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Environmental effects of nanoparticles on the ecological succession of gut

microbiota across zebrafish development

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DOI: 10.1016/j.scitotenv.2021.150963

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Environmental effects of nanoparticles on the ecological succession of gut microbiota

across zebrafish development
Pubo Chen a,1, Jie Huang b,1, Liuyu Rao b, Wengen Zhu b, Yuhe Yu b, Fanshu Xiao a,⁎, Huang Yu a, Yongjie Wu a,
Ruiwen Hu a, Xingyu Liu a, Zhili He a,c, Qingyun Yan a,⁎
a
Environmental Microbiomics Research Center, School of Environmental Science and Engineering, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Sun Yat-sen
University, Guangzhou 510006, China
b
Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
c
College of Agronomy, Hunan Agricultural University, Changsha 410128, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Nanoparticles exposure decreased the

microbial diversity of gut microbiota at
73–90 dph.
• nTiO2 altered the composition of the
microbial communities at 53–90 dph.
• Homogeneous selection mainly governed
the community assembly of fish
microbiomes.
• The microbial networks in the gut micro-
biota were destabilized by the nanoparti-
cles exposure.

a r t i c l e i n f o a b s t r a c t

Article history: The environmental stresses could significantly affect the structure and functions of microbial communities colo-
Received 20 August 2021 nized in the gut ecosystem. However, little is known about how engineered nanoparticles (ENPs), which have re-
Received in revised form 3 October 2021 cently become a common pollutant in the environment, affect the gut microbiota across fish development. Based
Accepted 9 October 2021
on the high-throughput sequencing of the 16S rRNA gene amplicon, we explored the ecological succession of gut
Available online xxxx
microbiota in zebrafish exposed to nanoparticles for three months. The nanoparticles used herein including tita-
Editor: Henner Hollert nium dioxide nanoparticles (nTiO2, 100 μg/L), zinc oxide nanoparticles (nZnO, 100 μg/L), and selenium
nanoparticles (nSe, 100 μg/L). Our results showed that nanoparticles exposure reduced the alpha diversity of
gut microbiota at 73–90 days post-hatching (dph), but showed no significant effects at 14–36 dph. Moreover,
Keywords: nTiO2 significantly (p < 0.05) altered the composition of the gut microbial communities at 73–90 dph
Environmental effects (e.g., decreasing abundance of Cetobacterium and Vibrio). Moreover, we found that homogeneous selection was
Zebrafish the major process (16.6–57.8%) governing the community succession of gut microbiota. Also, nanoparticles ex-
Engineered nanoparticles posure caused topological alterations to microbial networks and led to increased positive interactions to destabi-
Gut microbiota
lize the gut microbial community. This study reveals the environmental effects of nanoparticles on the ecological
High-throughput sequencing
succession of gut microbiota across zebrafish development, which provides novel insights to understand the gut
microbial responses to ENPs over the development of aquatic animals.
© 2021 Published by Elsevier B.V.

⁎ Corresponding authors.
E-mail addresses: xiaofansh@mail.sysu.edu.cn (F. Xiao), yanqy6@mail.sysu.edu.cn (Q. Yan).
1
These authors contributed equally.

https://doi.org/10.1016/j.scitotenv.2021.150963
0048-9697/© 2021 Published by Elsevier B.V.

Please cite this article as: P. Chen, J. Huang, L. Rao, et al., Environmental effects of nanoparticles on the ecological succession of gut microbiota
across zebrafi..., Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2021.150963
P. Chen, J. Huang, L. Rao et al.
1. Introduction
The gut microbiota in the intestinal lumen is presenting as an extra
“organ” of the host due to its essential roles such as metabolism and
pathogens defense (Lozupone et al., 2012; Zhang et al., 2021). Under-
standing how the environmental stresses affect gut microbiota is espe-
cially crucial to host health, immunity and development (Jin et al., 2017;
Stagaman et al., 2017). Although the rapid development of nanotech-
nology greatly benefits humans, it poses threats to environmental safety
(Patil et al., 2016). Engineered nanoparticles (ENPs) (e.g., titanium diox-
ide (nTiO2), Zinc dioxide (nZnO), and Selenium (nSe)) have been
widely used in the applications of medicine, food processing and
nutrition (Li et al., 2011; Singh et al., 2016). Recent researches have
shown that the concentration of ENPs in aquatic environments ranged
from ng/L to μg/L (Bundschuh et al., 2018; Peng et al., 2020). For
example, the concentration of nTiO2 in surface water reached about
2.2 μg/L (Sun et al., 2016). Moreover, ENPs can not only release to the
aquatic environments through atmospheric deposition, soil percolate,
or waste stream treatment (Fabrega et al., 2011; Turan et al., 2019),
but also can enter the food chain directly and enrich in the living
organism (Chen et al., 2017; Dudefoi et al., 2017). The accumulation of
ENPs in aquatic environments will inevitably cause harm to aquatic
organisms and humans when reaching a certain concentration (Abbas
et al., 2020).
As expected, many studies showed that exposure to ENPs and metal
ions released from nanoparticles could produce various biotoxicity ef-
fects on aquatic organisms such as fish, crustaceans, protozoa and
algae (Jiang et al., 2014; Lei et al., 2018; Patlolla et al., 2012). The toxic
effects of ENPs are facilitated by the generation of reactive oxygen spe-
cies (Ma et al., 2018; Wang et al., 2018), resulting in oxidative stress,
damage DNA and lipid peroxidation (Mao et al., 2016; Zheng et al.,
2019). Ingested ENPs are regarded as a major exposure route for the
fish gut ecosystem, which may negatively affect both aquatic organisms
and their gut microbiota (Clark et al., 2021; Evariste et al., 2019). Also,
ENPs can induce dysregulated microbiota and affect the health-
associated gut homeostasis due to its antibacterial activity (Van Den
Brûle et al., 2015; Rajkumar et al., 2016). For example, acute exposure
of nTiO2 or nZnO inhibited the growth of commensal beneficial
microorganisms through inducing gut inflammation to exacerbate
microbiota dysbiosis (Chen et al., 2018; Merrifield et al., 2013;
Miranda et al., 2016). Another study also reported that nAg treatment
on zebrafish for 35 days could change the composition and structure
of the gut microbial community (Ma et al., 2018). However, such
understanding is mainly restricted to a short period. Thus, there is an
urgent need to explore the impacts of ENPs on the gut microbiota in
aquatic animals over host development.
The environmental microbes surrounding the fish colonize into the
gut begins shortly after the fish hatched from the embryos (Bakke
et al., 2015; Vestrum et al., 2020). The early microbiota directly involves
fish's larval stage of gut maturation and immune system, and they are
also associated with the host health and fitness (Semova et al., 2012).
The symbiotic healthy microbiota also provides extra nutrition to fish
by producing fermentation outputs, which protects against colonization
by microbial pathogens (Holmes et al., 2011). But, gut microbiota
dysbiosis may cause immune-related reaction, such as the host immune
response (Dobranowski et al., 2019). In this respect, strategies for
assessing the risk of ENPs to the gut microbiota succession are essential.
The ecological processes and molecular ecological networks analysis
have also been useful tools to study the effects of exogenous stressors
on gut microbiota succession (Cheaib et al., 2020; Li et al., 2019).
Zebrafish has recently become a powerful model for studying host-
microbiota interactions (Burns et al., 2016; Yan et al., 2012). We hy-
pothesized that nanoparticle exposure would disturb the ecological suc-
cession of gut microbiota and largely depend on the fish development.
To test this hypothesis, we separately exposed zebrafish to nTiO2,
nZnO and nSe (100 μg/L) from larvae to adult. High-throughput
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sequencing of the 16S rRNA gene was performed to visualize the
dynamics of gut microbial communities. This study provides an
important insight on assessing the toxic aquatic environmental
impacts on the ecological succession of gut microbiota over fish
development.
2. Materials and methods
2.1. Preparation and characterization of nanoparticles suspensions
Three nanoparticles, including ZnO (99.9%, 30 ± 10 nm), TiO2
(99.8%, 5–10 nm) and Se (99.8%; 40 ± 10 nm) were purchased from
the Macklin Biochemical Co. Ltd. Shanghai, China. The powders of nano-
particles were suspended in filtered deionized water (Millipore, Biller-
ica, MA). The stock suspensions were then diluted immediately and
sonicated (50 W/L, 40 kHz) to prevent aggregation. Fresh nanoparticles
stock suspensions were renewed every 48 h to keep a relatively stable
concentration for the experiment. Although the diameter of three nano-
particles were approximately ranging from 5 to 40 nm, we measured
the size and size distribution of nanoparticles by dynamic light scatter-
ing (DLS) instruments using a Zetasizer Nano ZS (Malvern Panalytical
Ltd., Malvern, UK). Transmission electron microscopy (TEM) and X-
ray Diffraction (XRD) were used to visualize size and morphology of
nanoparticles.
2.2. Experimental design and sampling
To investigate the effect of nanoparticles on the gut microbiota suc-
cession from larvae to adult, we exposed larvae of zebrafish to different
types of nanoparticles (Fig. 1). Zebrafish were maintained in 20 L glass
tanks with full aerated tap water to avoid additional environmental per-
turbation, and the experiment was performed under stable conditions
(28 ± 0.5 °C, 14/10 h light/dark cycle). The hatched zebrafish (AB
strain) were randomly divided to 12 glass tanks and designated as con-
trol, nZnO, nTiO2 and nSe (triplicate tanks per treatment). The control
group only raised in the standard condition. Specifically, zebrafish
were fed with boiled egg yolk twice daily beginning at 8 dph, then fed
with newly hatched brine shrimp (Artemia nauplii) twice daily (9:00
and 15:00) after 15 dph.
Importantly, half of the exposure water was refreshed daily and the
whole exposure water was exchanged weekly using fresh water con-
taining nanoparticles to keep the consistent concentrations of nanopar-
ticles in each tank. The exposure water was removed by siphoning to
clear the possible deposited nanoparticles. There are four key develop-
mental events: the entire gut tract is first open and microbial coloniza-
tion of the lumen first occurs (4 dph), fish must rely on ingesting
external foods after the yolk sac consumed completely (10 dph), the
maturation of the adaptive immune system (21–35 dph), and sexual
maturity and dimorphism (75 dph). So, seven times of gut sampling
(7, 14, 24, 36, 53, 73, 90 dph) were performed accordingly. At each sam-
pling time, 3 replicates of fish were collected from each tank, and we to-
tally got 84 samples. After record the body weight and length, each
zebrafish was immediately anesthetized, dissected aseptically and col-
lected the gut tissues, subsequently stored at −80 °C for DNA extraction.
All experimental protocols involved in the fish experiment were sup-
ported by the Institutional Animal Care and Use Committee of the
Institute of Hydrobiology, Chinese Academy of Sciences (Approval ID:
Keshuizhuan 08529).
2.3. Histological analysis
To compare the potential effects of nanoparticles on gonad at 90
dph, we cut gonad sections using (5-μm thickness) Leica RM2235 mi-
crotome. After deparaffinization and rehydration, thin sections were
mounted on glass slides and then colored with eosin. The stained sec-
tions were imaged with an ECLIPSE Ni\\U microscope (Nikon, Tokyo,
P. Chen, J. Huang, L. Rao et al.
Fig. 1. Experimental design and the key developmental events during the exposure of nTiO2 (100 μg/L), nZnO (100 μg/L), and nSe (100 μg/L). Exposures lasted three months, and gut
samples were collected at seven sampling times (i.e., 7, 14, 24, 36, 53, 73, 90 dph).
Japan) and the photographic documentation was recorded by a Digit
Sight DS-Fi2 camera.
2.4. 16S rRNA gene sequencing analysis
The genomic DNA of gut microorganisms were extracted by using
the PowerFecal® Kit (Mo Bio, CA, USA). The 16S rRNA gene (V3-V4 re-
gions) was then amplified by the primers set 338F (5′-ACTCCTACGGG
AGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The
50 μL amplification system including 50 ng DNA, 1× buffer with dNTP
and Taq polymerase, 0.2 mM forward and reserve primers. The PCR
was then performed by a pre-denatured (95 °C) for 5 min, then 30 cycles
of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s, and finally extended at
72 °C for an additional 10 min. Negative controls were also included in
determining the absence of contamination. PCR products were tested
by agarose gel (1%) electrophoresis. Each of the amplified products
was quantified and then combined equally. The target band was puri-
fied by QIAquick Gel Extraction Kit (Qiagen, CA, USA), re-quantified
and then prepared to create sequencing libraries. The libraries were se-
quenced on the HiSeq 2500 platform (Illumina, CA, USA) using a
2 × 250 bp kit in Guangdong Magigene Biotechnology Co., Ltd.
Illumina sequence reads were analyzed using the public pipeline
(http://mem.rcees.ac.cn:8080/) (Feng et al., 2017). In brief, the se-
quences were first assembled using FLASH (Magoč and Salzberg,
2011), and the low-quality sequences such as those less than 140 bp
and moving-window (5 bp) quality score less than 20 were not in-
cluded in subsequent analysis. The final sequences were assigned to op-
erational taxonomy units (OTUs) by UPARSE at a 97% similarity
threshold. The generated OTU table including all the 84 samples was
rarefied to 15,058 reads per sample for subsequent analysis. Taxonomy
classification in this paper was performed according to the GreenGene
database (http://greengenes.lbl.gov/).
2.5. Ecological process analysis
We calculated the major ecological processes to disentangle the effects
of nanoparticles on the gut microbial community assembly (Dini-
Andreote et al., 2015; Stegen et al., 2013). The dynamics of phylogenetic
and taxonomic diversity were measured with the null model-based
beta nearest taxon indices (β-NTI) and Bray-Curtis-based Raup-Crick
(RCBray) metrics, respectively. The β-NTI > 2 represented heterogeneous
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selection, and β-NTI < −2 indicated homogeneous selection. The |β-
NTI| < 2 and RCBray < −0.95 were denoted as the homogenizing
dispersal, whereas |β-NTI| < 2 and RCBray > 0.95 were interpreted as
the dispersal limitation. If the |β-NTI| < 2 and |RCBray| < 0.95, that
means no dominant assembly process as described above (Stegen et al.,
2013).
2.6. Network construction and characterization
In order to determine the gut microbial interactions in response to
nanoparticles exposure, we constructed the networks for gut microbi-
ota based on Spearman correlation among OTUs at three stages (7–14,
24–36 53–90 dph). Each network was constructed with OTUs in more
than 2/3 samples. The connectivity of nodes was represented by its
within-module connectivity (Zi, the degree to how one node connected
to other nodes within the same module) and among-module connectiv-
ity (Pi, the degree to the node connected to different modules). Node
topological properties were classified into module hubs (Zi > 2.5,
Pi ≤ 0.62; critical to its module coherence), connectors (Zi ≤ 2.5,
Pi > 0.62), network hubs (Zi > 2.5, Pi > 0.62) and peripherals
(Zi < 2.5, Pi ≤ 0.62). Analysis of global network properties was
performed using the Molecular Ecological Network Analyses (MENA)
Pipeline (http://ieg2.ou.edu/MENA/) (Deng et al., 2012). The module
network diagrams were visualized with Cytoscape 3.8.2.
2.7. Statistical analysis
We carried out a multivariate regression tree (MRT) analysis to esti-
mate the association between the gut microbial community and impact
factors (e.g., developmental stage, nanoparticles exposure) as previ-
ously described (Xiao et al., 2021). This method has previously been ap-
plied to establish a model for species-environment relationships, and it
is therefore appropriated for datasets of various forms of ecological in-
teraction. Mantel and partial Mantel tests have been widely used to ex-
plore associations between community structure and environmental
factors based on both Bray–Curtis and Jaccard distances.
The indexes of Shannon and Chao1 were used to represent the
alpha-diversity and calculated by using the package of VEGAN in R
(v.4.0.5). In order to visualize the whole patterns of microbial communi-
ties, we performed non-metric multidimensional scaling (NMDS).
Heatmap was generated based on the relative abundance of the major
P. Chen, J. Huang, L. Rao et al. Science of the Total Environment xxx (xxxx) xxx

genera using the AUTOMAP package. The community dissimilarities

were tested by the analysis of similarity (ANOSIM) and permutational
multivariate analysis of variance (PERMANOVA) using the VEGAN
package in R (Oksanen et al., 2007). To identify putative bacterial
bioindicators at different nanoparticles exposure, linear discriminant
analysis (LDA) and effect size (LEfSe) analyses were performed
(Segata et al., 2011). Significance tests were performed by Tukey test
through one-way analysis (ANOVA) to measure whether differences
between treatments were significant or not.

3. Results

3.1. Phenotypic analyses of zebrafish during the nanoparticle exposure

Compared to the control, the bodyweight of zebrafish in the nTiO2

and nZnO exposure groups had a significant decrease (p < 0.05) after
53 days of exposure. We also found that body length was significantly
(p < 0.05) decreased by nZnO exposure. At 90 days, no significant
difference in the body length of zebrafish among the four groups
(Table S1). At the last sampling, the gonad tissues of the four groups
were stained for sectioning, considering the zebrafish has reached
sexual maturity within about three months (Fig. S1). There was no
effect on gonads development observed after 90 days of nanoparticles
exposure.

3.2. Characterization of nTiO2, nZnO, nSe

The toxicity of nanoparticles is mainly determined by their par-

ticle size. Therefore, we employed TEM and DLS to evaluate size
and structure characterization of three nanoparticles (Fig. S2).
Briefly, TEM analysis revealed the nanoparticles were often linked
into aggregates and isolated particles were rare. According to DLS
results, the average size distribution of nTiO2, nZnO and nSe were
122 nm, 220 nm, and 295 nm, respectively. nSe showed a relatively
high degree of agglomeration, but their average diameters are all
600 < nm.
Fig. 2. The variation of alpha-diversity of zebrafish gut microbiota as indicated by the
Shannon (A) and Chao1 (B). * means significant difference (ANOVA, p < 0.05) among
3.3. Effects of the nanoparticles on microbial diversity and structure in the
treatments. Data presented as mean ± SE (standard error, n = 3).
zebrafish gut

Total 1,355,480 high-quality sequencing reads were obtained from

Illumina-HiSeq sequencing of 84 gut samples. Both Shannon and
Chao1 indexes showed a downward trend within exposure groups
after 53 dph (Fig. 2). Particularly, only nTiO2 significantly (p < 0.05)
decreased both Shannon and Chao1 indexes of gut microbiota after
53 dph compared to the control, suggesting that gut microbiota
incurred more exposure and risk. However, the diversity of microbial
communities associated with each exposure group showed no
significant (p > 0.05) changes than the control before 53 dph, except
in 7 dph of nZnO exposure. Overall, these results indicated that the
nanoparticles exposure did not change the gut microbiota diversity
before 53 dph.
To identify differences among the gut microbial communities of
different groups, the NMDS plots could be divided into three
developmental stages (7–14 dph, 24–36 dph, 53–90 dph), indicat-
ing development stage shifted the structure of gut microbial com-
munities (stress = 0.14, ANOSIM's R2 = 0.57, p < 0.001) (Fig. 3).
The PERMANOVA test further confirmed the significant difference
(p < 0.05, Table 1). Specifically, microbial communities clustered
more tightly at 24–36 dph, while 24–36 and 53–90 dph were al-
most separated with NMDS. At 53–90 dph, three exposure groups
and the control group were almost separated from the controls by
the NMDS, and nTiO 2 exposure increased the difference mostly
among treatment groups in the microbial structure. It implied
that nanoparticle exposure increased the difference among Fig. 3. A non-metric multidimensional scaling (NMDS) ordination based on the Bray-
groups in the microbial structure. Curtis distance of gut microbiota across zebrafish development in different treatments.

4
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Table 1 enriched in the nTiO2 exposure group, indicated the nTiO2 exposure
Permutational multivariate analysis of variance (PERMANOVA) showing the community would disturb the gut microbiota. Moreover, exposed to nTiO2 also
differences of gut microbiota among zebrafish developmental stages.
depressed the Rhodobacter, Tepidmonas, Acinetobacter and Pseudomonas
Bray-Curtis Jaccard genus (0.5%, 0.7%, 0.4%, 0.3%, respectively, p < 0.05). Collectively,
F p F p statistical analysis of taxonomic composition changed after 53 dph
revealed several major differences in nTiO2 exposure group.
nTiO2 7–14 dph vs. 24–36 dph 2.20 0.068 3.59 0.003
7–14 dph vs. 53–90 dph 17.34 0.001 3.02 0.001 We further explored the nanoparticles-induced specific bacterial taxa
24–36 dph vs. 53–90 dph 27.20 0.001 6.08 0.001 by a LEfSe analysis at 53 (Fig. S4A), 73 (Fig. S4B) and 90 dph (Fig. S4C),
nZnO 7–14 dph vs. 24–36 dph 2.29 0.003 3.57 0.002 aimed to explain any potential biomarkers. The structure and predomi-
7–14 dph vs. 53–90 dph 2.06 0.003 2.44 0.003 nant taxonomic of microbiota were represented in a cladogram. Com-
24–36 dph vs. 53–90 dph 3.58 0.001 4.71 0.001
nSe 7–14 dph vs. 24–36 dph 3.70 0.005 4.21 0.002
pared with the control, exposure groups showed an increment of taxa
7–14 dph vs. 53–90 dph 5.94 0.001 3.12 0.001 that belong to phylum Proteobacteria, such as Promicromonospora,
24–36 dph vs. 53–90 dph 11.31 0.003 5.69 0.001 Rhodobacter, Legionellales, and Tatlockia (LDA score ≥ 3.5). The taxonomic
branch of Bacillaceae and Gemmata in the exposure groups was overrep-
resented in comparison with the control, indicating that those taxa
might represent potential biomarkers for nanoparticle exposure.
3.4. Environmental effects of nanoparticles on composition of gut
microbiota 3.5. The succession of gut microbiota affected by the nanoparticle exposure

Key taxonomic of zebrafish gut microbial communities were signifi-
cantly altered under nanoparticles exposure. In the taxonomy analysis,
the gut microbial communities were dominated by Proteobacteria
(50.3–75.2%), Fusobacteria (6.1–31.3%), Actinobacteria (3.8–9.3%),
Firmicutes (2.4–4.8%) and Bacteroidetes (1.5–4.1%) accounted for up-
wards of 90.1 to 99.3% percent of those communities at the phylum
level of gut microbial communities (Fig. S3). Specifically, the relative
abundances of Fusobacteria after nTiO2 exposures ranged from 58.7%
to 82.6% at 53–90 dph, which exhibited a higher abundance than the
control (p < 0.05). In contrast, the relative abundance of Proteobacteria
was significantly reduced from 22.2% to 11.1% (p < 0.05) by the nTiO2
disturbance.
The heatmap further showed the top 25 genera in different treatment
groups (Fig. 4A). The most abundant genera across all samples were
Cetobacterium, Rhodobacter, Tepidmonas, Acinetobacter, Pseudomonas and
Vibrio at 53–90 dph (Fig. 4B). Among these, the relative abundance of
Cetobacterium and Vibrio after nTiO2 exposure was significantly higher
than the control (72.7% and 2.9%, respectively; p < 0.001). This is espe-
cially important because the pathogenic genus Vibrio was significantly
As nanoparticles exposure exhibited a less pronounced effect on the
diversity and structure of zebrafish gut microbiota before 53 dph, we
next wanted to determine the different factors that might contribute
to the microbiota succession (i.e., developmental stage, food and nano-
particles exposure). The recursive partitioning analysis used for the
multiple regression trees (MRT) with 12 environmental variables con-
structed the tree with 13 branches, showing 25.3% of the variance in di-
versity. Especially, the estimations of trees were first divided by
developmental stage (25.3%), indicating that nanoparticles exposure
appeared not to affect the gut microbiota succession before 53 dph. In
comparison, the nanoparticles exposure explained a half proportion of
variance at a later stage (12.5% at 53 dph). These differences were also ob-
served for phylogenetic diversity (PD) and Shannon index (p < 0.05)
among three developmental stages (Fig. 5). The stages of 24–36 dph
had the highest alpha-diversity. Mantel tests indicated that nanoparticles
exposure significantly affected the microbial succession (p < 0.05), and
partial Mantel tests confirmed the relationships between microbial diver-
sity and nanoparticles when controlling the effects of host development
or food (p < 0.05, Table S3).

Fig. 4. The top 25 genera showed in the heatmap (A), and the relative abundance of key genera at 53–90 dph (B). Different letters above the bars indicated significant difference among
groups (ANOVA, p < 0.05), whereas the same letter suggested no significant difference. Data presented as mean ± SE (Standard error, n = 9).

5
P. Chen, J. Huang, L. Rao et al.
Fig. 5. The multivariate regression tree (MRT) analysis performed based on the Bray-Curtis distance with interactions of different factors (i.e., developmental stage, nanoparticles
exposure). The alpha-diversity of phylogenetic diversity (PD) and Shannon were given according to zebrafish development. The variations among stages were tested through an
ANOVA with least-significant-difference (LSD) tests. The presence of different letters denotes significant differences, and the same letter indicates no significant difference.
3.6. Deterministic processes governing the gut microbiota assembly
The Bray-Curtis distance based null model test indicated that fish gut
microbiota was significantly (p < 0.05) different from the null random
expectation (Table S2). The phylogenetic turnover analysis demon-
strated that selection was one of the essential processes in community
assembly (Fig. 6). During the exposure of nanoparticles, the selection
process was much more pronounced in juvenile and adult stages,
while dispersal played a vital role in assembling gut microbiota in lar-
vae. Regarding nonselective processes (RCbray), dispersal limitation
played a principal role in all stages, except at 53–90 dph when homoge-
neous dispersal became more dominant. For example, at 7–14 dph, the
proportion of dispersal limitation (55.0%) and undominated (21.6%)
processes were the major organizing forces in gut microbial communi-
ties. Moreover, the homogeneous selection could result in community
composition being driven towards similar under consistent environ-
mental conditions, accounting for 16.6–57.8% at two stages (24–36
dph and 53–90 dph). Therefore, the gut microbiota assembly was
mainly driven by homogeneous selection from 24 to 90 dph.
3.7. Network stability in fish gut microbial communities
To determine how different nanoparticles affected microbial net-
work properties, we selected modules associated with at least two
nodes and visualized the main modules with more than ten nodes. Mi-
crobial interactions were divided into negative links (blue lines) and
positive links (red lines). The positive links responsible for 47.4–76.2%
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Science of the Total Environment xxx (xxxx) xxx
were observed at different treatments (Fig. 7). The microbial interac-
tions among three developmental stages in exposure groups had higher
positive connections than those in control. Moreover, the network prop-
erties contained modules with modularity (M) values > 0.285 in all
groups (Table 2). Particularly, exposure groups of microbial community
networks were lower in modularity than the control (Fig. 7, Table 2), in-
dicating that microbial communities in nanoparticles exposure environ-
ments are in an unstable state. During the nanoparticle exposure,
Proteobacteria dominated all of the large modules and coexisted with
Actinobacteria.
The taxa of module hubs and connectors were regarded as keystone
because they are thought to play a crucial role in network topology
(Deng et al., 2012). As shown in Zi-Pi plots, these connectors mainly
concentrated in 53–90 dph, demonstrating that the microbial commu-
nity needed more connections to maintain stability (Fig. S5A and B).
Furthermore, the exposure group at 24–36 dph had the less module
hubs than the control, which implied networks had sparse interactions
within modules due to disturbance of nanoparticles. Comparing se-
quences of keystone OTUs indicated that some Proteobacteria and
Firmicutes members were considered as the dominant keystone taxa
in all networks (Table S4).
4. Discussion
Recently, an extensive use of ENPs results in serious contamination
in aquatic environments (Hochella et al., 2019). However, the response
of gut microbiota of fish to nanoparticles pollution during their life cycle
P. Chen, J. Huang, L. Rao et al.
Fig. 6. The quantified major ecological processes governing the gut microbial
communities. The percentages (numbers on the individual bars) were given the relative
contribution of each process to the community succession, and the remaining parts
attributed to undominated process.
remains unknown. Here, we explored the effects of nanoparticles on the
ecological succession of gut microbiota across zebrafish development.
Our results indicated that exposure to nanoparticles altered the compo-
sition of gut microbiota and decreased the stability of interaction
networks.
Consistent with a previous study, we found that the nanoparticle ex-
posure reduced the microbial diversity of gut microbiota at 73–90 dph,
suggesting that the nanoparticle exposure decreases the diversity of mi-
crobial community in the gut (Chen et al., 2021). Similarly, nanoparti-
cles (i.e., Ag and TiO2) or persistent organic pollutants (i.e., bisphenol
A and benzo[a]pyrene) could decrease the diversity of the microbial
community in the gut ecosystem (Cattò et al., 2019; Chen et al., 2018;
DeBofsky et al., 2021). The NMDS analysis showed that microbial com-
munities were exclusively grouped among different nanoparticles ex-
posure treatment. These results reflected that nanoparticles exposure
treatment contributed to the variations in gut microbiota which was
in accordance with the findings about antibiotic-induced variations in
the pig gut microbiota (Mateos et al., 2018). However, most of such
studies were mainly restricted to a short-term experiment and focused
on adult fish.
We detected remarkable changes in microbial composition at 53
dph, indicating that the gut microbiota succession was also associ-
ated with host development (Li et al., 2014; Mach et al., 2015) due
to intrinsic (i.e., host physiological situation) and extrinsic factors
(i.e., environmental exposures) (Burns et al., 2017; Tsolis and
Bäumler, 2020). Our results showed that Proteobacteria was a pre-
dominant phylum in gut before 53 dph. A recent study also indicated
that Proteobacteria were the most dominant phylum in gut microbi-
ota at the larvae stage of the lake sturgeon (Abdul Razak and Scribner,
2020). Additionally, the majority of gut microbes interacted with gut
tissues as a symbiont with beneficial effects on the host, which were
crucial for the early stages (Bates et al., 2006; Daisley et al., 2020). At
7
Science of the Total Environment xxx (xxxx) xxx
the genus level, the nTiO2 exposure increased the relative abundance
of Vibrio, which may result in inflammation and tissue damage to the
gut (Jia et al., 2019). Here, we suspected that the perturbation of gut
microbial composition in zebrafish probably induced the imbalance of
the metabolic ability, causing the reduction of body weight and length
after nTiO2 exposure. We also employed LefSe software to analyze
differences and predicted the potential biomarkers among different
treatment at 53–90 dph. The biomarker of Gemmata was identified to
have a strong hydrolyzing ability and promote the breakdown of or-
ganic matter (Ma et al., 2020). Although the composition of the micro-
bial community did not vary substantially at early stages,
microorganisms in the gut might have different responses due to the
bioaccumulation and biotoxicity of nanoparticles.
The ecological and evolutionary processes were quantified to deter-
mine the influence of nanoparticles exposure on microbial assembly
and turnover. Our results indicated that homogeneous selection was
the most predominant ecological process governing the gut microbiota
at 24–36 dph, which resulted in the similar community composition
under consistent environmental filtering (Ge et al., 2021). This may ex-
plain why juvenile zebrafish showed more similar gut microbiota than
other stages. A recent study on the gut microbiota of Tibetan herdsmen
also revealed that the homogeneous selection was the major process
overriding the effects of natural environmental changes (Li et al.,
2018). In contrast, the dispersal limitation was only important at the
7–14 dph, suggesting the dispersal limitation also generated composi-
tional differences (Moeller et al., 2017). This work showed the dynamics
in assembly processes of gut microbiota, allowing us to consider the im-
pact of nanoparticles on microbial succession from an ecological insight.
Co-occurrence network analysis might play an essential role in re-
vealing the interactions of microbial taxa under nanoparticle exposure
(Cai et al., 2021). We found that nanoparticles exposure destabilized
gut microbial community networks with less complexity, less modular-
ity, and more positive interactions. This finding raises a critical question
that whether another environmental disturbance is consistent with de-
creased network stability in the gut microbiota. A previous study sug-
gested that zebrafish exposed to diethylhexyl phthalate also reduced
the network connectivity and decreased stability (Buerger et al.,
2020). Moreover, it is well accepted that a stable microbial community
is often associated with more negative interactions and increased net-
work modularity (Coyte et al., 2015; Hernandez et al., 2020). Also, we
found that keystone taxa in the gut microbial communities were in-
creased under the nanoparticles exposure. One possible explanation
was that a common adaptive driving force (i.e., host immunity re-
sponse) recruited these distinct keystone taxa to withstand the distur-
bance, resulting in more connections with other modules (Widder
et al., 2014). As a result, the gut microbial communities under nanopar-
ticles exposure may be more sensitive with a vulnerable modularity.
Multiple factors including host developmental stages, environmen-
tal changes and food can impact the succession of gut microbiota
(Xiao et al., 2021). Zebrafish may open the mouth around 3 dph,
allowing microbial colonization of their gastrointestinal tracts in
which simultaneously serve as a habitat for diverse microorganisms
(Brinkmann et al., 2020). Then the diverse colonized microbiota in
turn contributes to host health and development (Rawls et al., 2006).
That means the beneficial relationship between gut microbes and the
host could affect the microbial community assembly at early stages.
Therefore, the relative instability of the gut microbiota at the early
stages could be caused by gut development. Additionally, the nanopar-
ticle characteristics including size, agglomeration, released metal ions
and shape in suspension determine their toxicological assessment (Cai
et al., 2020; Hedberg et al., 2019). The gut microbial alpha-diversity
did not decrease at 7–36 dph because nanoparticles are likely to ag-
glomerate in water, which may lead to limited exposure to the small
size of larval and juvenile zebrafish. Similarly, a previous study also ob-
served the limited toxicity of agglomerated nanoparticles to larval
zebrafish (Chen et al., 2011). Also, the nanoparticles may have indirect
P. Chen, J. Huang, L. Rao et al. Science of the Total Environment xxx (xxxx) xxx

Fig. 7. Highly connected modules within zebrafish gut microbial networks at different nanoparticles exposure. Colors of nodes indicate different major phyla. Red links indicate positive
correlations between two individual nodes, whereas blue links indicate negative correlations between nodes. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)

effects on the gut microbiota. For example, the distribution of the nano-
particle agglomerates would inhibit nutrient availability in the environ-
ment (Agans et al., 2019). In other words, nanoparticles would enter the
gut to inhibit zebrafish nutrient absorption from the water environ-
ment. Although the main toxicity of nanoparticles comes from itself or
from the metal ions released is still a matter of debate (Sengul and
Asmatulu, 2020), the possible effects of the metal ions released on the
gut microbes cannot be disregarded. As a result, gut community
assembly may be governed by both nanoparticles toxicity and host
development.
In conclusion, this study elucidated the effects of nanoparticles on
the gut microbiota throughout the life cycle of zebrafish. First, nanopar-
ticles exposure reduced the microbial diversity of gut microbiota after
53 dph and nTiO2 shape both composition and structure of gut

Table 2
Topological properties of the empirical molecular ecological networks in comparison to the random networks.

Empirical networks Random networksa

2
Similarity Modularity R of Average Average clustering Harmonic Average clustering Harmonic Modularity
threshold power-law degree coefficient geodesic distance coefficient (avgCC) geodesic distance
(avgK) (avgCC) (HD) (HD)

7–14 dph Control 0.920 0.509 0.672 6.648 0.332 3.535 0.127 ± 0.012 2.634 ± 0.030 0.308 ± 0.007
Exposure 0.740 0.285 0.722 9.686 0.522 2.222 0.262 ± 0.020 2.106 ± 0.013 0.224 ± 0.008
24–36 dph Control 0.920 0.823 0.831 3.255 0.256 6.233 0.013 ± 0.006 4.406 ± 0.044 0.579 ± 0.008
Exposure 0.640 0.625 0.852 4.253 0.243 3.429 0.058 ± 0.011 2.996 ± 0.039 0.439 ± 0.007
53–90 dph Control 0.810 0.681 0.871 3.833 0.284 4.406 0.041 ± 0.011 3.158 ± 0.042 0.482 ± 0.010
Exposure 0.570 0.292 0.630 13.789 0.494 2.156 0.372 ± 0.015 1.988 ± 0.013 0.157 ± 0.005
a
Given are the mean ± standard derivations that generated from 100 times of randomly rewired networks.

8
P. Chen, J. Huang, L. Rao et al.
microbial community at 53–90 dph. Second, the gut microbiota
assembly was mainly driven by homogeneous selection due to host
development and nanoparticles exposure. Third, the gut microbial
networks showed more positive links, lower modularity after nanopar-
ticles exposure. These findings enhance our understanding of the effects
of nanoparticles pollution on the gut microbial community across
zebrafish development.
CRediT authorship contribution statement
Pubo Chen: Conceptualization, Software, Writing – original draft. Jie
Huang: Conceptualization, Investigation. Liuyu Rao: Methodology.
Wengen Zhu: Methodology. Yuhe Yu: Conceptualization. Fanshu
Xiao: Visualization, Data curation. Huang Yu: Visualization, Writing –
review & editing. Yongjie Wu: Writing – review & editing. Ruiwen
Hu: Writing – review & editing. Zhili He: Writing – review & editing.
Qingyun Yan: Conceptualization, Methodology, Writing – review &
editing.
Declaration of competing interest
All authors declare no conflict of interest. None of the materials pre-
sented in this manuscript has been previously published, nor are they
under consideration for publication elsewhere.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (31802350, 92051120), the Innovation Group Project of
Southern Marine Science and Engineering Guangdong Laboratory
(Zhuhai) (311021006), the Fundamental Research Funds for the Central
Universities (19lgzd28), the Hundred Talents Program through Sun
Yat-sen University (18821107), and the Youth Innovation Promotion
Association of the Chinese Academy of Sciences (2019333).
Data availability
The raw sequencing data can be found at the National Centre for
Biotechnology Information (NCBI) Sequence Read Archive (SRA)
[Accession number: PRJNA757184].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.150963.
References
Abbas, Q., Yousaf, B., Ullah, H., Ali, M.U., Ok, Y.S., Rinklebe, J., 2020. Environmental trans-
formation and nano-toxicity of engineered nano-particles (ENPs) in aquatic and ter-
restrial organisms. Crit. Rev. Environ. Sci. Technol. 50, 2523–2581.
Abdul Razak, S., Scribner, K.T., 2020. Ecological and ontogenetic components of larval lake
sturgeon gut microbiota assembly, successional dynamics, and ecological evaluation
of neutral community processes. Appl. Environ. Microbiol. 86, e02662-19.
Agans, R.T., Gordon, A., Hussain, S., Paliy, O., 2019. Titanium dioxide nanoparticles elicit
lower direct inhibitory effect on human gut microbiota than silver nanoparticles.
Toxicol. Sci. 172, 411–416.
Bakke, I., Coward, E., Andersen, T., Vadstein, O., 2015. Selection in the host structures the
microbiota associated with developing cod larvae (Gadus morhua). Environ.
Microbiol. 17, 3914–3924.
Bates, J.M., Mittge, E., Kuhlman, J., Baden, K.N., Cheesman, S.E., Guillemin, K., 2006. Distinct
signals from the microbiota promote different aspects of zebrafish gut differentiation.
Dev. Biol. 297, 374–386.
Brinkmann, B.W., Koch, B.E., Spaink, H.P., Peijnenburg, W.J., Vijver, M.G., 2020. Colonizing mi-
crobiota protect zebrafish larvae against silver nanoparticle toxicity. Nanotoxicology 14,
725–739.
Buerger, A.N., Dillon, D.T., Schmidt, J., Yang, T., Zubcevic, J., Martyniuk, C.J., Bisesi Jr., J.H.,
2020. Gastrointestinal dysbiosis following diethylhexyl phthalate exposure in
zebrafish (Danio rerio): altered microbial diversity, functionality, and network con-
nectivity. Environ. Pollut. 265, 114496.
9
Science of the Total Environment xxx (xxxx) xxx
Bundschuh, M., Filser, J., Lüderwald, S., McKee, M.S., Metreveli, G., Schaumann, G.E.,
Metreveli, G., Schaumann, G.E., Schulz, R., Wagner, S., 2018. Nanoparticles in the en-
vironment: where do we come from, where do we go to? Environ. Sci. Eur. 30, 1–17.
Burns, A.R., Stephens, W.Z., Stagaman, K., Wong, S., Rawls, J.F., Guillemin, K., Bohannan,
B.J., 2016. Contribution of neutral processes to the assembly of gut microbial commu-
nities in the zebrafish over host development. ISME J. 10, 655–664.
Burns, A.R., Miller, E., Agarwal, M., Rolig, A.S., Milligan-Myhre, K., Seredick, S., Steve, S.,
Karen, G., Brendan, J.M.B., 2017. Interhost dispersal alters microbiome assembly
and can overwhelm host innate immunity in an experimental zebrafish model.
Proc. Natl. Acad. Sci. U. S. A. 114, 11181–11186.
Cai, X., Liu, X., Jiang, J., Gao, M., Wang, W., Zheng, H., Xu, S., Li, R., 2020. Molecular mech-
anisms, characterization methods, and utilities of nanoparticle biotransformation in
nanosafety assessments. Small 16, 1907663.
Cai, S., Wang, H., Tang, J., Tang, X., Guan, P., Li, J., Xu, R., 2021. Feedback mechanisms of
periphytic biofilms to ZnO nanoparticles toxicity at different phosphorus levels.
J. Hazard. Mater. 416, 125834.
Cattò, C., Garuglieri, E., Borruso, L., Erba, D., Casiraghi, M.C., Cappitelli, F., Zanchi, R., 2019.
Impacts of dietary silver nanoparticles and probiotic administration on the microbi-
ota of an in-vitro gut model. Environ. Pollut. 245, 754–763.
Cheaib, B., Seghouani, H., Ijaz, U.Z., Derome, N., 2020. Community recovery dynamics in
yellow perch microbiome after gradual and constant metallic perturbations.
Microbiome 8, 1–19.
Chen, T.H., Lin, C.Y., Tseng, M.C., 2011. Behavioral effects of titanium dioxide nanoparticles
on larval zebrafish (Danio rerio). Mar. Pollut. Bull. 63, 303–308.
Chen, H., Zhao, R., Wang, B., Cai, C., Zheng, L., Wang, H., Feng, W., 2017. The effects of
orally administered Ag, TiO2 and SiO2 nanoparticles on gut microbiota composition
and colitis induction in mice. NanoImpact 8, 80–88.
Chen, L., Guo, Y., Hu, C., Lam, P.K.S., Lam, J.C.W., Zhou, B., 2018. Dysbiosis of gut microbiota
by chronic coexposure to titanium dioxide nanoparticles and bisphenol A: implica-
tions for host health in zebrafish. Environ. Pollut. 234, 307–317.
Chen, P., Huang, J., Rao, L., Zhu, W., Yu, Y., Xiao, F., Chen, X., Yu, H., Wu, Y., Xu, K., Zheng, X.,
Hu, R., He, Z., Yan, Q., 2021. Resistance and resilience of fish gut microbiota to silver
nanoparticles. mSystems 6, e00630-21.
Clark, N.J., Clough, R., Boyle, D., Handy, R.D., 2021. Quantification of particulate ag in rain-
bow trout organs following dietary exposure to silver nitrate, or two forms of
engineered silver nanoparticles. Environ. Sci. Nano. 8, 1642–1653.
Coyte, K.Z., Schluter, J., Foster, K.R., 2015. The ecology of the microbiome: networks, com-
petition, and stability. Science 350, 663–666.
Daisley, B.A., Chmiel, J.A., Pitek, A.P., Thompson, G.J., Reid, G., 2020. Missing microbes in
bees: how systematic depletion of key symbionts erodes immunity. Trends Microbiol.
28, 1010–1021.
DeBofsky, A., Xie, Y., Challis, J.K., Jain, N., Brinkmann, M., Jones, P.D., Giesy, J.P., 2021. Re-
sponses of juvenile fathead minnow (Pimephales promelas) gut microbiome to a
chronic dietary exposure of benzo [a] pyrene. Environ. Pollut. 278, 116821.
Deng, Y., Jiang, Y., Yang, Y., He, Z., Luo, F., Zhou, J., 2012. Molecular ecological network
analyses. BMC Bioinformatics 13, 1–20.
Dini-Andreote, F., Stegen, J.C., Van Elsas, J.D., Salles, J.F., 2015. Disentangling mechanisms
that mediate the balance between stochastic and deterministic processes in microbial
succession. Proc. Natl. Acad. Sci. U. S. A. 112, E1326–E1332.
Dobranowski, P.A., Tang, C., Sauvé, J.P., Menzies, S.C., Sly, L.M., 2019. Compositional
changes to the ileal microbiome precede the onset of spontaneous ileitis in SHIP de-
ficient mice. Gut Microbes 10, 578–598.
Dudefoi, W., Moniz, K., Allen-Vercoe, E., Ropers, M.-H., Walker, V.K., 2017. Impact of food
grade and nano-TiO2 particles on a human intestinal community. Food Chem.
Toxicol. 106, 242–249.
Evariste, L., Barret, M., Mottier, A., Mouchet, F., Gauthier, L., Pinelli, E., 2019. Gut microbi-
ota of aquatic organisms: a key endpoint for ecotoxicological studies. Environ. Pollut.
248, 989–999.
Fabrega, J., Luoma, S.N., Tyler, C.R., Galloway, T.S., Lead, J.R., 2011. Silver nanoparticles: be-
haviour and effects in the aquatic environment. Environ. Int. 37, 517–531.
Feng, K., Zhang, Z., Cai, W., Liu, W., Xu, M., Yin, H., Wang, A., He, Z., Deng, Y., 2017. Biodi-
versity and species competition regulate the resilience of microbial biofilm commu-
nity. Mol. Ecol. 26, 6170–6182.
Ge, Y., Jing, Z., Diao, Q., He, J.Z., Liu, Y.J., 2021. Host species and geography differentiate
honeybee gut bacterial communities by changing the relative contribution of com-
munity assembly processes. mBio 12, e00751-21.
Hedberg, J., Blomberg, E., Odnevall Wallinder, I., 2019. In the search for nanospecific ef-
fects of dissolution of metallic nanoparticles at freshwater-like conditions: a critical
review. Environ. Sci. Technol. 53, 4030–4044.
Hernandez, D.J., David, A.S., Menges, E.S., Searcy, C.A., Afkhami, M.E., 2020. Environmental
stress destabilizes microbial networks. ISME J. 15, 1–13.
Hochella, M.F., Mogk, D.W., Ranville, J., Allen, I.C., Luther, G.W., Marr, L.C., Yang, Y., 2019.
Natural, incidental, and engineered nanomaterials and their impacts on the earth sys-
tem. Science 363, eaau8299.
Holmes, E., Li, J.V., Athanasiou, T., Ashrafian, H., Nicholson, J.K., 2011. Understanding the
role of gut microbiome-host metabolic signal disruption in health and disease. Trends
Microbiol. 19, 349e359.
Jia, P., Sun, T., Junaid, M., Xiong, Y., Wang, Y., Liu, L., Pu, S., Pei, D., 2019. Chronic exposure
to graphene oxide (GO) induced inflammation and differentially disturbed the intes-
tinal microbiota in zebrafish. Environ. Sci. Nano. 6, 2452–2469.
Jiang, H.S., Qiu, X.N., Li, G.B., Li, W., Yin, L.Y., 2014. Silver nanoparticles induced accumula-
tion of reactive oxygen species and alteration of antioxidant systems in the aquatic
plant Spirodela polyrhiza. Environ. Toxicol. Chem. 6, 1398–1405.
Jin, Y., Wu, S., Zeng, Z., Fu, Z., 2017. Effects of environmental pollutants on gut microbiota.
Environ. Pollut. 222, 1–9.
P. Chen, J. Huang, L. Rao et al.
Lei, C., Sun, Y., Tsang, D.C., Lin, D., 2018. Environmental transformations and ecological ef-
fects of iron-based nanoparticles. Environ. Pollut. 232, 10–30.
Li, M., Zhu, L., Lin, D., 2011. Toxicity of ZnO nanoparticles to Escherichia coli: mechanism
and the influence of medium components. Environ. Sci. Technol. 45, 1977–1983.
Li, J., Ni, J., Li, J., Wang, C., Li, X., Wu, S., Zhang, T., Yu, Y., Yan, Q., 2014. Comparative study
on gastrointestinal microbiota of eight fish species with different feeding habits.
J. Appl. Microbiol. 117, 1750–1760.
Li, H., Li, T., Li, X., Wang, G., Lin, Q., Qu, J., 2018. Gut microbiota in tibetan herdsmen re-
flects the degree of urbanization. Front. Microbiol. 9, 1745.
Li, H., Zhou, R., Zhu, J., Huang, X., Qu, J., 2019. Environmental filtering increases with ele-
vation for the assembly of gut microbiota in wild pikas. Microb. Biotechnol. 12,
976–992.
Lozupone, C.A., Stombaugh, J.I., Gordon, J.I., Jansson, J.K., Knight, R., 2012. Diversity, stabil-
ity and resilience of the human gut microbiota. Nature 489, 220–230.
Ma, Y., Song, L., Lei, Y., Jia, P., Lu, C., Wu, J., Strauss, P.R., Pei, D., 2018. Sex dependent effects
of silver nanoparticles on the zebrafish gut microbiota. Environ. Sci. Nano. 5,
740–751.
Ma, J., Chen, Q., O’Connor, P., Sheng, G.D., 2020. Does soil CuO nanoparticles pollution alter
the gut microbiota and resistome of Enchytraeus crypticus? Environ. Pollut. 256,
113463.
Mach, N., Berri, M., Estellé, J., Levenez, F., Lemonnier, G., Denis, C., Lepage, P., 2015. Early-
life establishment of the swine gut microbiome and impact on host phenotypes. En-
viron. Microbiol. Rep. 7, 554–569.
Magoč, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of short reads to improve
genome assemblies. Bioinformatics 27, 2957–2963.
Mao, L., Liu, C., Lu, K., Su, Y., Gu, C., Huang, Q., Petersen, E.J., 2016. Exposure of few layer
graphene to Limnodrilus hoffmeisteri modifies the graphene and changes its bioaccu-
mulation by other organisms. Carbon 109, 566–574.
Mateos, I., Combes, S., Pascal, G., Cauquil, L., Barilly, C., Cossalter, A.M., Oswald, I.P., 2018.
Fumonisin-exposure impairs age-related ecological succession of bacterial species in
weaned pig gut microbiota. Toxins 10, 230.
Merrifield, D.L., Shaw, B.J., Harper, G.M., Saoud, I.P., Davies, S.J., Merrifield, R.D., Henry, T.B.,
2013. Ingestion of metal-nanoparticle contaminated food disrupts endogenous mi-
crobiota in zebrafish (Danio rerio). Environ. Pollut. 174, 157–163.
Miranda, R.R., Damaso Da Silveira, A.L.R., De Jesus, I.P., Grötzner, S.R., Voigt, C.L., Campos,
S.X., Garcia, J.R.E., Randi, M.A.F., Oliveira, R.C.A., Filipak, N.F., 2016. Effects of realistic
concentrations of TiO2 and ZnO nanoparticles in Prochilodus lineatus juvenile fish.
Environ. Sci. Pollut. Res. 23, 5179–5188.
Moeller, A.H., Suzuki, T.A., Lin, D., Lacey, E.A., Wasser, S.K., Nachman, M.W., 2017. Dis-
persal limitation promotes the diversification of the mammalian gut microbiota.
Proc. Natl. Acad. Sci. U. S. A. 114, 13768–13773.
Oksanen, J., Kindt, R., Legendre, P., O’Hara, B., Stevens, M.H.H., Oksanen, M.J., Suggests,
M.A.S.S., 2007. The vegan package. Community Ecol. 10, 719.
Patil, S.S., Shedbalkar, U.U., Truskewycz, A., Chopade, B.A., Ball, A.S., 2016. Nanoparticles
for environmental clean-up: a review of potential risks and emerging solutions. Envi-
ron. Technol. Innov. 5, 10–21.
Patlolla, A.K., Berry, A., May, L., Tchounwou, P.B., 2012. Genotoxicity of silver nanoparticles
in Vicia faba: a pilot study on the environmental monitoring of nanoparticles. Int.
J. Environ. Res. Public Health 5, 1649–1662.
Peng, L., Fu, D., Qi, H., Lan, C.Q., Yu, H., Ge, C., 2020. Micro-and nano-plastics in marine en-
vironment: source, distribution and threats—a review. Sci. Total Environ. 698,
134254.
10
View publication stats
Science of the Total Environment xxx (xxxx) xxx
Rajkumar, K., Kanipandian, N., Thirumurugan, R., 2016. Toxicity assessment on
haemotology, biochemical and histopathological alterations of silver nanoparticles-
exposed freshwater fish Labeo rohita. Appl. Nanosci. 6, 19–29.
Rawls, J.F., Mahowald, M.A., Ley, R.E., Gordon, J.I., 2006. Reciprocal gut microbiota trans-
plants from zebrafish and mice to germ-free recipients reveal host habitat selection.
Cell 127, 423–433.
Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S., Huttenhower, C.,
2011. Metagenomic biomarker discovery and explanation. Genome Biol. 12, 1–18.
Semova, I., Carten, J.D., Stombaugh, J., Mackey, L.C., Knight, R., Farber, S.A., Rawls, J.F., 2012.
Microbiota regulate intestinal absorption and metabolism of fatty acids in the
zebrafish. Cell Host Microbe 3, 277–288.
Sengul, A.B., Asmatulu, E., 2020. Toxicity of metal and metal oxide nanoparticles: a review.
Environ. Chem. Lett. 18, 1659–1683.
Singh, P.K., Jairath, G., Ahlawat, S.S., 2016. Nanotechnology: a future tool to improve qual-
ity and safety in meat industry. J. Food Sci. Technol. 53, 1739–1749.
Stagaman, K., Burns, A.R., Guillemin, K., Bohannan, B.J., 2017. The role of adaptive immu-
nity as an ecological filter on the gut microbiota in zebrafish. ISME J. 11, 1630–1639.
Stegen, J.C., Lin, X., Fredrickson, J.K., Chen, X., Kennedy, D.W., Murray, C.J., 2013. Quantify-
ing community assembly processes and identifying features that impose them. ISME
J. 7, 2069–2079.
Sun, T.Y., Bornhöft, N.A., Hungerbühler, K., Nowack, B., 2016. Dynamic probabilistic
modeling of environmental emissions of engineered nanomaterials. Environ. Sci.
Technol. 50, 4701–4711.
Tsolis, R.M., Bäumler, A.J., 2020. Gastrointestinal host-pathogen interaction in the age of
microbiome research. Curr. Opin. Microbiol. 53, 78–89.
Turan, N.B., Erkan, H.S., Engin, G.O., Bilgili, M.S., 2019. Nanoparticles in the aquatic envi-
ronment: usage, properties, transformation and toxicity—a review. Process. Saf. Envi-
ron. Prot. 130, 238–249.
Van Den Brûle, S., Ambroise, J., Lecloux, H., Levard, C., Soulas, R., De Temmerman, P.J.,
Lison, D., 2015. Dietary silver nanoparticles can disturb the gut microbiota in mice.
Part. Fibre. Toxicol. 13, 1–16.
Vestrum, R.I., Attramadal, K.J., Vadstein, O., Gundersen, M.S., Bakke, I., 2020. Bacterial com-
munity assembly in Atlantic cod larvae (Gadus morhua): contributions of ecological
processes and metacommunity structure. FEMSMicrobiol. Ecol. 96, fiaa163.
Wang, X., Yang, F., Zhao, J., Xu, Y., Mao, D., Zhu, X., Luo, Y., Alvarez, P.J.J., 2018. Bacterial
exposure to ZnO nanoparticles facilitates horizontal transfer of antibiotic resistance
genes. NanoImpact 10, 61–67.
Widder, S., Besemer, K., Singer, G.A., Ceola, S., Bertuzzo, E., Quince, C., Sloan, W.T., Rinaldo,
A., Battin, T.J., 2014. Fluvial network organization imprints on microbial co-
occurrence networks. Proc. Natl. Acad. Sci. U. S. A. 111, 12799–12804.
Xiao, F., Zhu, W., Yu, Y., He, Z., Wu, B., Wang, C., Shu, L., Li, X., Yin, H., Wang, J., Juneau, P.,
Zheng, X., Wu, Y., Li, J., Chen, X., Hou, D., Huang, Z., He, J., Xu, G., Xie, L., Huang, J., Yan,
Q., 2021. Host development overwhelms environmental dispersal in governing the
ecological succession of zebrafish gut microbiota. npj Biofilms Microbiomes 7, 1–12.
Yan, Q., Van Der Gast, C.J., Yu, Y., 2012. Bacterial community assembly and turnover
within the intestines of developing zebrafish. PLoS ONE 7, e30603.
Zhang, T., Zhu, X., Guo, J., Gu, A., Li, D., Chen, J., 2021. Toxicity assessment of nano-ZnO ex-
posure on the human intestinal microbiome, metabolic functions, and resistome
using an in vitro colon simulator. Environ. Sci. Technol. 55, 6884–6896.
Zheng, M., Lu, J., Lin, G., Su, H., Sun, J., Luan, T., 2019. Dysbiosis of gut microbiota by dietary
exposure of three graphene-family materials in zebrafish (Danio rerio). Environ.
Pollut. 254, 112969.