Effects of polystyrene microplastics on the composition of the microbiome and metabolism in larval zebrafish

Chemosphere 217 (2019) 646e658

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Effects of polystyrene microplastics on the composition of the microbiome and metabolism in larval zebrafish Zhiqin Wan, Caiyun Wang, Jiajie Zhou, Manlu Shen, Xiaoyu Wang, Zhengwei Fu, Yuanxiang Jin* College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China

h i g h l i g h t s
Polystyrene MPs induced gut microbiota dysbiosis in larval zebrafish.
Polystyrene MPs disturbed the transcription of genes related to glucose and lipid metabolism in larval zebrafish.
Polystyrene MPs caused metabolism disorder in larval zebrafish.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 August 2018 Received in revised form 9 November 2018 Accepted 11 November 2018 Available online 12 November 2018

Microplastics are major pollutants in marine environment and may have health effects on aquatic organisms. In this study, we used two sizes (5 and 50 mm diameter) of fluorescent and virgin polystyrene microplastics to analyze the adverse effects on larval zebrafish. In our study, we evaluated the effects on larval zebrafish after exposure to 100 and 1000 mg/L of two sizes of polystyrene microplastics for 7 days. Our results show that polystyrene microplastics could cause alterations in the microbiome at the phylum and genus levels in larval zebrafish, including changes in abundance and diversity of the microbiome. In addition, metabolomic analysis suggested that exposure to polystyrene microplastics induced alterations of metabolic profiles in larval zebrafish, and differential metabolites were involved in energy metabolism, glycolipid metabolism, inflammatory response, neurotoxic response, nucleic acid metabolism, oxidative stress. Polystyrene microplastics also significantly decreased the activities of catalase and the content of glutathione. In addition, the results of gene transcription analysis showed that exposure to polystyrene microplastics induced changes in glycolysis-related genes and lipid metabolism-related genes, confirming that polystyrene microplastics disturbed glycolipid and energy metabolism. Taken together, the results obtained in the present study indicated that the potential effects of environmental microplastics on aquatic organisms should not be ignored. © 2018 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Polystyrene microplastic Microbiome Metabolism disorder Larval zebrafish

1. Introduction In the past 60 years, global plastic production has rapidly risen and has reached nearly 300 million tons in 2013 (Bouwmeester et al., 2015; Janajreh et al., 2015). Concerns about marine environment occurred when plastics entered the marine environment through indiscriminate disposal. Recently, microplastics pollution has been considered a great environmental problem (Welden and Cowie, 2016). Microplastics are defined as plastic debris smaller

* Corresponding author. 18, Chaowang Road, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China. E-mail address: [email protected] (Y. Jin). https://doi.org/10.1016/j.chemosphere.2018.11.070 0045-6535/© 2018 Elsevier Ltd. All rights reserved.

than 5 mm (Moore, 2008). Microplastics mainly come from manufactured products such as drug vectors, sunscreens, and cosmetics or degradation of large plastic debris by UV-radiation, mechanical abrasion, and biological degradation (Peters and Bratton, 2016; Andrady, 2011). Now, pollution caused by microplastics is very widespread, and it has appeared in rivers, sediments, sewages, soil, and all over the world's major seas. (Wright et al., 2013b; Eriksen et al., 2014; Woodall et al., 2014; Lusher et al., 2015; Imhof et al., 2013; Yang et al., 2015; Rillig, 2012; Ali et al., 2017). Many studies have shown that microplastics can be ingested by a variety of marine organisms such as copepods, nematodes, brown shrimp, crabs, mussels and fish, such as zebrafish and red tilapia. (Browne et al., 2008; Watts et al., 2014, 2015; Devriese et al., 2015; Peters

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and Bratton, 2016; Ding et al., 2018; Lei et al., 2018). Microplastics can also even be transferred through food chain, which will increase the potential health risks to humans (Set€ al€ a et al., 2014) On the contrary, there are some studies showing that microplastic have no adverse effects on several toxic biomarkers in aquatic animals (Kaposi et al., 2014; Cauwenberghe et al., 2015). Hence, widespread use of microplastics leads to serious environmental pollution and greater exposure risk for wildlife and humans. A number of previous studies have shown that microplastics can accumulate in crab gill or gut, the circulatory system of mussels, and gut, gills, liver and brain of red tilapia, etc (Watts et al., 2014; Browne et al., 2008; Ding et al., 2018). Accumulations of microplastics in organisms can cause all kinds of adverse reactions, such as decreased weight and survival rate, neurotoxicity, oxidative damage, alterations in lipid and energy metabolism, genetic toxicity and immune response (Besseling et al., 2013; Rochman et al., 2014; Cole et al., 2015; Avio et al., 2015; Lu et al., 2016; Ding et al., 2018; Zhang et al., 2018). Previous studies have indicated that microplastics can affect marine bacterial assemblages (McCormick et al., 2014; Harrison et al., 2011; Loblle and Cunliffe, 2011). Recently, our studies proved that exposure to different sizes of microplastics could induce gut microbiota dysbiosis, which resulted in inflammation, intestinal barrier dysfunction and lipid metabolism alteration in adult zebrafish and mice, respectively (Jin et al., 2018, 2019; Lu et al., 2018). However, there is no study focused on the effects caused by microplastics on the composition of microbiome and metabolism in larval zebrafish. Zebrafish (Danio rerio) are common tropical fish that has become an important model for toxicology studies because they are easy to feed and breed (Spitsberqen and Kent, 2003; Jin et al., 2010; Jin et al., 2015a, b). A previous study had shown that different sizes of microplastics could inhibit zebrafish larvae locomotion and significantly reduce larvae body length (Chen et al., 2017). In this study, we exposed the larval zebrafish to different sizes and concentrations of polystyrene microplastics for 7 days to explore the potential toxicity of microplastics. We exposed larval zebrafish to 5 and 50 mm diameter spherical polystyrene microplastics in water and determined whether they could induce microbiota dysbiosis, metabolism dysbiosis and oxidative stress. These results obtained in our study offer new insights into the potential health risks in aquatic organisms caused by microplastics. 2. Materials and methods

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and 14:10 dark/light cycles according to the zebrafish breeding protocol. Tap water (pH 7.2e7.6; hardness 39.9e61.0 mg CaCO3/L) was aerated for 24 h to activate carbon filtration. The fish were fed with Artemia nauplii twice a day and fed with red worms once a day at noon.

2.2. Experimental fish After oviposition, embryos were collected and staged using standard procedures as outlined by Westerfield (1993). To eliminate potential differences between breeding groups, the embryos were mixed gently before exposing them to polystyrene microplastics so that every experimental group contained a mixture of embryos from different breeders. The embryos were then transferred to different glass beakers containing different concentrations of polystyrene microplastics (experimental groups) or tap water (control group) at 2 h post fertilization (hpf). The suspensions of polystyrene microplastics were prepared using ultraviolet-treated dechlorinated tap water and sonicated prior to exposure. In all experiments, water was dechlorinated and filtered through activated carbon prior to use, and the embryos were incubated in the incubator with 14 h light/10 h dark cycles and the temperature of the incubator was maintained at 28 ± 1  C. 100% exposure solution was renewed daily in all experiments. No feeding during exposure. All experiments were performed in accordance with the Guiding Principles for the Use of Animals of Zhejiang University of Technology.

2.3. Characteristics and ingestion of the polystyrene microplastics To determine whether polystyrene microplastics could enter larval zebrafish, the fluorescently labeled different sizes of polystyrene microplastics (5- and 50-mm) were used to examine the ingestion of polystyrene microplastics in larval zebrafish. Briefly, 2hpf fertilized embryos were exposed to 100 mL 5- and 50-mm fluorescently labeled polystyrene microplastics at the concentrations of 0 and 1000 mg/L in glass beakers (size: 250 mL) and then incubated at ambient temperature (28 ± 1  C) with 14 h light/10 h dark cycles. After 7 d exposure, the larval zebrafish were washed with ultra-pure water. Fluorescently labeled polystyrene microplastics in the digestive organs of the larval fish were observed with a positive laser scanning confocal microscope (Olympus FV1000).

2.1. Chemical materials and experimental fish Two types of 5- and 50-mm polystyrene microplastics were used in this study, including fluorescent and pristine polystyrene microplastics. Pristine polystyrene microplastics were purchased from Microspheres-Nanospheres (New York, USA), and the catalog numbers were 100,231-10 and 100,259-10, and the corresponding product IDs were C-PS-0.5 and C-PS-50.0. Fluorescent polystyrene microplastics were purchased from Phosphorex (Shang Hai, China), and the catalog numbers were ZZS-2106B and ZZS-2112C, and lot number are 35002 and 10214. The microplastics come wet in solution and the medium of delivered PS is deionized water. The initial concentration of pristine polystyrene microplastics is 25 mg/ mL, and the initial concentration of fluorescent polystyrene microplastics is 10 mg/mL. Both of the polystyrene microplastics particles were used as received. Fluorescently labeled polystyrene microplastics were used to observe the distribution of polystyrene microplastics in the gut of zebrafish. The zebrafish (AB strain) were purchased from Jin Yu Cheng Long Fishery Technology Co., Ltd (Wuhan, China). Wild type AB strain zebrafish (Danio rerio) were kept at standard laboratory conditions with a temperature of 28  C

2.4. DNA extraction and qPCR amplification Fertilized embryos were exposed to 0, 100, and 1000 mg/L 5- and 50-mm polystyrene microplastics. About 40 fertilized embryos were placed in each glass beakers (size: 250 mL) with 100 mL of the above-mentioned exposure solution (five beakers for each exposure group). After 7 days of exposure, about 15 larval zebrafish were randomly selected from each beaker as one sample (five samples for each exposure group). The total DNA of larvae was prepared with the magnetic bead DNA isolation kit (Hangzhou Foreal Nanotechnology, China) according to the manufacturer's instructions. The extracted DNA was quantified by ultraviolet spectroscopy and stored at 80  C for further study. The prepared genomic DNA was amplified by PCR (polymerase chain reaction) with specific bacterial phyla-primers, and the primer sequences are shown in Table S1 (Cho et al., 2012; Engevik et al., 2013; Jin et al., 2016c). The PCR protocol was as performed as follows: 50 C for 2 min; 95 C for 10 min; 95 C for 15 s, 56 C for 30 s, and 72 C for 1 min for 40 cycles; and 72 C for 10 min.

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2.5. 16S rRNA gene sequencing analysis The main composition of the microbiomes in larval zebrafish were were analyzed by qPCR. Next, we selected the control groups, 1000 mg/L 5 and 50 mm polystyrene MP-treated groups for 16S rRNA gene sequencing analysis. DNA concentration was monitored by Qubit® dsDNA HS Assay Kit, and 20e30 ng DNA was used to generate amplicons. After the DNA quality was verified, a series of PCR primers (designed by Jin Weizhi) were used to amplify two highly variable regions including V3 and V4 on the 16S rRNA of the bacterial using 30e50 ng of DNA as a template. The V3 and V4 regions were amplified using an upstream primer (sequence: 50 CCTACGGRRBGCASCAGKVRVGAAT-30 ) and a downstream primer (sequence: 50 -GGACTACNVGGGTWTCTAATCC-30 ). At the same time, indexed adapters were added to the ends of the 16S rRNA amplicons to generate indexed libraries ready for downstream NGS sequencing on Illumina Miseq. DNA libraries concentration were validated by Qubit3.0 Fluorometer. Quantify the library to 10 nM, DNA libraries were multiplexed and loaded on an Illumina MiSeq instrument according to manufacturer's instructions (Illumina, San Diego, CA, USA). Sequencing was performed using PE250/300 paired-end; image analysis and base calling were conducted by the MiSeq Control Software (MCS) embedded in the MiSeq instrument. The QIIME data analysis package was used for 16S rRNA data analysis. The forward and reverse reads were joined and assigned to samples based on barcode and truncated by cutting off the barcode and primer sequence. The data volume and sequencing quality of all sample sequencing data (PF data) were calculated, and the original data statistics of sample sequencing are shown in table S2. Quality filtering on joined sequences was performed and sequence which did not fulfill the following criteria were discarded: sequence length <200 bp, no ambiguous bases, mean quality score 20. Then the sequences were compared with the reference database (RDP Gold database) using UCHIME algorithm to detect chimeric sequence, and then the chimeric sequences were removed. The effective sequences were used in the final analysis. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH (1.9.6) against the Silva 132 database pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at confidence threshold of 0.8. The RDP classifier uses the Silva 132 database which has taxonomic categories predicted to the species level. Sequences were rarefied prior to calculation of alpha and beta diversity statistics. Alpha diversity indexes were calculated in QIIME from rarefied samples using for diversity the Shannon index, for richness the Chao1 index. Beta diversity was calculated using weighted and unweighted UniFrac and principal coordinate analysis (PCoA) performed. Unweighted Pair Group Method with Arithmetic mean (UPGMA) tree from beta diversity distance matrix was built. 2.6. metabolite analysis Fertilized embryos were exposed to 0, and 1000 mg/L 5- and 50mm polystyrene microplastics. About 120 fertilized embryos were placed in each glass beakers (size: 500 mL) with 200 mL of the above-mentioned exposure solution (six beakers for each exposure group). After 7 days of exposure, about 100 larval zebrafish were randomly selected from each beaker as one sample (five samples for each exposure group) and stored at 80  C until GC-MS analysis. The sample pretreatment method was as follows: accurately weigh 30 mg of sample into a 1.5-mL centrifuge tube with addition of 20 mL of internal standard (L-2-chloro-phenylalanine, 0.3 mg/mL, methanol configuration) and 600 mL of methanol-water solution (v:v) ¼ 4:1); After adding two small steel balls, place the sample in

the refrigerator at 80c for 2 min, and then grind it in the grinder (60hz, 2 min); ultrasonically extract in an ice water bath for 10 min; incubate at 20  C for 30 min; apply low-temperature centrifugation for 15 min (13,000 rpm, 4  C); 400 mL of supernatant was loaded into a glass-derived flask; the quality control sample (QC) was prepared by equal volume mixing of the extracts of all the samples, and the volume of each QC was the same as the sample; the samples were evaporated with a centrifugal concentrator dryer; 80 mL of pyridine solution of methoxyamine hydrochloride (15 mg/ mL) was added to a glass-derived vial and vortexed for 2 min, and the oxidation reaction was performed in a shaking incubator at 37  C for 90 min; after removing the sample, 80 mL of BSTFA (containing 1% TMCS) derivatizing reagent and 20 mL of N-hexane were added, and the mixture was vortexed for 2 min and then reacted at 70  C for 60 min; remove the sample and place it at ambient temperature for 30 min for GC-MS metabolomic analysis. After derivatization, the sample was injected into the GC-MS system in splitless mode for analysis. The injection volume was 1 mL. The sample was separated by an HP-5MS capillary column and then subjected to mass spectrometry detection. The inlet temperature was 280  C, and high-purity helium was used as the carrier gas. The carrier gas flow rate was 6.0 mL/min. GC-/MS raw data were preprocessed by ChromaTOF (v 4.34, LECO, St Joseph, MI) software. The normalized data matrix was imported into the SIMCA-Pþ14.0 software package (Umetrics, Umeå, Sweden). Principal component analysis (PCA) was used to observe the overall distribution between the samples and the stability of the whole analysis process. Then, the partial least squares analysis (O) PLS-DA was used to distinguish the overall differences in the metabolic profiles between the groups. 2.7. RNA isolation and gene transcription analysis To determine the expressions of glycolysis-related genes (GK, HK 1, PK, PEPCKc) and lipid metabolism-related genes (Aco, Cpt1, Pparg, Ppara, Fas, FABP6, Apo, Dgat, Srebp1a) induced by polystyrene microplastics, fertilized embryos were exposed to 0,100, and 1000 mg/L 5- and 50-mm polystyrene microplastics. About 40 fertilized embryos were placed in each glass beakers (size: 250 mL) with 100 mL of the above-mentioned exposure solution (five beakers for each exposure group). After 7 days of exposure, about 20 larval zebrafish were randomly selected from each beaker as one sample (five samples for each exposure group). Then, total RNA was isolated using TRIzol reagent (Takara, China), and cDNA synthesis was performed by the kit (Toyobo) according to the manufacturer's protocol. The SYBR Green system (Toyobo, Japan) and Eppendorf MasterCycler® ep RealPlex 2 system (Wesseling-Berzdorf, Germany) were used for RT-qPCR analysis. The b-actin transcript was used as a housekeeping gene. The primer sequences are shown in Table S3. The cDNA amplification program was performed as the following protocol: degeneration for 1 min at 95  C, followed by 40 cycles at 95  C for 15 s and 60  C for 1 min (Chen et al., 2015; Wang et al., 2018). 2.8. Measurements of the content of GSH and activities of SOD and CAT In order to examine whether polystyrene microplastics can cause oxidative stress in larval zebrafish, content of GSH and activities of SOD and CAT were checked after 7 dpf in response to microplastics. Fertilized embryos were exposed to 0, 100, and 1000 mg/L 5- and 50-mm polystyrene microplastics. About 40 fertilized embryos were placed in each glass beakers (size: 250 mL) with 100 mL of the above-mentioned exposure solution (five beakers for each exposure group). After 7 days of exposure, about 30

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larval zebrafish were randomly selected from each beaker as one sample (five samples for each exposure group) to determine the glutathione (GSH) content and superoxide dismutase (SOD) and catalase (CAT) enzyme activities. Each sample was added to 9 vol of normal saline in a ratio by weight (g); volume (ml) ¼ 1:9, then were ground on ice with a grinding rod. The mixture was centrifuged at 4000 rpm at 4  C for 15 min, and the supernatant was collected for use. The resulting supernatant was used to determine SOD and CAT activities and GSH levels using commercial kits according to the manufacturers' instructions. The kits for determining the activities of SOD and CAT and the GSH level were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.9. Data analysis The data from each polystyrene microplastics treated group were compared with that of the control group. And prior to conducting the statistical comparisons, the data were assessed for the normality and homogeneity of variances using the KolmogorovSmirnov one-sample test and Levene's test, respectively. As the assumptions were met, the differences between two groups were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's or Fisher's protected least significant difference tests using SPSS 13.0 (Chicago, Illinois, USA). The values are expressed as the mean ± SEM. Differences with a p-value less than 0.05 were considered to be significant. 3. Results 3.1. Characteristics and ingestion of polystyrene microplastics The morphology of 5- and 50-mm polystyrene microplastics in water is a dispersed sphere (Fig. 1A). Both sizes of polystyrene microplastics were ingested by zebrafish larvae. No fluorescent signal was observed in the gut and tissue in the control fish (Fig. 1B). The 5- mm polystyrene microplastics can enter the tissue of larval fish (Fig. 1C). When the larval fish were exposed to 50- mm polystyrene microplastics, strong fluorescent signal was observed in the gut (Fig. 1D), indicating that 50- mm polystyrene microplastics were only stained in the gut. 3.2. Effects of polystyrene microplastics on the composition of microbiome in larval zebrafish at phylum level In the larval zebrafish, polystyrene microplastics significantly changed the structure of microbiome at the phylum level (Fig. 2). The relative abundances of Bacteroidetes decreased remarkably in the larval zebrafish after exposure to 1000 mg/L 5- and 50-mm polystyrene microplastics for 7 d when compared with the control group (Fig. 2). In contrast, the relative abundance of Firmicutes increased significantly in larval zebrafish after exposure to 1000 mg/ L 50- mm polystyrene microplastics. In addition, a significant decrease in g-Proteobacteria was also observed when exposed to 100 and 1000 mg/L 5-mm and 1000 mg/L 50-mm polystyrene microplastics (Fig. 2). 3.3. Effects of polystyrene microplastics on the composition of microbiome analyzed by 16S rRNA gene sequencing In order to further analyze the changes in the composition of microbiome in the larval zebrafish after exposure to 1000 mg/L 5and 50-mm polystyrene microplastics, the high throughput sequencing of 16S rRNA was used to determine the changes in the composition of microbiome. As shown in Fig. 3 A, the composition of microbiome at the phylum level changed after exposure to

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different sizes of microplastics (Fig. 3A). Alpha diversity metrics were analyzed using a Kruskal-Wallis test with a Dunn's pairwise multiple comparisons test. As shown in Fig. 3C, the Chao 1 index also suggested a decrease in richness of gut microbiota after exposure to 5- and 50-mm polystyrene microplastics, (Fig. 3C), and Chao 1 decreased significantly after exposure to 5-mm polystyrene microplastics. In addition, some indicators related to species richness have been added in table S4. Furthermore, through the UniFrac principal coordinates analysis, it was found that the main components of microbiome in larval zebrafish were shifted by both polystyrene microplastics treatments (Fig. 3D). In addition, at genus level, the composition of microbiome of zebrafish also changed after exposure to 1000 mg/L 5- and 50-mm polystyrene microplastics for 7 days (Fig. 3B). The significantly different genus-level taxa were evaluated with a non-parametric test with a multi-test correction, and the results indicated that the abundance of microorganisms, including Sphaerotilus, Haliangium and Leptothrix decreased significantly, whereas the abundance of Methyloversatilis, Polynucleobacter, Legionella and Ottowia increased significantly after exposure to 5-mm polystyrene microplastics. The abundance of Pseudomonas decreased significantly, whereas the abundance of Flectobacillus and Methylophilus increased significantly after exposure to 50-mm polystyrene microplastics. In addition, the abundance of Methylobacterium decreased significantly after exposure to two sizes of polystyrene microplastics. 3.4. Metabolomic alterations induced by different sizes of polystyrene microplastics Changes in metabolic profiles of larval zebrafish after exposure to polystyrene microplastics were determined. Moreover, the PCA model was used to further visualize the difference between the treatments and control. As shown in PCA score plots, larval zebrafish exposed to two sizes of polystyrene microplastics could be clearly separated from the control group (Fig. 4A and B). Moreover, samples treated with 5-mm and 50-mm polystyrene microplastics were also successfully separated (Fig. 4C). These findings suggested that exposure to polystyrene microplastics strikingly affected the metabolic profiles of larval zebrafish and that these two sizes of polystyrene microplastics induced different metabolomic profiles. Multidimensional analysis of OPLS-DA was used to screen for differences between groups of metabolites (VIP>1, p value <0.05). Compared with the control, exposure to 1000 mg/L 5-mm polystyrene microplastics induced changes in 78 metabolites, and exposure to 1000 mg/L 50-mm polystyrene microplastics induced changes in 121 metabolites, including carbohydrates, fatty acids, amino acids, nucleic acid and others. These differential metabolites were involved in energy metabolism, glycolipid metabolism, inflammatory response, neurotoxic response, nucleic acid metabolism, oxidative stress and others. In order to elucidate the variations of individual metabolites among the treatment groups, we selected some common differential metabolites and generated heat maps based on z scores (Fig. 4D). For the group that was exposed to 1000 mg/L 5-mm polystyrene microplastics, the levels of pyruvic acid, glutamine, creatine, beta-alanine, squalene, pyruvic acid, nicotinic acid, linolenic acid, glucose, cholesterol, gallic acid, spermidine, methylmalonic acid, epsilon-Caprolactam, 20 -deoxyguanosine, uridine, adenosine, xanthine, melatonin, alphatocopherol, mannitol, 2-Ketovaleric acid, sorbose, D-Talose, sitosterol, scopoletin, alpha-ketoisocaproic acid and gly-pro significantly increased, while the levels of sarcosine, 2-monoolein, aconitic acid, zymosterol, putrescine, s-carboxymethylcysteine, 5,6dimethylbenzim-idazole, 6-hydroxynicotinic acid, lactobionic acid, ascorbate, tagatose, prostaglandin A2, norvaline, androsterone, maleic acid and dioctyl phthalate significantly decreased when

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Fig. 1. Characteristics and ingestion of polystyrene microplastics. (A) The morphology of 5- and 50-mm polystyrene microplastics in water detected by scanning electron microscope (SEM). Images of different sizes of fluorescently labeled polystyrene microplastics ingested by larval zebrafish: (B) control, (C) 5-mm, and (D) 50-mm. The concentration of both fluorescently labeled polystyrene microplastics was 1000 mg/L.

compared with the control group (Fig. 4D). However, for the group that was exposed to 1000 mg/L 50-mm polystyrene microplastics, the levels of succinic acid, pyruvic acid, creatine degr, glucose, 2monopalmitin, methyl palmitoleate, fructose-6-phosphate, ribose-5-phosphate, nicotinic acid, 3-phosphoglycerate, serine, gallic acid, noradrenaline, aspartic acid, hydroquinone, glycine, ribose, lyxonic acid, 1,4-lactone, uridine, adenosine, uracil, xanthine, hypoxanthine, 5-aminovaleric acid lactam, mannitol, octanal, phloroglucinol, sophorose, sorbose, D-Talose, trans-4hydroxy-L-proline, pelargonic acid, 2-Methylglutaric acid, scopoletin, alpha-ketoisocaproic acid, gly-pro and urea significantly increased, while the levels of sarcosine, inosine, creatine, myoinositol, taurine, aconitic acid, zymosterol, phosphomycin, s-carboxymethylcysteine, azelaic acid, 3,5-Dihydroxyphenylglycine, 3-

Cyanoalanine, DL-Anabasine, 2-aminophenol, adenine, 6Hydroxynicotinic acid, ascorbate, alpha-Aminoadipic acid, androsterone, Octadecanol, Lyxose, indole-3-acetic acid, maleic acid, saccharopine, norvaline, and cytidine-50 -monophosphate significantly decreased when compared with the control group (Fig. 4E). Moreover, we also summarized the pathways of the main differential metabolites and found that they had a significant impact on glucose metabolism, amino acid metabolism and nucleic acid metabolism pathways (Fig. 5). 3.5. Effects of polystyrene microplastics on the transcription of genes related to glycolysis and lipid metabolism As shown by GC/MS analysis, some metabolites related to

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Fig. 2. Effects of polystyrene microplastics exposure on the composition of microbiome at phylum level in larval zebrafish detected by real-time quantitative PCR. (A) Relative abundance of the bacteria at the phylum level in larval zebrafish after exposure to 100 and 1000 mg/L 5-mm polystyrene microplastics for 7 days. (B) Relative abundance of the bacteria at the phylum level in larval zebrafish after exposure to 100 and 1000 mg/L 50-mm polystyrene microplastics for 7 days. Data are expressed as the mean ± SEM of five replicates (n ¼ 5). Statistically significant differences compared to the control group are indicated by asterisks: *p < 0.05; and **p < 0.01.

Fig. 3. Effects of polystyrene microplastics exposure on the composition of microbiome in larval zebrafish, which was detected by 16S rRNA gene sequencing. (A) The composition of microbiome at the phylum level between the control group and 1000 mg/L 5- or 50-mm polystyrene microplastics-treated groups. (B) Heat map of specimens showing changes in main identified bacteria at the genus taxonomic level after exposure to 1000 mg/L 5- or 50-mm polystyrene microplastics. (C) Chao 1 index of the diversity of microbiome after polystyrene microplastics exposure. (D) UniFrac principal coordinates analysis (PCA) estimates of the microbiome in larval zebrafish in control and 1000 mg/L 5- or 50-mm polystyrene microplastics-treated group.

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Fig. 4. Metabolomic alterations induced by different sizes of polystyrene microplastics exposure. Score plots of principal coordinates analysis (PCA) for 5-mm polystyrene microplastics and the control group (A), 50-mm polystyrene microplastics and the control group (B) or two sizes of polystyrene microplastics and the control group (C). Heat map for the differential metabolites in different pathways identified between the control and 5- or 50-mm polystyrene microplastics-treated group based on z-scores (D and E).

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Fig. 5. Possible pathway analysis of differential metabolites in larval zebrafish induced by 1000 mg/L 5- or 50-mm polystyrene microplastics. The red arrows indicate that the metabolites increased significantly by polystyrene microplastics exposure, and the blue arrows indicate that the metabolites increased significantly by polystyrene microplastics exposure when compared to the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

carbohydrates and lipids changed significantly after exposure to polystyrene microplastics. Thus, we further determined the mRNA transcriptional levels of glycolysis- and lipid metabolism-related genes in larval zebrafish. It was observed that the mRNA levels of cytosolic phosphoenol pyruvate carboxykinase (Pepckc) and glucokinase (Gk) tended to increase in larval zebrafish after exposure to polystyrene microplastics. On the contrary, the transcriptional levels of pyruvate kinase (PK) and hexokinase 1 (HK1) tended to decrease when exposed to 100 and 1000 mg/L 5- and 50-mm polystyrene microplastics. Notably, the mRNA levels of HK1 decreased significantly (Fig. 6A). In addition, for the transcription of lipid metabolism-related genes, the mRNA levels of acyl-CoA oxidase (Aco), carnitine palmitoyltransferase 1 (Cpt1), fatty acid binding protein 6 (Fabp6), and diacylgycerol acyltransferase (Dgat) tended to decrease in larval zebrafish when exposed to polystyrene microplastics. And the mRNA levels of Aco decreased significantly after exposure to 100 and 1000 mg/L 50-mm or 1000 mg/L 5-mm polystyrene microplastics. The transcriptional levels of Fabp6 decreased significantly when exposed to 100 and 1000 mg/L 50-mm polystyrene microplastics. The transcriptional levels of Dgat decreased remarkably after exposure to 100 and 1000 mg/L 5- and 50-mm polystyrene microplastics. On the contrary, the mRNA levels of acetyl-CoA carboxylase

1 (Acc1), sterol regulatory element binding protein 1a (SREBP1a), fatty acid synthase (Fas) and apolipoprotein (Apo) tended to increase compared with the control group. And the transcriptional levels of Acc1 and Apo increased remarkably after exposure to 100 mg/L 50mm polystyrene microplastics. Furthermore, the mRNA levels of peroxisome proliferator-activated receptor gamma (PPARg) increased significantly in larval zebrafish when exposed to 1000 mg/L 50-mm polystyrene microplastics (Fig. 6B).

3.6. Effects on content of GSH, and activities of SOD and CAT The results of GC/MS also indicated that some metabolites related to oxidative stress were changed after exposure to polystyrene microplastics, so we measured the content or activities of antioxidant enzymes (GSH, SOD, and CAT). As shown in Fig. 7, the content of GSH decreased significantly after exposure to 100 and 1000 mg/L 5- and 50-mm polystyrene microplastics. Besides, the activity of CAT decreased significantly in larval zebrafish when exposed to 1000 mg/L 5- or 50-mm polystyrene microplastics. However, there was no significant change in the activity of SOD between the control group and both sizes of polystyrene microplastics-treated groups.

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Fig. 6. Effects of different sizes of polystyrene microplastics exposure on the transcription of the genes related to glucose and lipid metabolism in larval zebrafish. (A) Transcription of the genes related to glycolysis. (B) Transcription of the genes related to lipid metabolism. Data are expressed as the mean ± SEM of five replicates (n ¼ 5). Statistically significant differences compared to the control group are indicated by asterisks: *p < 0.05.

4. Discussion More and more studies have shown that microplastics in water can be ingested by aquatic organisms (Peters and Bratton, 2016; Lei et al., 2018; Devriese et al., 2015; Watts et al., 2014; Browne et al., 2008; Ding et al., 2018). Recently, the negative influence of microplastics on aquatic organisms has increasingly concerned by researchers. For example, Cole M et al. reported that polystyrene microplastics impede feeding in copepods and decreased reproductive output (Cole et al., 2015). Ding et al. proved that microplastics caused oxidative damage and neurotoxicity in red tilapia (Ding et al., 2018). Lu et al. (2016) found that microplastics could induce alterations in metabolic profile in adult zebrafish liver. However, little attention has been paid to the influence of microplastics intake on larval zebrafish. Recently, our studies suggested that 1000 mg/L 5- and 50-mm polystyrene microplastics could

induce microbiota dysbiosis and inflammation in the gut after 21 days of exposure (Jin et al., 2018; Lu et al., 2018). Though a previous study had shown that ingestion of microplastics could cause intestinal blockage (Jovanovi c, 2017), the specific mechanism of microplastics on the intestinal tract is still not clear. In this study, we demonstrated for the first time that different sizes of polystyrene microplastics can induce microbiome dysbiosis in zebrafish larvae. Gut microbiota is composed by varieties of microorganisms that inhabit the intestines of animals (Sekirov et al., 2010; Xia et al., 2018a, b). Many studies have proved that gut microbiota is vital to the health of the host and plays important roles in nutrition, development, resistance against invasive pathogens, and regulation of immune responses (Ley et al., 2005; Dethlefsen et al., 2008; Sanz et al., 2010; Zhang et al., 2019). Especially, gut microbiota is closely related to the metabolism of host. Supporting this idea, a large

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Fig. 7. Effects of different sizes of polystyrene microplastics exposure on the content of GSH and the activities of CAT and SOD in larval zebrafish. Data are expressed as the mean ± SEM of five replicates (n ¼ 5). Statistically significant differences compared to the control group are indicated by asterisks: *p < 0.05; and **p < 0.01.

number of previous studies had shown that microbiome played very important roles in energy regulation and metabolism (Ley et al., 2006; Allahham et al., 2012; Neis et al., 2015). For example, Neis et al. proved that the gut microbiota was involved in the utilization and catabolism of several amino acids originating from both alimentary and endogenous proteins, and these amino acids could serve as precursors for the synthesis of metabolic end products produced by the microbiota including SCFAs (Neis et al., 2015). Recently, Nieuwdorp et al. suggested there were three major pathways via which intestinal microbiota could affect human metabolism: host metabolism and energy balance were influenced by an interplay of nutrients, bile acids, intestinal microbiota, and composition of the epithelial mucus layer; the processes in the intestinal lumen exert their effects systemically on metabolism via production of gut hormones (including glucose-dependent insulinotropic polypeptide and glucagon-like peptide), affecting (para) sympathetic tone and regulation of immune cell action (Nieuwdorp et al., 2014). According to previous studies, the gut microbiota could be influenced by chemicals in environment, such as antibiotics, heavy metals and pesticides, etc., and further lead to the pervasive effects in the host (Lu et al., 2014; Xu et al., 2014; Joly Condette et al., 2015; Jin et al., 2016a, b; Jin et al., 2017; Wu et al., 2018). As far as we know, there is no research on whether polystyrene microplastics can induce the gut microbiota disorders in the early developmental stage of zebrafish. In fact, some studies have demonstrated that different microplastics could interact with microbes in the environment (Harrison et al., 2011; Loblle and Cunliffe, 2011; Zettler et al., 2013; McCormick et al., 2014). In our study, the composition of the microbiome in zebrafish larvae changed at phylum and genus levels when exposed to difference sizes of polystyrene microplastics. At phylum levels, compared with the control group, the relative abundances of Bacteroidetes decreased remarkably in the larval zebrafish after exposure to 1000 mg/L 5- and 50-mm

polystyrene microplastics for 7 d. In contrast, the relative abundance of Firmicutes increased significantly in larval zebrafish after exposure to 1000 mg/L 50-mm polystyrene microplastics. Previous studies have shown that obese individuals had an imbalance in the ratio of Bacteroidetes and Firmicutes (Ley et al., 2006; Turnbaugh et al., 2006). Bacteroidetes in the gut microbiota is directly related to lean body mass: levels are reduced with obesity (Ley et al., 2006; Turnbaugh et al., 2009; Ley, 2010). Alternately, fiber consumption and the weight loss-promoting effects of monounsaturated fatty acids (oleic acid) and polyphenols are also associated with a rise in Bacteroidetes (Rastmanesh, 2011; Simoes et al., 2013; Mujico et al., 2013). These results observed that Bacteroidetes and Firmicutes affected energy metabolism, glucose metabolism and lipid metabolism. In addition, a significant decrease in g-Proteobacteria was also observed when exposed to 100 and 1000 mg/L 5- mm and 1000 mg/L 50- mm polystyrene microplastics. A pervious study had shown that the levels of g-Proteobacteria were also directly associated with changes in liver fat (Spencer et al., 2011). Otherwise, at genus levels, some altered bacteria were closely related to metabolism, disease and inflammation although the function of some varied bacteria is not clear yet. For example, legionellae are intracellular parasites of freshwater protozoa, and these bacteria could cause respiratory disease (Fields et al., 2002). Leptothrix can cause multiple liver abscesses (Harris, 1933). Flectobacillus can produce sphingolipids and sphingolipids, which are involved in signal transduction pathways that mediate cell growth, differentiation, multicellular function and cell death (Batrakov et al., 2000; Merrill et al., 1997). Polynucleobacter is related to a genetic disorder in glycogenolysis (Vannini et al., 2007). Pseudomonas can cause cystic fibrosis lung infection (Oliver et al., 2000). Methylobacterium, which is significantly decreased in the two treatment groups, can stimulate a proinflammatory response that may aggravate and perpetuate the pathological processes

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underlying atherosclerosis in rheumatoid arthritis (Curran et al., 2014). These results indicated that different sizes of polystyrene MPs induced microbiota dysbiosis in zebrafish larvae. Here, it was also observed that exposure to polystyrene microplastics could also cause the changes in many metabolites in zebrafish larvae. The significant changed metabolites were mainly involved in energy metabolism, such as glutamine, sarcosine, pyruvic acid and creatine. Glutamine could provide energy to small intestine epithelial cells, and glutamine synthesis and the glutamine-cycle are related to brain energy metabolism (Fleminq et al., 1997; Zwinqmann and Butterworth, 2005). As an energy precursor, creatine could provide energy to muscle and nerve cells (Wyss and Wallimann, 1994; Brewer and Wallimann, 2000). Thus, it is possible that these findings indicated that exposure to polystyrene microplastics may disturb energy metabolism. Previous results have shown that microplastics could deplete energy reserves in marine worms and copepods and influence the feeding activities of crabs (Cedervall et al., 2012; Wright et al., 2013a; Watts et al., 2015). In this study, linolenic acid, cholesterol, squalene and zymosterol changed after exposure to 5-mm polystyrene microplastics. Taurine and various lipids (2-Monopalmitin, methylpalmitoleate, etc.) changed after exposure to 50-mm polystyrene microplastics. These metabolites are all related to lipid metabolism, indicating that lipid metabolism in larval zebrafish was significantly influenced after exposure to polystyrene microplastics. Moreover, the glucose, pyruvic and aconitic acid also changed significantly after exposure to different sizes of polystyrene microplastics. Fructose-6-phosphate, ribose-5-phosphate and fumaric acid increased after exposure to 50-mm polystyrene microplastics. Pyruvic and fructose-6-phosphate are important intermediates for glycolysis, and aconitic acid, and fumaric acid are essential to TCA. Consistent with this, the RT-qPCR results showed that HK1 was significantly decreased after two different size polystyrene microplastics treatments. As a key enzyme in glucose metabolism, the first step in metabolism of glucose is usually phosphorylation, which is catalyzed by hexokinase (Wilson, 2003). In addition, the mRNA levels of Aco, Fabp6 and Dgat were significantly decreased after exposure to polystyrene microplastics. AcylCoA oxidase (Aco) plays an important role in lipid b-oxidation, and fatty acid binding protein 6 (Fabp6) is commonly regarded as a bile acid binding protein and important in maintaining bile acid homeostasis (Oaxaca-Castillo et al., 2007; Raj et al., 2015). DGAT is essential to triacylglycerol synthesis and lipid droplets in adipocytes (Harris et al., 2011). All of these changes indicate that polystyrene microplastics could affect glycolipid metabolism in zebrafish larvae. Based on metabolomic analysis and gene expression analysis of glycolysis-related genes and lipid metabolismrelated genes, we proved that polystyrene microplastics can cause energy metabolism and glucose and lipid metabolism disorders in larval zebrafish. A previous study showed reduced food consumption of crabs that had ingested microplastics (Watts et al., 2015). Thus, we thought that ingestion of polystyrene microplastics could cause satiety and reduce food intake of larval zebrafish directly. In addition, it was observed that 5- and 50-mm polystyrene microplastics induced different metabolomic profiles. Compared to the control group, the treatment group exposure to 50-mm polystyrene microplastics induced more metabolic differences than the 5-mm polystyrene microplastics-treated group. But we cannot simply conclude that 50-mm polystyrene microplastics are more toxic than 5-mm. In fact, it is generally thought that smaller microplastics are more toxic because smaller beads have a larger specific surface area and more possibility of being absorbed by cells (Lu et al., 2016; Choi and Hu, 2008; Jeong et al., 2016). In addition, it was observed that 5- and 50-mm polystyrene microplastics could also induce oxidative stress in zebrafish larvae.

As shown in Fig. 7, the activity of CAT and the content of GSH decreased significantly after exposure to the two sizes of polystyrene microplastics. CAT and GSH are sensitive markers for assessing oxidative damage caused by chemicals in the environment. These enzymes are responsible for eliminating reactive oxygen species (ROS) (Deng et al., 2014). Consistent with this, GC-MS results also showed significant changes in oxidative stress-related metabolites. For example, melatonin, glutathione and alphatocopherol increased after exposure to 5-mm microplastics. Ascorbate and 6-hydroxynicotinic acid decreased after exposure to 5and 50-mm polystyrene microplastics. The increase in pyruvic was due to the two sizes of polystyrene microplastics. These metabolites are mainly used as antioxidants (Schulz et al., 2000; Ramanathan et al., 2002; Rodriguez et al., 2004). These results indicated that exposure to polystyrene microplastics might lead to an imbalance in the antioxidant defense system in zebrafish larvae. A previous study had suggested the potential neurotoxicity of microplastics to freshwater fish (Ding et al., 2018). Consistent with this, in our study, exposure to 5-mm polystyrene microplastics caused changes in the levels of spermidine, epsilon-caprolactam, methylmalonic acid and 5, 6-dimethylbenzimidazole, which could induce neurotoxicity to the body (Doyle and Shaw, 1994; Hogeveen et al., 2008). And after exposure to 50-mm microplastics, aspartate increased and taurine decreased. It acts as a neurotransmitter substance (Wiklund et al., 1982). These results provided the possibility that polystyrene microplastics may induce neurotoxicity, which may be associated with changes in some metabolites in larval zebrafish. In conclusion, our results clearly observed that different sizes of polystyrene microplastics could induce microbiome dysbiosis and changed the metabolomic profiles in larval zebrafish. The altered microbiome and metabolites may be tightly related to the occurrence of energy metabolism disorder, oxidative stress, and neurotoxicity in larval zebrafish. These findings provide insights toward understanding the mechanism underlying microplastics-induced aquatic toxicity. Acknowledgements This work was supported by the National Natural Science Foundation of China (21777146), Zhejiang Provincial Natural Science Foundation of China (LR16B070002) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT17R97). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2018.11.070. References Ali, K., Abolfazl, G., Cheng, K.C., Vincent, L., Tamara, S.G., Babak, S., 2017. The presence of microplastics in commercial salts from different countries. Sci. Rep. 7, 46173. Allahham, S., Roelofsen, H., Rezaee, F., Weening, D., Hoek, A., Vonk, R., Venema, K., 2012. Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects. Eur. J. Clin. Invest. 42, 357e364. Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596e1605. Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., d'Errico, G., Pauletton, M., Barqelloni, L., Reqoli, F., 2015. Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environ. Pollut. 198, 211e222. Batrakov, S.G., Mosezhnyi, A.E., Ruzhitsky, A.O., Sheichenko, V.I., Nikitin, D.J., 2000. The polar-lipid composition of the sphingolipid-producing bacterium Flectobacillus major. Biochim. Biophys. Acta 1484, 225e240. Besseling, E., Weqner, A., Foekema, E.M., van den Heuvel-Greve, M.J., Koelmans, A.A., 2013. Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.). Environ. Sci. Technol. 47,

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