and F/B (b). CK indicates the control check (non-PCP treatment). The fish were exposed to 0, 10, 50, and 100 μg/L PCP for 7, 14, 21, and 28 days.

Figure 5. Correlations (Pearson correlation analysis) of genera (>1%) with physical indices (body length, body weight, liver weight, CAT, SOD, and protein concentration of liver tissue), enrichment of PCP in liver tissue, and intestine content samples (a), within physical indices and hepatic and intestinal tract PCP levels (b), and within the major genera in the intestinal tract (c) (stars, R > 0.5 or R < −0.5; P < 0.05).

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Figure 6. Network analysis of co-occurring bacteria for the intestinal content samples of goldfish (C. auratus) under PCP stress. A connection stands for a strong (R > 0.5) and significant (P < 0.05) correlation. For each panel, the size of each node is proportional to the number of connections (i.e., degree); the thickness of each connection between two nodes (i.e., edge) is proportional to the value of Pearson’s correlation coefficients ranging from 0.5 to 1. The co-occurrence generalists are shown in a black box: (a) network colored by phylum, (b) network colored by negatively correlated Bacteriodes or Cetobacterium, and (c) network colored by modularity class. The positive network of gut bacteria shows 23 nodes (genera) and 78 edges (average degree of 7.91). For the gut positive network, the average path length between nodes was 1.46 edges with a network diameter (the longest distance between nodes) of 3 edges, with a high clustering coefficient of 0.40. The average clustering coefficient (indicating the number of nodes embedded in their neighborhood and the degree to which they tend to cluster together) was 0.86, and the modularity index was 0.45.

of PCP in the intestinal tract and its effect on fish health is currently limited. This study revealed that intestinal tract was an important reservoir of PCP in fish bodies, and similar phenomena were also observed for polycyclic aromatic hydrocarbons40 and dibromodiphenyl ethers.41 The accumulation of PCP in intestinal tract did not affect the pH of the fish gut environment (Figure S7) but was found to induce gut microbial community shift, which was similar to effects of polychlorinated biphenyls,10,42 organochlorine pesticides,43 antibiotics,44 and insecticide chlorpyrifos.45 To the best of our knowledge, this is the first study revealing that PCP exposure induced the enhancement of Bacteroidetes and Bacteroides abundance and reduction of the F/B ratio in the fish gut. Different from PCP, cyclophosphamide,46 hexachlorobenzene,47 and subtherapeutic antibiotics44 can reduce gut microbial diversity and lead to obesity by decreasing the proportion of Bacteroidetes while increasing the proportion of Firmicutes in the microbial community.48,49 The capacity of

exposure on fish, including oxidative stress and damage,33,34 endocrine system disruption,35 and immunotoxicity.36 This study showed that the PCP exposure altered hepatic SOD and CAT activities, which was in accordance with a previous study indicating hydroxyl radical generation and antioxidant system disturbance in goldfish exposed to PCP.3 The hepatic oxidative damage induced by PCP was evidenced by the histopathological damage observed in this study, which may be related to fish growth inhibition.5,6 It has been reported that subchronic exposure to PCP can affect growth of earthworms37 and fish.5,38 The bioconcentration of PCP in the liver and intestinal tract of goldfish may account for the hepatic oxidative damage and fish growth inhibition. PCP, an uncoupler of oxidative phosphorylation, can distort the organism’s energy metabolism via ATP synthesis inhibition.4,39 The fish exposed to PCP commonly exhibited an increase in the level of oxygen consumption and a reduction in the level of stored lipids and body weight.4 However, information regarding the distribution 11899

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Environmental Science & Technology PCP dissimilatory iron reduction by Bacteroides spp. may partially contribute to the enhancement of the genus abundance.50,51 This study showed that Fusanobacteria and Proteobacteria dominated in fish gut, a finding also supported by previous studies.13,52 The increase in Bacteroidetes abundance and the reduction of the F/B ratio in the gut of the fish exposed to PCP may be responsible for the decrease in body weight and liver weight. Although Bacteroidetes and Firmicutes had relatively lower abundance than Fusanobacteria and Proteobacteria, they are well-known to be involved in lipid metabolism of mammals.53,54 Obese human adults subjected to a hypoenergic diet showed a significant increase in the fecal proportions of Bacteroidetes parallel to weight loss over a 1-year-long intervention.55 A significantly higher fecal F/B ratio in obese subjects than in lean ones has also been observed in mice56,57 and swine.58 The roles of the two phyla in lipid metabolism have been confirmed by an epidemiological investigation of pregnant women revealing that lean individuals have more Bacteroidetes in their gut microbiota, while obese individuals have more Firmicutes.59 Bacteroidetes and Firmicutes in fish gut may also play an essential role in fish obesity,60,61 since Li et al.62 revealed that transgenic obesity fish had a F/B ratio significantly higher than that of wild-type controls. Ni et al.13 also indicated that wild grass carp had a Bacteroidetes/ Fusobacteria ratio in their gut microbiota higher than that of the ryegrass-fed grass carp undergoing lipid metabolism disturbance. The alteration of intestinal Bacteroides and Firmicutes abundance induced by PCP exposure may affect fish growth mainly in two ways. (1) The transformation of bile acids in gut is mainly catalyzed by bile salt hydrolase secreted by Bacteroides, and deconjugation and dehydroxylation of bile salts increase their hydrophobicity and absorptivity, which is closely related with xenobiotic assimilation and lipid metabolism.63,64 (2) PCP exposure can induce the decrease in the butyrate-producing Firmicutes and the increase in propionateproducing Bacteroidetes, and propionate is able to inhibit lipid synthesis from acetate.65 This study revealed the core genera steadily occurring in fish gut under PCP exposure or not, and network analysis further indicated that the quantity of Microbacterium in fish gut was negatively correlated with Bacteroides abundance. Network analysis also showed that the abundance of Chryseobacterium, Arthrobacter, and Legionella (noncore genera revealed by the occupancy−abundance plot) had a negative correlation with Bacteroides abundance, and Chryseobacterium and Microbacterium were key bridges (keystone genera with the highest degree of association with other bacteria66) connecting different genera between modules I and III, revealing that the genera might also play crucial roles in fish growth. A highly interconnected subcluster of human gut or feces-related bacteria was detected, including Bif idobacterium, Streptococcus, Blautia, Lactococcus, and Lactobacillus.31 However, little information about their roles in lipid metabolism and body growth is available, indicating complicated interactions within the bacterial community and between the host and the microbiota. Future studies have to be conducted to find the more reliable biomarkers in intestinal microbiota, indicating the potential health damage to the hosts induced by environmental contaminants. In conclusion, PCP can accumulate in the fish liver and intestinal tract, resulting in fish growth inhibition and hepatic

oxidative and histopathological damage. Bioaccumulation of PCP in the intestinal tract can cause microbial community shift, Bacteroidetes abundance enhancement, and F/B ratio reduction, which may be responsible for the decrease in body weight and liver weight. The adaptive reproduction of Bacteroides under PCP stress may mainly contribute to the enhancement of Bacteroidetes abundance and disturbance of the co-occurrence network, by inhibiting the growth of other genera, including Chryseobacterium, Microbacterium, Arthrobacter, and Legionella. The results may help us understand the correlations of fish growth inhibition and lipid metabolism disturbance with the gut microbial community shift under PCP exposure and extend our knowledge regarding the roles of gut microbial community in toxicological mechanisms of environmental contaminants.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b02990. Determination of levels of pentachlorophenol (Method S1), DNA extraction (Method S2), 454 pyrosequencing and data processing (Method S3), bioinformatics analysis (Method S4), statistical analysis (Method S5), changes in goldfish phenotype indices after PCP exposure (Table S1), information on pyrosequencing data and biodiversity indices (Table S2), distribution information for each genus in two principal component axes from principal component analysis (Table S3), histopathological observation of liver and gill damage (Figure S1), percentages of unclassified sequences (Figure S2), rarefaction curves of the intestinal content samples (Figure S3), proportions of the sequencing reads assigned to each phylum (Figure S4), proportions of the sequencing reads assigned to each genus (Figure S5), Venn diagram illustrating the number of shared and unique genera among the samples (Figure S6), and pH in the digestive tract of the control and treatment goldfish (Figure S7) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, 163 Xianlin Rd., Nanjing 210023, China. Phone: +86-25-89680363. Fax: +86-25-89680363. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Major Project of Science & Technology of China (2012ZX07501-003-002) and National Natural Science Foundation of China (21377050).

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REFERENCES

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DOI: 10.1021/acs.est.5b02990 Environ. Sci. Technol. 2015, 49, 11894−11902

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DOI: 10.1021/acs.est.5b02990 Environ. Sci. Technol. 2015, 49, 11894−11902