Exposure of a Soil Collembolan to Ag Nanoparticles and AgNO3

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Exposure of a Soil Collembolan to Ag Nanoparticles and AgNO3 Disturbs Its Associated Microbiota and Lowers the Incidence of Antibiotic Resistance Genes in the Gut Dong Zhu,†,‡,⊥ Fei Zheng,†,‡,⊥ Qing-Lin Chen,†,‡ Xiao-Ru Yang,† Peter Christie,† Xin Ke,§ and Yong-Guan Zhu*,†,∥

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Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, 1799 Jimei Road, Xiamen 361021, China ‡ University of the Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China § Institute of Plant Physiology and Ecology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China ∥ State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China S Supporting Information *

ABSTRACT: Gut microbiota contribute to host health. Numerous recent studies have focused on the survival and reproduction of nontarget soil animals exposed to the toxicity of silver nanoparticles (AgNPs) but changes in the gut microbiota due to nanoparticle toxicity are largely unknown. Here, we examine some effects of AgNPs and silver nitrate (ionic Ag) on the gut microbiota of the common soil collembolan Folsomia candida using Illumina sequencing and concomitant changes in antibiotic resistance genes (ARGs) of the gut microbiota using highthroughput quantitative PCR. A large number of Ag accumulated in Agexposed individuals after 28 days and ionic Ag significantly inhibited the reproduction of the collembolan (by 19.3%). Exposure to AgNPs disturbed the composition of the collembolan gut bacterial community, resulting in dysbiosis of the gut microbiota. However, the dominant microbiota was shared among different treatments. In addition, AgNPs exposure did indeed reduce the incidence of ARGs in the collembolan gut microbiota. A weak relationship was identified between gut bacterial communities and ARG profiles. These results extend our knowledge regarding the role of the gut microbiota in assessing the soil ecotoxicology of AgNPs.

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soil fauna,17,18 but the changes in their gut microbiota under conditions of exposure to pollution remain poorly characterized. Only a few studies have explored the effects of antibiotics and microplastics on soil collembolan gut microbiota,19,20 and the impacts of potentially toxic elements on earthworm and isopod gut microbiota.21,22 However, different contaminants may exert different effects on soil animal gut microbiota.23−25 There is therefore a distinct knowledge gap regarding the effects of contaminant exposure on soil animal gut microbiota. Silver nanoparticles (AgNPs) have been widely used as bactericides in consumer products such as food packaging, kitchenware, textiles, and medicines.26 It has been estimated that thousands of tonnes of AgNPs have been released into the

INTRODUCTION

Soil fauna are very diverse and may represent 23% of the total diversity of known animals.1,2 They play important, but usually overlooked, roles in soil ecosystem services, including production services (e.g., nutrient cycling) and regulation services (e.g., carbon sequestration).2−5 Animal guts are commonly colonized by microbiota.6 These microorganisms exert an important influence on host immunity, metabolism, and health.7,8 Essential nutrient resources of many animals, especially arthropods, are offered by their gut microbiota.9,10 The gut microbiota of many animal species (e.g., mice,11 pigs,12 termites,13 honey bees,14 and fish15) have been studied to date but the gut microbiota of soil animals, and particularly soil microarthropods, remain poorly understood. Soil pollution has raised widespread public concern.16 In polluted soils the effects of contaminants on soil fauna consist mainly of direct influence on host physiology and indirect disturbance of the host gut microbiota.10 Most previous studies have focused on impacts of contaminants on the physiology of © XXXX American Chemical Society

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May 26, 2018 August 29, 2018 October 16, 2018 October 16, 2018 DOI: 10.1021/acs.est.8b02825 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology soil environment every year,27 largely by the application of sewage sludges and sewage irrigation,28 which have resulted in increasing AgNP concentrations in sludge-amended soils (∼0.036 mg Ag kg−1 yr−1 in the U.K.).29 Moreover, Ag concentrations in dry sewage sludge were found to range from 1.7 to 856 mg kg−1 in the U.S.A.30 Sewage sludges contain abundant nutrients19 that will be ingested by soil fauna. In this scenario, soil fauna will be exposed to high concentrations of AgNPs via the diet, suggesting that AgNPs entering soils may represent some risk to nontarget species (soil fauna). Studies on the effects of AgNPs on soil fauna have increased recently.27 However, these studies have focused mainly on changes in soil animal physiology or behavior (e.g., accumulation of Ag in animal tissues, avoidance, mortality, and reproduction).18,27 Previous studies show that AgNPs can disturb the gut microbial composition of mice and fish,24,31 but no study has been conducted to explore the impacts of AgNPs on soil animal gut microbiota. In addition, there are also no data available on the impact of ionic Ag on soil animal gut microbiota. The emergence and dispersal of antibiotic resistance genes (ARGs) have become a global public threat.32,33 Numerous studies demonstrate that metals can coselect antibiotic resistance34−37 and graphene (nanoscale) also has a significant influence on the composition and diversity of ARGs in the wider environment.38 In addition, a large number and high abundance of ARGs have been found in the soil animal gut microbiome.19 It is therefore also necessary to examine the characteristics of ARGs in soil animal gut microbiomes exposed to Ag. Collembolans are one of the most abundant groups of soil fauna. They can be found almost everywhere worldwide and make an important contribution to the processes and functions of the soil ecosystem.19,39,40 In the current study we tested the influence of AgNPs and ionic Ag on the gut microbiota of the model species Folsomia candida by 16S rRNA gene Illumina sequencing, a technique that has been used routinely in soil ecotoxicology studies. Our aims were (1) to confirm the accumulation of Ag in collembolan body tissues and shifts in the growth, reproduction and mortality of the collembolan, (2) to study the changes in the collembolan gut microbiota, (3) to depict the characteristics of ARGs in the collembolan gut microbiome by high-throughput qPCR, and (4) to explore the relationship between bacterial communities and ARG profiles following chronic exposure to AgNPs and ionic Ag. The results provide new insights into the ecological risk from nanometer materials to soil nontarget species, combined for the first time with shifts in the soil animal gut microbiota.

The yeasts were mixed with a certain amount of AgNPs or AgNO3 in ultrapure water to obtain two Ag-spiked foods (200 mg Ag kg−1 dried yeast based on the nominal concentration). The yeast was mixed directly with an equal amount of ultrapure water to serve as a control. The yeast mixtures were freeze-dried and ground before use. The AgNO3 (98.5−99.9% purity, high-grade) and AgNPs (50 nm) were obtained from Aladdin Industrial Corporation, Hangzhou, China. Characteristics of the AgNPs are shown in Figure S1 of the Supporting Information (SI). There was no coating on the AgNPs, and the TEM diameter of AgNPs was 50 nm. The result of the SEMEDS shows that the AgNPs were homogeneous in the yeast, and the TEM image shows that some AgNPs remain as nanoparticle forms and others have been agglomerated after adsorption in the yeast followed by freeze-drying. The Ag concentrations of the yeast were determined to be 0.26 ± 0.06, 205 ± 2.5 and 182 ± 7.4 mg kg−1 in unspiked, ionic Ag-spiked and AgNP-spiked foods (n = 3), respectively. The Ag concentrations used in our test were based on previous studies.42,43 Experimental Design. The test consisted of three exposure treatments (control, ionic Ag and AgNPs). Ten synchronized collembolans (10−12 days) were placed in the 9 mm Petri dishes and 5 mg of unspiked yeast or Ag-spiked yeast (200 mg kg−1) were added twice a week together with water replenishment. Each treatment was replicated six times to give a total of 18 dishes. We used numbers of living adults and juveniles as measures of survival and reproduction, respectively. After exposure for 28 days the numbers of living adults and juveniles produced were counted. Three adult collembolans from each replicate dish were then transferred to a new 3 mm Petri dish covered with a layer of wet filter paper for 48 h to empty their gut contents. These individuals were used to determine body weight and Ag concentration. Finally, the surplus adults in each replicate were used to isolate the gut microbial DNA. Two replicates were combined to give a composite sample to provide sufficient DNA. Thus, there were three replicates for the extraction of collembolan gut DNA in each treatment. Measurement of Ag Concentration. Approximately 15 mg yeast were weighed into 20 mL polytetrafluoroethylene tubes. Then 2 mL HNO3:H2O2:HF mixture (4:2:1, v/v/v) were added and the tubes were kept at room temperature for 2 h prior to sealing. Finally, the sealed tubes were transferred to a high-pressure steel digestion vessel for digestion in a drying oven at 105 °C for 6 h. The adult collembolans obtained were dried at 50 °C and their body weights were determined using an automatic electronic microbalance (Mettler Toledo XS3DU, Columbus, OH; precision ±1 μg). Silver concentrations of adult collembolan body tissues were determined based on the method of Zhu et al. (2017).44 Briefly, the weighed collembolans were digested at 100 °C for 8 h on a heating block with 1 mL of ultrapure nitric acid (Merck Millipore, 65%, Darmstadt, Germany) in small Pyrex tubes. The digests were diluted 50 times with ultrapure water and the Ag concentrations were determined with an ICP-MS (Agilent 7500cx, Agilent, Santa Clara, CA). A certified reference material (GBW10050(GSB-28), king prawns) was used to ensure quality assurance and quality control and was purchased from the Institute of Geophysical and Geochemical Exploration, Hefei, China. The recovery rates of Ag from the

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MATERIALS AND METHODS Preparation of Collembolan and Food. The model collembolan Folsomia candida (“Berlin strain”) was maintained in an incubator at 75% relative humidity and 20 ± 2 °C with an 8:16 h (light/dark, 800 lx) light regime for more than six years after it was originally obtained from Aarhus University in Denmark. Petri dishes (9 mm diameter) covered with a layer of plaster of Paris mixed with activated charcoal (8:1 w/w) were selected as animal culture vessels. Sufficient yeast (Angel Yeast Co., Ltd., Yichang, Hubei, China) as food and ultrapure water were added to the Petri dishes twice a week. Synchronized individuals (10−12 days old) were obtained as described by Zhu et al. 2016.41 B

DOI: 10.1021/acs.est.8b02825 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Figure 1. (a) Reproduction of the collembolan (number of living juveniles) and (b) Ag concentration of the body tissues (mean ± SE, n = 6) in all treatments after 28 days of exposure. The Ag concentration data were natural logarithm-transformed before statistical analysis. Different letters indicate significant differences between different treatments at the 0.05 level (ANOVA).

ARGs was counted to represent the ARG abundance in our test as described in a previous study.19 Amplification, High Throughput Sequencing, and Bioinformatics Analysis. The universal primer set (515F: forward GTGCCAGCMGCCGCGG and 907R: reverse CCGTCAATCMTTTRAGTTT) targeting the bacterial 16S rRNA gene V4−V5 hypervariable region was used to amplify the collembolan gut DNA which was embedded with unique barcodes. The conditions and reaction system of 16S rRNA gene amplification were as described previously.20 The amplification products were purified with a TIANGEN universal DNA purification kit (TIANGEN Biotech, Beijing, China), and their concentrations were determined using a Qubit Fluorometer (Invitrogen, Ghent, Belgium) with a Qubit dsDNA HS Assay kit. The purified products were pooled at equal content to construct a sequencing library, and the Illumina Hiseq 2500 platform (Novogene, Beijing, China) was selected for sequencing. All sequences obtained were analyzed following the online instructions of Quantitative Insights Into Microbial Ecology (QIIME).47 After filtering sequences to remove ambiguous nucleotides, primer sequences and low-quality reads, the clean reads were clustered into the operational taxonomic units (OTUs) using de novo cluster. Then UCLUST clustering was used to identify the OTUs at 97% sequence similarity.48 OTUs with at least two sequences were selected for further analysis. The most abundant sequence of each cluster was selected to represent the OTU. PyNAST was used to align the taxonomy of each representative sequence, and RDP Classifier 2.2 was adopted to assign taxonomic status and all referred to the Greengenes 13.8 database.49,50 The alpha diversity of the collembolan gut microbiota was calculated and principal coordinate analysis (PCoA) based on Bray−Curtis distances was conducted to compare the differences between collembolan gut bacterial communities in different treatments. All high throughput sequence data have been submitted to the National Center for Biotechnology Information Sequence Read Archive under accession number SRP140544. Statistical Analysis. Mean values ± standard error (SE) of all data were calculated using Microsoft Excel 2013. The IBM SPSS version 20 statistical software package was used to compare the differences between different samples at the 0.05 significance level by single factor analysis of variance (ANOVA). Observed species, Shannon diversity index, Chao

certified reference material following digestion ranged from 92% to 106%. DNA Extraction. The collembolans obtained were washed with 2.5% (m/v) sodium hypochlorite solution three times and sterilized water five times to minimize contamination with microbiota from the body surfaces. Guts of the collembolans were dissected with sterile forceps under a sterile dissecting microscope. About 10 collembolan guts were collected in a 1.5 mL centrifuge tube and 20 mL proteinase K and 180 mL ATL solutions were added for DNA extraction. The centrifuge tube was then vortexed for 5 min and incubated at 56 °C for 12 h. After incubation, total DNA was isolated from the collembolan guts using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines. Finally, the collembolan gut DNA was eluted with 50 μL AE solution. The DNA samples obtained were stored at −20 °C for further analysis. High Throughput Quantitative PCR (HT-qPCR). The characteristics of ARGs in the gut microbiome of the soil collembolan were investigated using the SmartChip Real-Time PCR System (Warfergen Inc., Fremont, CA) to perform highthroughput quantitative PCR reactions on the gut DNA obtained. The 296 primer pairs for HT-qPCR were included in this test (Table S1), and target genes of these primers consisted of a 16S rRNA gene, 285 ARGs (almost all classes of ARGs) and 10 mobile genetic elements (MGEs) comprising 1 clinical class 1 integron, 1 class 1 integron, and 8 transposases. The MGEs make an important contribution to the exchange of ARGs between microorganisms.45 Amplification of each primer pair was conducted in 100 nL reaction systems which included LightCycler 480 SYBR Green I Master Mix (Roche Inc., Indianapolis, IN), each primer, collembolan gut DNA template and nuclease-free PCR grade water. The conditions of amplification were: initially 95 °C for 10 min and then 40 cycles consisting of 95 °C for 30 s and 60 °C for 30 s. Three replicates were amplified in each primer pair along with a nontemplate control. All results of HT-qPCR for detected genes were analyzed using the SmartChip qPCR software (v 2.7.0.1). The sample was discarded when amplification efficiency was beyond the range (90−110%). ARGs of the sample were confirmed when three replicates had all been amplified. We used a threshold cycle (CT) of 31 as the detection limit of amplification in this test according to previous studies.19,46 The normalized copy abundance of C

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Environmental Science & Technology 1 and PD whole tree of collembolan gut microbiota were calculated with QIIME, and the inverse Simpson index and Shannon evenness score were obtained using R with vegan 2.4−3.51 The Adonis test (nonparametric multivariate analysis of variance) based on Bray−Curtis distances was used to identify significant differences in the collembolan gut OTU composition between different treatments. The Adonis test, HCLUST, nonmetric multidimensional scaling (NMDS) analysis, Mantel test and Procrustes test were conducted in the vegan 2.4−3 of R, and the heatmap was produced using R with the heatmap package.52 A Venn diagram of shared bacterial OTUs between different collembolan gut samples was drawn using the online Venny 2.1.0. The associated OTUs of collembolan gut samples were obtained via the ANOVA and ttest based on the Bayesian model (FDR-corrected, P < 0.05) in R version 3.4.1.53 Visualization of collembolan gut microbial associated OTUs was achieved by network analysis using Gephi 0.9.1. Indicator species analysis was performed in R with the indicspecies package which depicts species characteristic of the collembolan gut microbiota in different treatments.54 Additional graphics were produced with OriginPro 9.1.

Figure 2. Relative read abundance (mean ± SE, n = 3) of the collembolan gut microbial community at the phylum level in all treatments after 28 days of exposure. Different letters indicate significant differences between different treatments of the same phylum at the 0.05 level (ANOVA). Phyla with relative abundance 0.05, Figure S2). Ionic Ag exposure significantly decreased the reproduction of the collembolan compared to the control (F2,15 = 20.68, P < 0.001; Tukey HSD, P < 0.001), by 19.3%, but AgNPs had no significant influence (Figure 1a). The concentrations of Ag in the collembolan body increased significantly above the control (0.6 mg Ag kg−1) due to exposure to ionic Ag and AgNPs, reaching 59.7 and 33.7 mg kg−1, respectively (F2,15 = 1244, P < 0.001; Figure 1b). Bioaccumulation of Ag was higher in the ionic Ag exposure treatment. Collembolan Gut Microbial Taxonomic Composition. Overall, 1 339 496 high quality sequences were obtained and the number of reads per sample ranged from 69 659 to 239 271. A total of 5305 OTUs were identified and each sample had an average of 1480 OTUs. Proteobacteria (65.8%), Firmicutes (25.2%), and Actinobacteria (5.9%) were the dominant phyla in the collembolan gut microbiota, occupying 96.9% of the total reads (Figure 2). Brucellaceae (21.1%) and Pseudomonadaceae (32.6%) were the two predominant families in the gut microbiota, especially in Ag-exposed collembolans (Figure S3a). The abundance of Proteobacteria in AgNP-exposed collembolans was 2.3 times higher than in the control (P = 0.019), but the abundance of Firmicutes and Actinobacteria decreased significantly in the AgNP treatment (P < 0.05; Figure 2). Compared with the control, exposure to ionic Ag also significantly reduced the abundance of Actinobacteria in the gut microbiota (by 84.8%; P = 0.005; Figure 2). At family level the highest abundances of Rickettsiaceae (8.1%), Staphylococcaceae (19.9%) and Bacillaceae (20.1%) were found in the control gut microbiota, and the highest abundance of Enterococcaceae (14.4%) was recorded in AgNP-exposed collembolans (Figure S3a). Collembolan Gut Microbial Diversity and Community Structure. Regarding the alpha diversity of collembolan gut microbiota, all six diversity parameters (observed species, Shannon diversity index, Chao 1, PD whole tree, inverse

Simpson index and Shannon evenness score) showed no significant differences between treatments (ANOVA, P > 0.05), but a decreasing trend was observed within the ionic Ag treatment (Table S2). However, in the case of the collembolan gut microbial community structure the Adonis test indicates that Ag-exposed treatments differed significantly from the control (Figure 3a, P = 0.03). Principal coordinates analysis also shows that Ag-exposed collembolan gut samples were separated from the control on the primary axis (explaining 40.42% of the total variance), and their distribution was more diffuse (Figure 3a). Collembolan gut samples of the control were clustered together by HCLUST (Figure 3b). Shared Microbiota. A total of 91 shared OTUs were recorded among the control and the ionic Ag and AgNPs treatments, occupying 95% of total reads (Figure 4a). The lowest number of unique OTUs (51) occurred in the ionic Agexposed collembolans (Figure 4a). Among the shared dominant genera, the relative abundances of Microbacterium, Paracoccus, Wolbachia, and Staphylococcus in Ag-exposed collembolans were lower than in the control (ANOVA, P < 0.05; Figure S3b). The 16 most abundant families with >1% relative abundance in the gut microbiota were used to construct the associated network (ANOVA, P < 0.05 indicating association, P > 0.05 indicating shared; Figure 4b). Ten of the 16 abundant families were shared across all treatments, and four associated families were found in the control but no associated family was observed in Ag-exposed collembolans. Indicator species analysis also indicates that six characteristic OTUs were identified in the control, and the AgNP-exposed animals had five characteristic OTUs but no characteristic OTUs were found in the ionic Ag treatment (Figure 1). Composition and Diversity of ARGs. Overall, 41 unique ARGs (conferring resistance to eight classes of antibiotics: aminoglycoside, tetracycline, other, chioramphenicol, MLSB, sulfonamide, β-lactamase and vancomycin) and 1 transposase were detected across all gut samples (Figure 5). Ionic Ag exposure significantly decreased the number of ARGs and MGEs compared to the control (by 44.2%, P < 0.05) but there was no significant difference in AgNP-exposed collembolans (P > 0.05; Figure S4a). The abundance of ARGs and MGEs in the control was approximately three times higher than in Ag-

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DOI: 10.1021/acs.est.8b02825 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Figure 3. (a) Principal coordinates analysis (PCoA) of collembolan gut samples based on the relative abundance of bacterial OTUs using Bray− Curtis distances. Different colors and shapes indicate different treatments. The sample variation explained by the first two PCoA axes is listed in parentheses. (b) HCLUST based on Bray−Curtis distances reveals the differences between the collembolan gut microbiota of different treatments.

Figure 4. (a) Venn diagram shows the number of shared bacterial OTUs between collembolan gut samples of different treatments. (b) Network analysis of collembolan gut microbiota with the relative abundance of associated OTUs at the family level. OTUs with >1% abundance are presented in the network. The size of node represents numbers of connections, and increasing size indicates more connections.

exposed treatments (ANOVA, P < 0.01; Figure S4b). The heatmap clearly shows that the abundance of most ARGs was significantly reduced in Ag-exposed collembolans compared to the control, especially cmr, tetG, and tetPB (ANOVA, P < 0.05; Figure 5). The transposase (tnpA) was detected in the control only. There was a significant difference between ARG profiles of different treatments (Anosim test, P = 0.033). Nonmetric multidimensional scaling analysis also indicates that ARG profiles of control samples were clustered and separated from samples of the Ag exposure treatments (stress = 0.1126; Figure S5a). Relationship between Gut Bacterial Communities and ARG Profiles. The Procrustes analysis and Mantel test were used to evaluate relationships between collembolan gut bacterial communities and ARG profiles based on the Bray− Curtis distance (Figure S5b). No significant correlation was found between gut microbial communities (bacterial OTU data) and ARG profiles (P = 0.173). Similarly, the results of the Procrustes analysis (M2 = 0.5171, P = 0.0305) also indicate that the ARG profiles had a weak correlation with the collembolan gut bacterial communities (Figure S5b).

Table 1. Results of Indicator Species Analysis Revealing the Association between OTUs and Treatmenta indicator OTUs unassigned (2313) Bacillaceae (9650) Myxococcus Lactococcus Bacillaceae (7570) Gordonia Lactobacillales (3733) Stenotrophomonas Lactobacillales (7210) Comamonas Granulicatella

association (treatment) indicator value control control control control control control AgNPs AgNPs AgNPs AgNPs AgNPs

1.000 1.000 1.000 0.998 0.990 0.980 1.000 1.000 0.992 0.989 0.988

P-value 0.042 0.042 0.042 0.042 0.042 0.020 0.033 0.033 0.033 0.033 0.033

a

In the indicator OTUs column the name represents the taxonomic affiliation of the OTU according to the GenBank BLASTsearch. Numbers in parentheses are specific OTU identifiers. Results present only those OTUs whose relative abundance was >0.03% and significance P < 0.05.

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Figure 5. Heat map depicts the relative abundance of all detected ARGs and MGEs in each collembolan gut sample. The ARGs and MGEs are arranged according to their category.

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DISCUSSION Our study indicates that 28 days of dietary exposure to AgNPs or ionic Ag at 200 mg kg−1 resulted in no change in collembolan body weight or adult mortality but ionic Ag, in contrast to AgNPs, significantly decreased the reproduction of the collembolan. This suggests that dietary exposure to Ag has an adverse effect on reproduction and ionic Ag was more toxic than AgNPs. In addition, the study of Ag nanoparticles and AgNO3 dietary exposure also shows that ionic Ag has a higher toxicity to soil isopods.43 In the present study, higher Ag accumulation was found in collembolans exposed to ionic Ag than in those exposed to AgNPs (Figure 1b), and this may account for the higher toxicity of ionic Ag. In this study we have found for the first time that exposure to AgNPs and ionic Ag significantly altered the composition of the collembolan gut bacterial community, indicating that oral AgNP exposure can produce adverse effects on soil collembolan gut microbiota. Although it is easy to attribute this to the antibacterial activity of silver ions,24 the findings extend our knowledge to effects of AgNPs on nontarget organisms. Our results and previous studies show no overt toxicity (growth, mortality, and reproduction) in collembolans exposed to AgNPs at 200 mg kg−1.42,55 The gut microbial community makes a crucial contribution to host health54 and changes in the gut microbiota may therefore affect animal health (e.g., nutrient absorption and the immune system) through AgNP exposure. The gut microbiota may therefore be a more sensitive indicator of AgNP exposure than growth, mortality, or reproduction of the animals. Studies of other animals (e.g., mice24 and fish31) have also shown that exposure

to AgNPs disturbed their gut microbiota but had no significant impact on their growth. The relative abundance of the phylum Proteobacteria tended to increase in AgNP-exposed gut microbiota, occupying 89% (Figure 2). This suggests that the gut microbiota may be dysbiotic in the AgNP exposure treatment. It has been confirmed in many animal species that an increase in the abundance of Proteobacteria will result in an imbalanced gut microbiota. 56 Lower diversity and larger variation in collembolan gut microbiota have been found in Ag exposure treatments and this supports our suggestion. These results indicate that exposure to AgNPs can disturb the balance of the gut microbiota. In our study the relative abundance of Enterococcaceae was highest in the AgNP-exposed collembolans. It has been recently confirmed that Enterococcaceae include many important pathogens57 and thus AgNP exposure might increase pathogenic infection of collembolans. Our results show that no significant differences in collembolan gut microbiota relative read abundance were observed between AgNP and AgNO3 treatments. This might indicate a similar mode of action of AgNPs and AgNO3 on the gut microbiota. In other words, they may both transform into similar chemical species (ionic Ag) in the collembolan gut. However, their effects on collembolan reproduction were distinct. This might be because only a portion of AgNPs was transformed into ionic Ag in the collembolan guts compared to the AgNO3 treatment, and ionic Ag was more toxic than AgNPs. In addition, exposure to both AgNPs and ionic Ag significantly reduced the abundance of Wolbachia in the collembolan guts. It is well established that Wolbachia is a widespread bacterial endosymbiont of the collembolan F. candida,9 and a previous study F

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Environmental Science & Technology indicates that antibiotic treatment caused sterility of F. candida owing to the elimination of Wolbachia.58 Our reproduction result also indicates that exposure to Ag reduced the reproduction of the collembolan, suggesting that Ag exposure may affect collembolan reproduction by reducing the abundance of Wolbachia. Other work has reported the disruption of an endosymbiont of earthworms due to arsenic exposure.59 Nevertheless, the Venn diagram and network analysis both show that the dominant microbiota were shared among the different treatments, indicating that the core microbiota of the collembolan gut was not eliminated due to AgNP exposure. Our results indicate that the abundance and diversity of ARGs decreased in the Ag-exposed collembolan gut microbiota (Figure 5). This contrasts with antibiotics and graphene which can increase the incidence of ARGs in animal guts.19,38 This may be due to AgNPs being a nonantibiotic bactericide24 that can kill many types of antibiotic-resistant bacteria. A significant decrease in the phylum Actinobacteria was found in the Agexposed collembolan gut microbiota and this may partly explain the reduction in ARGs. Many antibiotics originating from the phylum Actinobacteria can usually lead to the production of resistant bacteria under natural conditions.19,60 This is also supported by a previous study showing the enrichment of ARGs following an increase in Actinobacteria due to exposure to antibiotics.19 Although MGEs were identified in the collembolan gut of the control in the present study, they were not detected in Ag-exposed collembolan guts, indicating that the absence of MGEs may also be an important explanation for the decrease in ARGs via exposure to AgNPs. The abundance of most common ARGs (e.g., cmr, tetG, and tetPB) was significantly reduced in AgNP-exposed collembolan guts. These ARGs are usually detected in diverse environments including the human gut.45,46 Furthermore, collembolans can be found in almost all soil environments and they play a key role in soil food webs.3,39,61 Thus, AgNPs might be able to reduce the ecological risk of ARGs in soil food webs. A weak relationship was identified between collembolan gut bacterial communities and ARG profiles in this study, in contrast to the strong correlations found in other environments (e.g., soils,62 groundwaters,63 sewage sludges,64 and the phyllosphere65). This might be explained largely by the core microbiota (Ochrobactrum, Microbacterium, Paracoccus, Wolbachia, and Staphylococcus) in the collembolan gut remaining unchanged following AgNP exposure, but the taxa of ARGs showed a distinct change (Figure 5). Despite the fact that the balance of the collembolan gut microbial community was disturbed due to AgNP exposure, the unique habitat of the collembolan gut (e.g., anaerobic and high organic environment) may have protected the core microbiota from any change. A previous study also found that ARG profiles were not correlated with microbial communities in drinking water.62 These results suggest that the relationship between ARG profiles and bacterial communities may be flexible, especially in the animal gut under pollutant stress. More studies may be required to further explore this relationship. In general, Ag can accumulate in collembolan body tissues via dietary exposure, resulting in a decrease in collembolan reproduction, and ionic Ag has a more toxic effect than AgNPs. Exposure to 200 mg kg−1 AgNPs (50 nm) with no coatings disturbs the composition of soil collembolan gut bacterial community, resulting in dysbiosis of the gut microbiota. However, the core microbiota of the collembolan gut are not

eliminated. In addition, AgNP exposure can reduce the incidence of ARGs in the collembolan gut microbiota. Since the characteristics of AgNPs (e.g., TEM diameter, composition, and coatings) play a key role in the toxicity of AgNPs, we should carefully consider the effects of AgNP characteristics in future studies. These results extend our knowledge regarding the effects of AgNPs on soil nontarget animals and may help us understand the role of AgNPs in the incidence of ARGs in the soil ecosystem.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b02825. Table S1: Information on 296 genes detected in the gene chip; Table S2: diversity parameters of the collembolan gut microbiota after 28 days of exposure; Figure S1: (a) Characteristic of AgNPs (50 nm) using transmission electron microscopy analysis, (b) b1 showing the characteristics of yeast using the transmission electron microscopy analysis, and b2 depicting the distribution of AgNPs in the yeast of b1 by SEMEDS, (c) the forms of AgNPs after adsorption in the yeast followed by freeze-drying (c1, ×10 000 magnification and c2, ×70 000 magnification); Figure S2: (a) number and (b) body weight of living adult collembolans (mean ± SE, n = 6) in all treatments after 28 days of exposure; Figure S3: (a) relative abundance of collembolan gut bacteria at family level and (b) shared dominant genus level in all treatments after 28 days of exposure; Figure S4: (a) number and (b) normalized abundance of ARGs and MGEs detected in collembolan gut samples of all treatments after 28 days of exposure (mean ± SE, n = 3); and Figure S5: (a) nonmetric multidimensional scaling (NMDS) analysis depicting the overall distribution pattern of ARGs in the collembolan gut microbiota of all treatments based on the Bray−Curtis distance, (b) procrustes test revealing the relationship between ARG profiles and collembolan gut bacterial community (16S rRNA gene OTU data) based on Bray−Curtis dissimilarity metrics (sum of squares M2 = 0.5171, r = 0.6949, P < 0.05, 9999 permutations) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 592- 6190997; fax: +86 592-6190977; e-mail: [email protected]. ORCID

Yong-Guan Zhu: 0000-0003-3861-8482 Author Contributions ⊥

D.Z. and F.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the National Natural Science Foundation of China (41571130063), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB15020302 and XDB15020402), and the National Key Research and Development Program of ChinaInternational G

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Environmental Science & Technology

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Collaborative Project from the Ministry of Science and Technology (Grant No. 2017YFE0107300).

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