Journal Pre-proof

Effects of titanium dioxide nanoparticle exposure on the gut

microbiota of pearl oyster (Pinctada fucata martensii)

Fengfeng Li, Yujing Lin, Chuangye Yang, Yilong Yan, Ruijuan

Hao, Robert Mkuye, Yuewen Deng

PII: S1532-0456(24)00074-7

DOI: https://doi.org/10.1016/j.cbpc.2024.109906

Reference: CBC 109906

To appear in: Comparative Biochemistry and Physiology, Part C

Received date: 2 February 2024

Revised date: 5 March 2024

Accepted date: 21 March 2024

Please cite this article as: F. Li, Y. Lin, C. Yang, et al., Effects of titanium dioxide

nanoparticle exposure on the gut microbiota of pearl oyster (Pinctada fucata martensii),

Comparative Biochemistry and Physiology, Part C (2023), https://doi.org/10.1016/

j.cbpc.2024.109906

This is a PDF file of an article that has undergone enhancements after acceptance, such

as the addition of a cover page and metadata, and formatting for readability, but it is

not yet the definitive version of record. This version will undergo additional copyediting,

typesetting and review before it is published in its final form, but we are providing this

version to give early visibility of the article. Please note that, during the production

process, errors may be discovered which could affect the content, and all legal disclaimers

that apply to the journal pertain.

? 2024 Published by Elsevier Inc.

Journal Pre-proof

Journal Pre-proof

Effects of titanium dioxide nanoparticle exposure on the gut microbiota of pearl

oyster (Pinctada fucata martensii)

Fengfeng Li a

, Yujing Lin a

, Chuangye Yang a, b, c, d*

, Yilong Yan a

, Ruijuan Hao e

,

Robert Mkuye a

, Yuewen Deng a, b, c, d, f

a. Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, China

b. Guangdong Science and Innovation Center for Pearl Culture, Zhanjiang 524088,

China

c. Pearl Breeding and Processing Engineering Technology Research Centre of

Guangdong Province, Zhanjiang, 524088, China

d. Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and

Healthy culture, Zhanjiang, 524088, China

e. Development and Research Center for Biological Marine Resources, Southern

Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang,

524088, China

f. Pearl Research Institute, Guangdong Ocean University, Zhanjiang, China

*Corresponding author

E-mail addresses: yangcy@gdou.edu.cn

Journal Pre-proof

Journal Pre-proof

Abstract

With the advancement of nanotechnology and the growing utilization of

nanomaterials, titanium dioxide (TiO2) has been released into aquatic environments,

posing potential ecotoxicological risks to aquatic organisms. In this study, the

toxicological effects of TiO2 nanoparticles were investigated on the intestinal health

of pearl oyster (Pinctada fucata martensii). The pearl oysters were subjected to a

14-day exposure to 5-mg/L TiO2 nanoparticle, followed by a 7-day recovery period.

Subsequently, the intestinal tissues were analyzed using 16S rDNA high-throughput

sequencing. The results from LEfSe analysis revealed that TiO2 nanoparticle

increased the susceptibility of pearl oysters to potential pathogenic bacteria infections.

Additionally, the TiO2 nanoparticles led to alterations in the abundance of microbial

communities in the gut of pearl oysters. Notable changes included a decrease in the

relative abundance of Phaeobacter and Nautella, and an increase in the Actinobacteria,

which could potentially impact the immune function of pearl oysters. The abundance

of Firmicutes and Bacteroidetes, as well as the expression of genes related to energy

metabolism (AMPK, PK, SCS-1, SCS-2, SCS-3), were down-regulated, suggesting

that TiO2 nanoparticles exposure may affect the digestive and energy metabolic

functions of pearl oysters. Furthermore, the short-term recovery of seven days did not

fully restore these levels to normal. These findings provide crucial insights and serve

as an important reference for understanding the toxic effects of TiO2 nanoparticles on

bivalves.

Keywords: TiO2 nanoparticles; Pinctada fucata martensii; Gut microbes; Energy

metabolism

Abbreviations

TiO2, titanium dioxide; AMPK, adenosine 5'-monophosphate (AMP)-activated protein

kinase; PK, pyruvate kinase; SCS, succinyl-CoA synthetase; CG, control group; EG,

exposure group; RG, recovery group; DNA, deoxyribonucleic acid; RNA, ribonucleic

acid; PCR, polymerase chain reaction; qPCR, quantitative PCR; ASV, amplicon

sequence variants.

Journal Pre-proof

Journal Pre-proof

1. Introduction

According to the European Commission, nanomaterial is the natural or artificial

material in the form of powder or agglomerate consisting of elementary particles, with

at least one having a three-dimensional size ranging from 1 to 100 nm. Additionally,

more than 50% of the total number of particles in the material fall within this size

range (Hubert et al., 2013). TiO2 nanoparticles are one of the most widely used

nanoparticles owing to their favorable chemical properties. They are used in various

sectors, including cosmetics, food additives, pharmaceuticals and hygiene, inks, and

wastewater treatment (Chen and Selloni, 2014). Consequently, their presence in the

environment is inevitable. Studies indicate a global rise in the environmental

concentration of TiO2 nanoparticles, with levels reaching 1.65 mg/L in certain regions

(Shi et al., 2016). Additionally, heavily polluted aquatic environments can exhibit

actual TiO2 nanoparticle concentrations of 0.5 mg/L, and projections estimate a future

concentration of 1.0 mg/L in 10 years (Lu et al., 2021). The increasing presence of

TiO2 nanoparticles poses a considerable threat to marine organisms, particularly

invertebrates like bivalve mollusks. Consequently, there is growing concern about the

physiological and ecological impacts of TiO2 nanoparticles on these organisms.

Recent studies have demonstrated that exposure to TiO2 nanoparticles in various

marine species leads to adverse physiological consequences, such as decreased

success in fertilization (Nielsen et al., 2008; Alessandra et al., 2016), constrained

metabolism and growth (Muller et al., 2014; Johari et al., 2013), delayed embryonic

development (Libralato et al., 2013), compromised immune responses (Ringwood et

al., 2009; Rocha et al., 2014), and disruptions in gut microbiome (Chen et al., 2022;

Duan et al., 2023).

The gut microbiome plays a crucial role in energy and nutrient transfer within the

body, influencing the host's ability to digest and adapt to environmental fluctuations

(Yan et al., 2020). Scientific investigations reveal that the composition of the

microbial group linked to the host is not haphazard; instead, it is generally influenced

by the host's phylogeny and living environment (Carrier et al., 2018; Brooks et al.,

2016; Cárdenas et al., 2014). Gut microbiota formation is usually influenced by

Journal Pre-proof

Journal Pre-proof

factors such as pollutants, salinity, temperature, and genetics (Chen et al., 2017;

Tasnim et al., 2017). Host exposure to environmental changes can dramatically affect

the composition of the gut microbiome even over a short period (Chen et al., 2017;

Xia et al., 2018). For instance, exposure of Larimichthys crocea to 100 nm

nanopolystyrene for 14 days resulted in a significant alteration in the composition of

its gut microbiota (Gu et al., 2020). Exposure to 10 and 104

items/L nanopolystyrene

(100 nm) significantly altered the abundance of dominant bacterial phyla

(Bacteroidetes, Proteobacteria, and Firmicutes) of the Larimichthys crocea gut

microbiota, with an increase in potentially pathogenic bacteria like Parabacteroides

and Alistipes (Gu et al., 2020). TiO2 nanoparticles also affect the ecological

succession of intestinal microbiota in zebrafish development (Chen et al., 2022).

Furthermore, pentachlorophenol and TiO2 nanoparticles impair gastrointestinal tract

productivity and digestibility in Mytilus coruscus and have deleterious effects on the

gut microbiota of nautilus (Chen et al., 2023). These findings collectively emphasize

the urgency for comprehensive investigations to elucidate the impacts of TiO2

nanoparticles on diverse organisms and ecosystems. At the same time, the changes in

the intestinal flora of marine bivalves exposed to TiO2 nanoparticles have attracted

much attention.

Pinctada fucata martensii, a species of pearl oysters found in Japan, India, southern

China, Southeast Asia, and Australia (Yang et al., 2019, 2020, 2021), holds great

economic value, producing up to 90% of marine pearls (He et al., 2020; Yang et al.,

2022; Wu et al., 2022). The major methods of offshore pearl oyster culture include

pile and raft culture (Yang et al., 2017). However, recent serious pollution in offshore

waters poses a great threat to the survival of pearl oysters. Evidence indicates that

nanoparticles tend to accumulate in the digestive systems of aquatic organisms across

various trophic levels (Aljaibachi et al., 2020; Qiao et al., 2019; Watts et al., 2014).

Yet, investigations into the consequences of nanopollutants on gut microbial

communities have primarily focused on only few species, such as zebrafish and

Larimichthys crocea (Gu et al., 2020; Xie et al., 2021; Zhou et al., 2023). The impact

of these nanopollutants on the gut microbiota of benthic sentinel species remains

Journal Pre-proof

Journal Pre-proof

inadequately explored (Li et al., 2024). Therefore, this study exposed pearl oysters to

a concentration of 5 mg/L of TiO2 nanoparticles for 14 days, followed by a 7-day

recovery period, and analyzed the dynamics of their intestinal microbiota.

Additionally, the mechanisms of nanoparticle toxicity were explored, providing a

toxicological basis for evaluating the long-term ecological risks of nanoparticles.

2. Materials and methods

2.1 TiO2 nanoparticles

TiO2 nanoparticles (5–10 nm; anatase) were purchased from Shanghai Macklin

Biochemical Technology (China). Prior to conducting exposure experiments, a

designated quantity of TiO2 nanoparticles was added into filtered seawater to prepare

a 1 g/L nanoparticle suspension. This suspension was processed in a 65-W ultrasonic

instrument (LC-JY92-TTN; Lichen Bonsi Instrument Technology, China) for 20 min,

followed by dilution to achieve the desired concentration of TiO2 nanoparticles for the

experiment (5 mg/L).

2.2 Exposures

Pearl oysters, sourced from the black color selection line breed (Deng et al., 2013),

were obtained from Leizhou Daqiuzhuang Aquaculture Farm (China). Samples were collected and chosen by randomly selecting pearl oysters with undamaged shells and

uniform size. After gently removing the adherents on the shell surface, they were

transferred to 300-L cylindrical drums for a temporary rearing period of seven days.

The experiment comprised three parallel groups, each consisting of 30 pearl oysters.

These groups were exposed to 5 mg/L TiO2 nanoparticles for 14 days, followed by a 7-day recovery period in seawater without additional TiO2 nanoparticles. Water was

changed daily during the experiment to maintain the concentration of nanoparticles in

the bucket, and Chlorella sp. (20,000 cells/mL) and Platymonas subcordiformis

(10,000 cells/mL) were fed every morning and evening. On days 0, 14, and 21 of the

experiment, three pearl oysters were randomly collected from each parallel group, and

the intestinal and gill tissues of each pearl oyster were extracted, immediately stored

in liquid nitrogen, and the samples obtained were named the control group (CG), the

exposure group (EG), and the recovery group (RG), respectively, and then preserved

Journal Pre-proof

Journal Pre-proof

at ?80°C for further analysis. No dead pearl oysters were found during the exposure

and recovery periods.

2.3 Determination of indicators

2.3.1 Bacterial community

Total DNA from the intestinal tissues was extracted using the

cetyltrimethylammonium bromide method. The efficacy of the reagent developed to

detect DNA in minimal sample quantities was demonstrated in the extraction of DNA

from a wide range of bacterial species. Nuclear-free water was used for blank. The

entire DNA content was eluted using 50 μL of elution buffer and subsequently stored

at ?80°C until PCR measurement.

For sequencing PCR amplification, universal primers were employed in a 25 μL

reaction containing 25 ng template DNA, 12.5 μL PCR Premix, 2.5 μL of each primer,

and the required amount of water for the PCR step. PCR conditions for amplifying

prokaryotic 16S fragments were as follows: initial denaturation at 98°C for 30 s;

denaturation at 98°C for 10 s, annealing at 54°C for 30 s, and extension at 72°C for 45 s (32 cycles); final extension at 72°C for 10 min. PCR products were identified

through electrophoresis on a 2% agarose gel. To exclude false positives during DNA

extraction, ultrapure water was used as a negative control instead of a sample solution.

PCR products were purified using AMPure XT beads (Beckman Coulter Genomics,

Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Amplicon

compounds were prepared for sequencing, and the size and number of amplicon

libraries were estimated using an Agilent 2100 Bioanalyser (Agilent, USA) and

Illumina Library Quantification Kit (Kapa Biosciences, Woburn, MA, USA).

Libraries were sequenced on a NovaSeq PE250 platform.

2.3.2 Expression of energy metabolism genes Five genes playing crucial roles in physiological activities were selected to evaluate

the gene expression levels in pearl oysters exposed to TiO2 nanoparticles, and

analyses were conducted on five energy metabolism genes, namely, AMPK, PK,

SCS-1, SCS-2, and SCS-3. The primers were designed based on P. f. martensii

genome data (Zheng et al., 2023) and are presented in Table 1. Total RNA was

Journal Pre-proof

Journal Pre-proof

isolated from the gill tissues using TRIzol (Thermo-Fisher Scientific, USA) and then

treated with RNase-free DNA (Promega, USA). cDNA was synthesized for qPCR

using mRNA (2 μg) from each isolated sample. Reactions were performed using the

DyNAmo Flash SYBR Green qPCR kit (Thermo-Fisher Scientific) on an Applied

Biosystems 7000/7500 Rapid Real-Time PCR System (Applied Biosystems, USA).

The qPCR cycling conditions were as follows: an initial denaturing step at 95 ?C for 2

min; then 40 cycles at 95 ?C for 15 s and 60 ?C for 60 s; and a final extension at 72 ?C

for 30 s.

2.4 Data analysis

2.4.1 Gut microbiological data

Samples were sequenced on the Illumina NovaSeq platform according to the

manufacturer's guidelines. Matching read endpoints were assigned to samples using

unique barcodes, and barcodes and primer sequences were cut and trimmed.

Paired-end reads were merged using the FLASH software. Raw reads were subjected

to qualitative filtering according to the FQTRIM program (v0.94) to obtain

high-quality clean labels under specific filtering conditions. Chimeric sequences were

filtered using the Vsearch program (v2.3.4). Alpha diversity and beta diversity were

calculated by normalized to the same sequences randomly. Then according to SILVA

(release 138) classifier, feature abundance was normalized using relative abundance

of each sample. Alpha diversity was analyzed using five metrics, including Chao1,

observed species, Goods coverage, Shannon, and Simpson, to assess the complexity

of species diversity in the sample, and all this indices in our samples were calculated

with QIIME2. Beta diversity were calculated by QIIME2, the graphs were drew by R

package. Blast was used for sequence alignment, and the feature sequences were

annotated with SILVA database for each representative sequence. Other plots were

generated using the R package (v3.5.2).

2.4.2 Genes related to energy metabolism

The reference gene used for calculating the relative expression levels of the target

genes was GAPDH, and this was done using the 2???CT method (Livak and

Schmittgen, 2001). Data were analyzed using SPSS 26.0 software. The normality and

Journal Pre-proof

Journal Pre-proof

heteroscedasticity of the data were tested using the Shapiro–Wilk and Levene tests,

respectively. One-way analysis of variance (ANOVA) and Tukey's post-hoc test were

employed to analyze significant differences between data obtained at the same time

point.

3. Result

3.1 Gut microbiota of pearl oysters

After sequencing and initial filtering of 27 samples, a total of 1,931,014 ASVs were

obtained. CG, EG, and RG were represented by 2736, 3267, and 3424 ASVs,

respectively, with 1655, 1619, and 1850 unique ASVs detected and a total of 595

ASVs were common across all groups (Fig.1A). The coverage of ASVs in all samples

was at 100%, indicating that the sequencing data of this experiment can fully interpret

the composition and abundance of microbial communities in each sample.

3.2 Analysis of bacterial community diversity

To determine changes in the diversity of intestine bacterial communities, abundance

Shannon, Simpson, Chao1, observed species, good-coverage, and Pielou-e indices

were used to analyze samples (Fig. 2). Significant differences were observed in

Shannon (Kruskal–Wallis test; p < 0.01) and Simpson (Kruskal–Wallis test; p < 0.01)

indices between CG, EG, and RG (Fig. 2). The abundance and the diversity of gut

microbiota in EG significantly increased compared to CG and RG (Kruskal–Wallis

test; p < 0.05; Fig. 2). However, no significant differences were found in Chao1,

observed species, Good-coverage, or Pielou-e indices between EG, CG, and RG

(Kruskal–Wallis test; p > 0.05; Fig. 2).

Beta diversity was employed to analyze differences and similarities in microbial

communities among the samples (Fig. 1B). Principle component analysis (PCoA)

revealed that samples from all treatments clustered together, with an overlap in EG

and RG, while CG maintained distance from the other treatments. TiO2 nanoparticle

exposures caused alterations in the intestinal microbiota, with 12.52% and 7.9%

variation explained by the two principal components (PC1 and PC2), respectively (Fig.

1B).

3.3 Community structure analysis

Journal Pre-proof

Journal Pre-proof

At the phylum taxonomy level, a list of the 30 most prevalent bacterial phyla found in

the gut of pearl oysters was compiled (Fig. 3A). In CG and EG, the top two phyla

were Proteobacterian (47.77% and 49.04%) and Firmicutes (22.2% and 14.66%). The

third most abundant phylum were Cyanobacteria (18.39%) in CG and

Planctomycetota (9.67%) in EG. In RG, the top three phyla were Proteobacterian

(45.29%), Cyanobacteria (33.46%), and Firmicutes (7.12%). Compared to CG, the

EG showed no significant change in the relative abundance of Proteobacteria (p >

0.05, Fig, 3B), a significant reduction in Cyanobacteria (p < 0.05, Fig. 3B), and a

significant increase in Planctomycetota and Actinobacteriota (p < 0.05, Fig. 3B). The

abundance of Bacteroidota and Firmicutes decreased in EG, but not significantly,

while it significantly decreased in RG compared to CG (p < 0.05, Fig. 3B).

At the genus taxonomy level, a list of the 30 most prevalent bacterial genera in the gut

of pearl oysters was compiled (Fig. 4A). In CG, the top three genera with the highest

relative abundance were Mycoplasma (18.64%), Chloroplast_unclassified (18.05%),

and Nautella (16.06%). In EG, the top three genera were Mycoplasma (13.19%),

Vibrio (10.53%), and Enterovibrio (9.07%). In RG, the top three genera were

Chloroplast_unclassified (33.34%), Enterovibrio (27.13%), and Mycoplasma (5.60%).

Additionally, at the genus level, the composition of the gut microbiota of pearl oysters

changed in EG compared to CG (Fig. 4B). Relative abundance of Mycoplasm in EG

did not change significantly (p > 0.05), but Nautella, Sulfitobacter, Lactococcus, and

Phaeobacter decreased significantly (p < 0.05), while Vibrio, Enterovibrio,

Pseudmonas, and Chlamydia increased significantly (p < 0.05). A significant decrease

in the Mycoplasma abundance was observed in RG compared to CG (p < 0.05), with

significant differences also noted in the abundance of Nautella, Sulfitobacter, Lactococcus, Phaeobacter, Vibrio, Enterovibrio, and Pseudmonas (p < 0.05).

3.4 Unique biomarkers

LEfSe analysis revealed 15 unique biomarkers present in CG (LDA > 4 and p < 0.05;

Fig. 5). The analysis highlighted Nautella and Mycoplasma as the main

microorganisms present in CG at the genetic level. In EG, 20 unique microorganisms

were detected, with Brevundimonas, Ralstonia, and Blastopirellula identified at the

Journal Pre-proof

Journal Pre-proof

genus level (Fig. 5A). In RG, 15 unique microorganisms were observed, and the

cladeogram illustrated Enterovibrio as the unique microorganism at the genetic level (Fig. 5A).

3.5 KEGG functional classification statistics

PICRUST2 analysis indicated significant impacts of TiO2 nanoparticle stress on the

intestinal microbiome function of pearl oysters (Fig. 6). At level 2, \"Lipid

Metabolism\", \"Folding, Sorting, and Degradation\", \"Glycan Biosynthesis and

Metabolism\", and \"Transport and Catabolism\" were significantly up-regulated, while

\"Membrane Transport\", \"Excretory System\", \"Nervous System\", \"Immune System

Diseases\", \"Metabolism of Other Amino Acids\", \"Energy Metabolism\", and \"Enzyme

Families\" were significantly down-regulated in EG compared to CG (Fig. 6A). At

level 3, \"Lysosome\" and \"Steroid hormone biosynthesis\" were most noticeably

up-regulated, while \"Transporters\" and \"RNA transport\" were most noticeably

down-regulated in EG compared to CG (Fig. 6B).

After seven days of recovery, at level 2, \"Excretory system\",

\"Nervous System\",

\"Immune System Diseases\", \"Energy Metabolism\", \"Folding, Sorting, and

Degradation\", and \"Enzyme Families\" were significantly up-regulated (Fig. 6C),

whereas \"Lipid Metabolism\", \"Transport and Catabolism\", and \"Transport and

Catabolism\" were significantly down-regulated in RG compared to EG (Fig. 6C). At

level 3, \"RNA transport\" and \"Glutathione metabolism\" were significantly

up-regulated, while \"Lysosome\" and \"Steroid hormone biosynthesis\" were

significantly down-regulated in RG compared to EG (Fig. 6D).

3.6 Expression of energy metabolism genes

As shown in Fig 7, the expression levels of AMPK, PK, SCS-2, and SCS-3, which are

related to energy metabolism, were significantly affected by TiO2 nanoparticle

exposure (p < 0.05), leading to a notable down-regulation. Even after a 7-day short

recovery period, the expression of AMPK, PK, SCS-2, and SCS-3 in RG still

significantly lower than that in CG (p < 0.05). SCS-1 expression did not differ

substantially in EG compared to CG (p > 0.05), but it significantly decreased in RG

compared to CG (p < 0.05).

Journal Pre-proof

Journal Pre-proof

4. Discussion

Previous studies have demonstrated the close association between the gut microbiota

of bivalves and their overall health (Liao et al., 2020; Zheng et al., 2021). The gut

microbiota plays a crucial role in the breakdown and utilization of proteins,

carbohydrates, vitamins, and non-essential amino acids, as well as its regulation of

essential physiological processes such as host development and immune function (Keerthisinghe et al., 2020; Claus et al., 2011). Prior investigations focused on aquatic

organisms have revealed that exposure to microplastics, heavy metals, and ammonia

can induce disruptions in the gut microbial community (Jin et al., 2017; Qiao et al.,

2019; Zhang et al., 2021). Such disruptions can lead to impaired nutrient absorption

and compromised immune system functionality (Wan et al., 2019; Wang et al., 2022;

Xu et al., 2021). Moreover, disturbance of the gut microbiome and subsequent

imbalances exacerbate the detrimental effects of contaminant toxicity, ultimately

leading to alterations in the permeability of the host's intestinal tract (Wang et al.,

2022; Zhang et al., 2021). This, in turn, can cause inflammation, abnormal behavior,

and even mortality.

4.1 Impact of TiO2 nanoparticles on the potential risk of pathogenicity

The experimental treatment groups were analyzed using LEfSe to detect taxonomic

variations in the relative abundance of bacteria and unique biomarkers. This revealed

a distinct abundance of specific genera in pearl oysters exposed to a high

concentration of TiO2 nanoparticles. In EG, Brevundimonas, Ralstonia, and

Blastopirellula emerged as genus-level biomarkers in pearl oysters. Notably,

Brevundimonas and Ralstonia are recognized as opportunistic pathogens, leading to

bacteremia, especially in immunosuppressed patients (Zhang et al., 2020; Chi et al.,

2004; Tejera et al., 2016). Additionally, the pathogenic bacterium Ralstonia was more

abundant in EG than in CG. Although Ralstonia has been reported as an opportunistic

pathogen in various environments, including the gut (Payne et al., 2022; Dam et al.,

2020), its occurrence in aquatic animals has been less reported. Therefore, there is a

need for further studies on these potential pathogenic bacteria in the gut of pearl

oysters within the aquaculture context. Blastopirellula, representing a planktonic

Journal Pre-proof

Journal Pre-proof

bacteria species, demonstrated resistance against antibacterial substances generated by

macroalgae or other bacteria within the biofilm consortium due to the absence of

peptidoglycan in their cell wall. Moreover, these species exhibit considerable efficacy

in safeguarding against potential microbial intrusion (Lage and Bondoso, 2014). In

RG, Enterovibrio emerged as a genus-level biomarker in pearl oysters. Wu et al.

(2015) highlighted the high toxicity of Enterovibrio to zebrafish. This bacterium

promotes the production of indole, a toxin harmful to intestinal lactic acid bacteria in

excessive amounts (Wu et al., 2015; Pascual et al., 2009). This suggests that a 14-day

exposure to TiO2 nanoparticles may disrupt pearl oyster intestinal bacteria, disturbing

intestinal homeostasis and heightening the risk of pathogenic bacterial infection.

Additionally, differences in bacterial taxa with the potential risk of pathogenicity were

observed among the three groups. At the phylum level, members of the phylum

Dependentiae have been identified as pathogens in diverse aquatic protests (Chen et

al., 2022; Deeg et al., 2019). At the genus level, Chlamydia, recognized as a

pathogenic bacterium, can infect a wide range of hosts (Burnard et al., 2017) and may

serve as vectors of disease transmission, constituting zoonoses (Taylor-Brown and

Polkinghorne, 2017). In aquatic animals, the abundance of Chlamydia significantly

increased when Nile tilapia was exposed to high concentrations of porous plastics,

indicating detrimental effects on the intestinal health of Oreochromis niloticus (Zhang

et al., 2022). Additionally, Chlamydia can cause epithelioid cysts in fish, which can

lead to fish mortality (Sood et al., 2019). Pseudomonas, a spoilage bacterium

(Skírnisdóttir et al., 2021), appeared in the gut of pearl oysters after exposure to

nanoplastics treatment (Zhou et al., 2023). Alterations in the gut microbial community,

including increased numbers of Pseudomonas, may be associated with gut

inflammation and tissue damage (Zhou et al., 2023). The increase in the abundance of

pathogenic bacteria of the Chlamydia genus and the Dependentiae phyla in EG

increased the potential risk of disease in pearl oysters compared to CG. Although the

abundance of Chlamydia and Dependentiae did not significantly decrease after a

short-term recovery, suggesting that TiO2 nanoparticles increased the risk of disease in

pearl oysters, posing a further threat to their health. Exposure to TiO2 nanoparticles

Journal Pre-proof

Journal Pre-proof

resulted in an increased pathogenic bacteria abundance while simultaneously

diminishing the levels of beneficial bacteria in pearl oysters. At the genus level, EG

showed a significant decrease in Sulfitobacter and Lactococcus. Previous studies have

highlighted Sulfitobacter as a probiotic capable of inhibiting the growth of fish

pathogenic bacteria (Sharifah and Eguchi, 2012). Lactococcus has been recognized as

a potential probiotic in aquaculture and animals. Liu et al. (2015) isolated a variety of

lactic acid bacteria from the intestinal tracts of 27 species of fish, Chinese Ming

shrimp, Philippine hagfish, and white scallop, and tested their bacteriostatic activity in

vitro and found that Lactococcus lactis exhibited a significant bacteriostatic effect in

vitro. Our study observed a decrease in the abundance of both Lactococcus and

Sulfitobacter in pearl oysters following a 14-day exposure to TiO2 nanoparticles. This

decrease indicates the inhibitory effects of TiO2 nanoparticles on bacteria belonging to

these genera (Pan et al., 2023). Exposure of pearl oysters to TiO2 nanoparticles can

lead to alterations in the composition of the microbial community that affect the

function of the gut flora. Previous research has highlighted the critical roles played by

specific taxa of gut microbes in maintaining the normal physiological functions of

organisms. A reduction in the abundance of such functionally important gut microbes

may make pearl oysters more susceptible to disease (Kong et al., 2022). The observed

decrease in the abundance of specific bacterial genera associated with beneficial

functions, coupled with an increase in the abundance of harmful bacterial genera,

strongly suggested that TiO2 nanoparticles cause intestinal disorders in pearl oysters

and increase the risk of pathogenicity.

4.2 Impact of TiO2 nanoparticles on digestive and energy metabolism

According to Zha et al. (2018), the abundance of dominant flora can be influenced by

the environment, consequently impacting the quality and quantity of mussels. Our

study focused on the gut microbiota of pearl oysters, revealing Proteobacteria,

Cyanobacteria, and Firmicutes as the predominant phyla. The occurrence of TiO2

nanoparticles exhibited distinct impacts on the diversity of these primary microbiota

species in the digestive tract of pearl oysters. Specifically, Cyanobateria and

Firmicutes exhibited a substantial decline, while Proteobacteria remained unaltered.

Journal Pre-proof

Journal Pre-proof

Firmicutes play a pivotal role in expediting the development of lipid droplets in the

intestinal lining and liver, enhancing fat uptake and influencing nutrient metabolism

(Semova et al., 2012; Liu et al., 2022). Bacteroidetes, known for their role in

facilitating carbohydrate fermentation and metabolism of carbohydrates, bile acids,

and steroids, contribute to host energy supply and immune enhancement (Zhang et al.,

2014; Binda et al., 2018.). Additionally, Bacteroidetes can serve as an indicator for

aquatic animal health (Foysal et al., 2019), and an increase in their abundance has

been shown to improve the health status of species such as Atlantic salmon (Dehler et

al., 2017). Bacteroidetes and Firmicutes are generally thought to dominate the

bacterial communities in the digestive tracts of aquatic animals (Song et al., 2018).

Our study indicated a continual decrease in the relative abundance of Bacteroidetes

and Firmicutes during TiO2 nanoparticle exposure and a brief recovery, suggesting

that TiO2 nanoparticle exposure negatively affected the digestive capacity of pearl

oysters. The relative abundance of Firmicutes in the intestines of Eriocheir sinensis

infected with white spot syndrome virus has also been reported to reduce, suggesting

a decline in their health status (Ding et al., 2017). Importantly, the Firmicutes to

Bacteroidetes ratio serves as a significant biomarker for gut dysbiosis (Grigor'eva,

Irina N., 2020), affecting nutrient metabolism and aquaculture animal growth (Kong

et al., 2022; Chen et al., 2021). Previous studies on Micropterus salmoides exposed to

100 μg/L polyethylene microplastics resulted in a decrease in thick-walled

phylum/anabolic phylum ratio after seven days of exposure and an increase after 19 days of exposure, suggesting that polyethylene microplastics may affect energy,

glucose, and lipid metabolism (Chen et al., 2022). This persistent decrease in

Firmicutes, Bacteroidetes, and Firmicutes/Bacteroidetes ratios suggests that TiO2

nanoparticle exposure adversely affects the digestion of pearl oysters, and these

negative effects persist even after a short recovery period of seven days. Previous

findings on pearl oysters exposed to TiO2 nanoparticles at 5 mg/L indicated a decrease

in digestive enzyme activities after 14 days, and a brief recovery period of seven days

did not fully restore these activities to normal levels (Li et al., 2024). This may be

indicative of digestive system failure (Romano et al., 2018). Dai et al. (2017)

Journal Pre-proof

Journal Pre-proof

combined intestinal microbiome composition with the growth of razor clam

Sinonovacula constricta and explained that intestinal microbiota may participate in

intermediary metabolism by affecting enzyme activities. Additionally, KEGG function

prediction revealed a decrease in \"Energy Metabolism\" due to TiO2 nanoparticles,

which may be related to the reduced abundance of digestion-related bacteria (e.g.,

Firmicutes and Bacteroidetes). The analysis of the expression of genes closely related

to energy metabolism (AMPK, PK, and SCS) revealed down-regulation after

exposure to TiO2 nanoparticles. These findings indicate that TiO2 nanoparticles can

potentially disrupt the equilibrium of intestinal gut microbiota and microflora,

consequently impacting digestive processes and energy metabolism in pearl oysters.

4.3 Impact of TiO2 nanoparticles on the immune function

Actinobacteria, a diverse group comprising numerous beneficial bacteria, actively

contribute to the production of secondary metabolites, serving as antibiotics to combat

intrusive pathogens (Fernandes et al., 2014). Furthermore, Actinobacteria possess the

ability to stimulate immune cells, triggering inflammatory reactions and regulating

autoimmune responses (Azman et al., 2017; Zhang et al., 2022). Previous studies with

Crucian carp and Cyprinus carpio exposed to polystyrene microplastics demonstrated

a significant increase in the relative abundance of Actinobacteria in the gut (Chen et

al., 2022; Zhang et al., 2022). Similarly, exposure of Mytilus coruscus to TiO2

nanoparticles resulted in an increased abundance of Actinobacteria in the gut (Li et al.,

2024). In our study, the increase in Actinobacteria abundance in EG may be attributed

to nanoparticle invasion, disrupting the intestinal barrier function. This, in turn,

increased the sensitivity of pearl oysters to immune stimuli, enhancing their immune

function (Li et al., 2024). The abundance of actinomycetes returned to normal levels

once the source of stimulation disappeared and briefly recovered. This trend exhibits

the potential imbalance of gut microbiome and risk of diseases (Shin et al., 2015).

Additionally, the genera Phaeobacter and Nautella are known for their ability to

synthesize and break down unsaturated fatty acids, decompose intricate organic

substances, and generate bioactive compounds with potential antimicrobial

characteristics (Luo and Moran, 2014; Ooi et al., 2019). Certain members within these

Journal Pre-proof

Journal Pre-proof

genera display antimicrobial attributes that could potentially enhance the host's

immunity against pathogens (Vezzulli et al., 2018). However, the abundance of

Phaeobacter and Nautella significantly decreased in EG compared to CG, indicating

that TiO2 nanoparticle invasion reduced the host's ability to defend itself against

pathogens, and transient recovery restored neither of them to the normal level. LYS, a

lysosomal enzyme, plays a crucial role in the host's defense against pathogenic

bacteria (Olsen et al., 2003). KEGG function prediction showed an increase in the

\"lysozyme\" pathway after 14 days of TiO2 nanoparticle exposure and a subsequent

decrease after seven days of recovery in this study. These findings align with our

previous study, indicating that the innate immune system was activated under TiO2

nanoparticle exposure (Li et al., 2024).

5. Conclusion

In this study, changes in the gut microflora of Pinctada fucata martensii after

exposure to TiO2 nanoparticles were analyzed to explore the potential threat of

nanoparticles to their health. The results reveal that exposure to TiO2 nanoparticles

affects the intestinal microflora of pearl oysters, resulting in a decrease in the

abundance of digestive-related beneficial flora (e.g., Firmicutes and Bacteroidetes), an

increase in the abundance of several pathogenic bacteria (e.g., Chlamydia,

Pseudomonas and Dependentiae), and a decrease in the abundance of immune-related

beneficial flora (e.g., Phaeobacter and Nautella). Additionally, there was a decrease in

the functioning of related digestive pathways, such as energy metabolism and enzyme

families. These findings suggest that TiO2 nanoparticle stress may lead to intestinal

dysfunction, decreased digestive metabolism, and adverse effects on immune function

in Pinctada fucata martensii.

CRediT authorship contribution statement

Fengfeng Li: Methodology and Writing – original draft. Chuangye Yang:

Conceptualization, Funding acquisition, Writing–original draft. Yujing Lin, Yilong

Yan, and Ruijuan Hao: Methodology. Robert Mkuye: Writing–review & editing.

Yuewen Deng: Funding acquisition, Project administration.

Journal Pre-proof

Journal Pre-proof

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial

or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the Science and Technology Program of Guangdong

Province (Grant No. 2023A1515011769), National Natural Science Foundation of

China (Grant No. 32102817), Special Fund for Guangdong Province's Science and

Technology Innovation Strategy (Grant No. pdjh2024a191), Students Innovation and

Entrepreneurship Training Program of Guangdong Ocean University (Grant No.

CXXL2023003 and CXXL2022015), Department of Education of Guangdong

Province (Grant No. 2021KCXTD026), the earmarked fund for CARS-49, the

program for scientific research start-up funds of Guangdong Ocean University

(060302022304), and Hengli Biosciences Excellence Project of Guangdong Ocean

University (Grant No. B23335-4). We are very grateful to Marine Pearl Science and

Technology Backyard in Leizhou of Guangdong for collecting samples. The authors

would like to thank TopEdit (www.topeditsci.com) for its linguistic assistance during

the preparation of this manuscript. 16S rDNA high-throughput sequencing was

assisted by Biotree Biotech Co., Ltd. (Shanghai, China).

References

Alessandra, G., Raffaele, B., Isabella, B., 2016. Spermiotoxicity of nickel

nanoparticles in the marine invertebrate Ciona intestinalis (ascidians).

Nanotoxicology 10(8), 1096-1104.

Aljaibachi, R., Laird, W.B., Stevens, F., Callaghan, A., 2020. Impacts of polystyrene

microplastics on Daphnia magna: A laboratory and a mesocosm study. Science

of the Total Environment 705, 135800.

Azman, A.S., Othman, I., Fang, C.M., Chan, K.G., Goh, B.H., Lee, L.H., 2017.

Antibacterial, anticancer and neuroprotective activities of rare actinobacteria

Journal Pre-proof

Journal Pre-proof

from mangrove forest soils. Indian Journal of Microbiology 57(2), 177-187.

Baker, T., Tyler, C., Galloway, T., 2014. Impacts of metal and metal oxide

nanoparticles on marine organisms. Environmental Pollution 186, 257-271.

Barmo, C., Ciacci, C., Canonico, B., Fabbri, R., Cortese, K., Teresa, B., Marcomini, A., Pojana, G., Gallo, G., Canesi., 2013. In vivo effects of n-TiO2 on digestive

gland and immune function of the marine bivalve Mytilus galloprovincialis.

Aquatic Toxicology 132, 9-18.

Binda, C., Lopetuso, L.R., Rizzatti, G., Gibiino, G., Cennamo, V., Gasbarrini, A .,

2018. Actinobacteria: A relevant minority for the maintenance of gut homeostasis.

Digestive and Liver Disease 50(5), 421-428.

Brooks, A.W., Kohl, K.D., Brucker, R.M., van Opstal, E.J., Bordenstein, S.R., 2016.

Phylosymbiosis: relationships and functional effects of microbial communities

across host evolutionary history. Plos Biology 14(11), 2000225.

Burnard, D., Polkinghorne, A., 2017. Chlamydial infections in wildlife-conservation

threats and/or reservoirs of 'spill-over' infections? Veterinary Microbiology 196,

78-84.

Cárdenas, C,A., Bell, J.J., Davy, S.K., Hoggard, M., Taylor, M.W., 2014. Influence of

environmental variation on symbiotic bacterial communities of two temperate

sponges. Fems Microbiology Ecology 88(3), 516-527.

Carrier, T.J., Reitzel, A.M., 2018. Convergent shifts in host-associated microbial

communities across environmentally elicited phenotypes. Nature

Communications 9(1), 952.

Chen, C.Y., Chen, P.C., Weng, F,CH., Shaw, G,TW.,Wang, D,Y., 2017. Habitat and

indigenous gut microbes contribute to the plasticity of gut microbiome in oriental

river prawn during rapid environmental change. Plos One 12(7), 0181427.

Chen, J.J., Rao, C.Y., Yuan, R.J., Sun, D.D., Guo, S.Q., Li, L.L., Yang, S., Qian, D.D .,

Lu, R.H., Cao, X.L., 2022. Long-term exposure to polyethylene microplastics

and glyphosate interferes with the behavior, intestinal microbial homeostasis, and

metabolites of the common carp (Cyprinus carpio L.). Science of the Total

Environment 814, 152681.

Journal Pre-proof

Journal Pre-proof

Chen, M.S., Yue, Y.H., Bao, X.X., Feng, X.J., Ou, Z.Z., Qiu, Y.M., Yang, K.L., Yang, Y., Yu, Y.Y., Yu, H., 2022. Effects of polystyrene nanoplastics on oxidative stress,

histopathology and intestinal microbiota in largemouth bass (Micropterus

salmoides). Aquaculture Reports 27, 101423.

Chen, P.B., Huang, J., Rao, L.Y., Zhu, W.G., Yu, Y.H., Xiao, F.S., Yu, H., Wu, Y.J., Hu,

R.W., Liu, X.Y., He, Z.L., Yan, Q.Y., 2021. Environmental effects of

nanoparticles on the ecological succession of gut microbiota across zebrafish

development. Science of the Total Environment 806(4), 150963.

Chen, Q.S., Wei, T.L., Yang, B., Li, S.Y., Ge, L.J., Zhou, A.G., Xie, S.L., 2022. The

impact of deleting the mitfa gene in zebrafish on the intestinal microbiota

community. Gene 846, 146870.

Chen, X., Huang, W., Liu, C.H., Song, H.T., Waiho, K., Lin, D.H., Fang, J.K.H., Hu,

M.H., Kwan, K.Y., Wang, Y.J., 2023. Intestinal response of mussels to nano-TiO2

and pentachlorophenol in the presence of predator. Science of the Total

Environment 867, 161456.

Chen, X.B., Selloni. A., 2014. Introduction: titanium dioxide (TiO2) nanomaterials.

Chemical reviews 114(19), 9281-2.

Chen, X.W., Lu, D.Y., Li, Z.H., Yue, W.C., Wang, J., Jiang, X.Y., Han, H., Wang, C.H.,

2021. Plant and animal-type feedstuff shape the gut microbiota and metabolic

processes of the Chinese mitten crab Eriocheir sinensis. Frontiers in Veterinary

Science 8, 589624.

Chi, C.Y., Fung, C.P., Wong, W.W., Liu, C.Y., 2004. Brevundimonas bacteremia: Two

case reports and literature review. Scandinavian Journal Of Infectious Diseases

36(1), 59-61.

Claus, S.P., Ellero, S.L., Berger, B., Krause, L., Bruttin, A., Molina, J., Paris, A., Want, E.J.,Waziers, I., Cloarec, O., Richards, S.E., Wang, Y., Dumas, M.E., Ross, A .,

Rezzi, S., Kochhar, S., Van Bladeren, P., Lindon, J.C., Holmes, E., Nicholson, J.K., 2011. Colonization-induced host-gut microbial metabolic interaction. Mbio

2(2), 00271-10.

Dai, W.F., Dong, Y.H., Ye, J., Xue, Q.G., Lin, Z.H., 2022. Gut microbiome

Journal Pre-proof

Journal Pre-proof

composition likely affects the growth of razor clam Sinonovacula constricta.

Aquaculture 550, 737847.

Dam, C.T.M., Booth, M., Pirozzi, I., Salini, M., Smullen, R., Ventura, T., Elizur, A.,

2020. Alternative feed raw materials modulate intestinal microbiota and its

relationship with digestibility in yellowtail kingfish Seriola lalandi. Fishes 5(2),

14.

Deeg, C.M., Zimmer, M.M., George, E.E., Husnik, F., Keeling, P.J., Suttle, C.A.,

2019. Chromulinavorax destructans, a pathogen of microzooplankton that

provides a window into the enigmatic candidate phylum Dependentiae. Plos

Pathogens 15(5), 1007801.

Dehler, C.E., Secombes, C.J., Martin, S.A.M., 2017. Environmental and physiological

factors shape the gut microbiota of Atlantic salmon parr (Salmo salar L.).

Aquaculture 467, 149-157.

Deng, Y.W., Fu, S., Lu, Y.Z., Du, X.D., Wang, Q.H., Huang, H.L., Liu, D., 2013.

Fertilization, hatching, survival, and growth of third-generation colored pearl

oyster (Pinctada martensii) stocks. Journal of Applied Aquaculture 25(2),

113-120

Ding, Z.F., Cao, M.J., Zhu, X.S., Xu, G.H., Wang, R.L., 2017. Changes in the gut

microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White

spot syndrome virus (WSSV) infection. Journal of Fish Diseases 40(11),

1561-1571.

Duan, Y.F., Yang, Y.K., Zhang, Z., Xing, Y.F., Li, H., 2023. Toxicity of titanium

dioxide nanoparticles on the histology, liver physiological and metabolism, and

intestinal microbiota of grouper. Marine Pollution Bulletin 187, 114600.

Fernandes, T.A.R., da Silveira, W.B., Lopes Passos, F.M., L., Zucchi, T.D., 2014.

Laccases from Actinobacteria—what we have and what to expect. Advances in

Microbiology 4, 285-296..

Foysal, M.J., Alam, M., Momtaz, F., Chaklader, M.R., Siddik, M.A.B., Cole, A.,

Fotedar, R., Rahman, M.M., 2019. Dietary supplementation of garlic (Allium

sativum) modulates gut microbiota and health status of tilapia (Oreochromis

Journal Pre-proof

Journal Pre-proof

niloticus) against Streptococcus iniae infection. Aquaculture Research 50(8),

2107-2116.

Grigor'eva, I.N., 2020. Gallstone disease, obesity and the Firmicutes/Bacteroidetes

ratio as a possible biomarker of gut dysbiosis. Journal of Personalized Medicine

11(1), 13.

Gu, H.X., Wang, S.X., Wang, X.H., Yu, X., Hu, M.H., Huang, W., Wang, Y.J., 2020.

Nanoplastics impair the intestinal health of the juvenile large yellow croaker

Larimichthys crocea. Journal of Hazardous Materials 397, 122773.

He, C.Z., Hao, R.J., Deng, Y.W ., Yang, C.Y., Du, X.D., 2020. Response of pearl

oyster Pinctada fucata martensii to allograft-induced stress from lipid

metabolism. Fish & Shellfish Immunology 98, 1001-1007.

Hubert, R., Birgit, S., Hermann, S., 2013. The European Commission's

recommendation on the definition of nanomaterial makes an impact.

Nanotoxicology 7(7), 1195-11997.

Jin, Y.X.,Wu, S.S., Zeng, Z.Y., Fu, Z.W., 2017. Effects of environmental pollutants on

gut microbiota. Environmental Pollution 222, 1-9.

Jin, YX., Xia, J.Z., Pan, Z.H., Yang, J.J., Wang, W.C., Fu, Z.W ., 2018. Polystyrene

microplastics induce microbiota dysbiosis and inflammation in the gut of adult

zebrafish. Environmental Pollution 235, 322-329.

Johari, S.A., Kalbassi, M.R., Soltani, M., Yu, I.J., 2013. Toxicity comparison of

colloidal silver nanoparticles in various life stages of rainbow trout (Oncorhynchus mykiss). Iranian Journal of Fisheries Sciences 12(1), 76-95.

Keerthisinghe, T.P., Wang, F., Wang, M.J., Yang, Q., Li, J.W., Yang, J.F., Xi, L., Dong, W., Fang, M.L., 2020. Long-term exposure to TET increases body weight of

juvenile zebrafish as indicated in host metabolism and gut microbiome.

Environment International 139, 105705.

Kong, N., Han, S., Fu, Q., Yu, Z.C., Wang, L.L., Song, L.S., 2022. Impact of ocean

acidification on the intestinal microflora of the Pacific oyster Crassostrea gigas.

Aquaculture 546, 737365.

Labille, J., Slomberg, D., Catalano, R., Robert, S., Apers-Tremelo, M., Boudenne, J.,

Journal Pre-proof

Journal Pre-proof

Manasfi, T., Radakovitch, O., 2020. Assessing UV filter inputs into beach waters

during recreational activity: A field study of three French Mediterranean beaches

from consumer survey to water analysis. Science of the Total Environment 706,

136010.

Lage, O.M., Bondoso, J., 2014. Planctomycetes and rnacroalgae, a striking

association. Frontiers in Microbiology 5, 267.

Li, F.F., Xie, Y.F., Yang, C,Y., Ye, Q.X., Wang, F.Y., Liao, Y.S., Mkuye, R., Deng,

Y.W., 2024. The physiological responses to titanium dioxide nanoparticles

exposure in pearl oysters (Pinctada fucata martensii). Marine Environmental

Research 195,106345.

Li, Z.Q., Li, L., Sokolova, I., Shang, Y.Y., Huang, W., Khor, W., Fang, J.K.H., Wang,

Y.J., Hu, M.H., 2024. Effects of elevated temperature and different crystal

structures of TiO2 nanoparticles on the gut microbiota of mussel Mytilus

coruscus. Marine Pollution Bulletin 199, 115979.

Liao, Y.S., Cai, C.X., Yang, C.Y., Zheng, Z., Wang, Q.H., Du, X.D., Deng, Y.W., 2020.

Effect of protein sources in formulated diets on the growth, immune response,

and intestinal microflora of pearl oyster Pinctada fucata martensii. Aquaculture

Reports 16, 100253.

Libralato, G., Minetto, D., Totaro, S., Micetic, I., Pigozzo, A., Sabbioni, E.,

Marcomini, A., Ghirardini, A.V., 2013. Embryotoxicity of TiO2 nanoparticles to

Mytilus galloprovincialis (Lmk). Marine Environmental Research 92, 71-78.

Liu, J., Lin, J.F., Guo, L,Q., Ye, Z,W., Fang, Z,G., Guo, X.Y., Li, Z.L., 2015. Studies

on the diversity and bacteriostatic activity of lactic acid bacteria from aquatic

sources. Fisheries Science 34(6), 1003-1111 (in Chinese).

Liu, Y.Y., Cheng, J.X., Xia, Y.Q., Li, X.H., Liu, Y., Liu, P.F., 2022. Response

mechanism of gut microbiome and metabolism of European seabass (Dicentrarchus labrax) to temperature stress. Science of the Total Environment

813, 151786.

Livak, K. J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using

real-time quantitative PCR and the 2? ΔΔCT method. Methods 25(4), 402-408.

Journal Pre-proof

Journal Pre-proof

Lu, J., Wang, P., Tian, S.Y., Qian, W., Huang, Y.X., Wang, Z.Y., Zhu, X.S., Cai, Z.H.,

2021. TiO2 nanoparticles enhanced bioaccumulation and toxic performance of

PAHs via trophic transfer. Journal of Hazardous Materials 407, 124834.

Luo, H.W., Moran, M.A., 2014. Evolutionary Ecology of the Marine Roseobacter

Clade. Microbiology and Molecular Biology Reviews 78(4), 573-587.

Meng, X.L., Tian, X., Nie, G.X., Wang, J.L., Liu, M., Jiang, K.Y., Wang, B.J., Guo, Q.Q., Huang, J.R., Wang, L., 2015. The transcriptomic response to copper

exposure in the digestive gland of Japanese scallops (Mizuhopecten yessoensis).

Fish & Shellfish Immunology 46(2), 161-167.

Muller, E.B., Hanna, S.K., Lenihan, H.S., Miller, R., Nisbet, R.M., 2014. Impact of

engineered zinc oxide nanoparticles on the energy budgets of Mytilus

galloprovincialis. Journal of Sea Research 94, 29-36.

Nielsen, H.D., Berry, L.S., Stone, V., Burridge,T.R., Fernandes, T.F., 2008.

Interactions between carbon black nanoparticles and the brown algae Fucus

serratus: Inhibition of fertilization and zygotic development. Nanotoxicology

2(2), 88-97.

Olsen, O.M., Nilsen, I.W., Sletten, K., Myrnes, B., 2003. Multiple invertebrate

lysozymes in blue mussel (Mytilus edulis). Comparative Biochemistry and

Physiology B-Biochemistry & Molecular Biology 136(1), 107-115.

Ooi, M.C., Goulden, E.F., Smith, G.G, Bridle, A.R., 2019. Haemolymph microbiome

of the cultured spiny lobster Panulirus ornatus at different temperatures.

Scientific Reports 9(1), 1677.

Pan, Y.T., Qian, J., Ma, X.W., Huang, W., Fang, J.K.H., Arif, I., Wang, Y.J., Shang, Y.Y., Hu, M.H., 2023. Response of moulting genes and gut microbiome to

nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus.

Marine Environmental Research 191, 106128.

Pascual, J., Macián, M.C., Arahal, D.R., Garay, E., Pujalte, M.J., 2009. Description of

Enterovibrio nigricans sp nov., reclassification of Vibrio calviensis as

Enterovibrio calviensis comb. nov and emended description of the genus

Enterovibrio Thompson et al. 2002. International Journal of Systematic and

Journal Pre-proof

Journal Pre-proof

Evolutionary Microbiology 59(4), 698-704.

Payne, C.J., Turnbull, J.F., MacKenzie, S., Crumlish, M., 2022. The effect of

oxytetracycline treatment on the gut microbiome community dynamics in

rainbow trout (Oncorhynchus mykiss) over time. Aquaculture 560, 738559.

Qiao, R.X., Deng, Y.F., Zhang, S.H., Wolosker, M.B., Zhu, Q.D., Ren, H.Q., Zhang,

Y., 2019. Accumulation of different shapes of microplastics initiates intestinal

injury and gut microbiota dysbiosis in the gut of zebrafish. Chemosphere 236,

124334.

Qiao, R.X., Sheng, C., Lu, Y.F., Zhang, Y., Ren, H.Q., Lemos, B., 2019. Microplastics

induce intestinal inflammation, oxidative stress, and disorders of metabolome

and microbiome in zebrafish. Science of the Total Environment 662, 246-253.

Ringwood, A.H., Levi-Polyachenko, N., Carroll, D.L., 2009. Fullerene Exposures

with Oysters: Embryonic, Adult, and Cellular Responses. Environmental Science

& Technology 43(18), 7136-7141.

Ringwood, A.H., McCarthy, M., Levi-Polyachenko, N., Carroll, D.L., 2010.

Nanoparticles in Natural Aquatic Systems and Cellular Toxicity. Comparative

Biochemistry and Physiology Part A: Molecular & Integrative Physiology 157

(S1): S53-S53.

Rocha, T.L., Gomes, T., Cardoso, C., Letendre, J., Pinheiro, J.P., Sousa, V.S., Teixeira, M.R., Bebianno, M.J., 2014. Immunocytotoxicity, cytogenotoxicity and

genotoxicity of cadmium-based quantum dots in the marine mussel Mytilus

galloprovincialis. Marine Environmental Research 101, 29-37.

Romano, N., Ashikin, M., Teh, J.C., Syukri, F., Karami, A., 2018. Effects of pristine

polyvinyl chloride fragments on whole body histology and protease activity in

silver barb Barbodes gonionotus fry. Environmental Pollution 237, 1106-1111.

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 12(3), 277-288.

Sharifah, E.N. Eguchi, M., 2012. Benefits of live phytoplankton, Chlorella vulgaris,

as a biocontrol agent against fish pathogen Vibrio anguillarum. Fisheries Science

Journal Pre-proof

Journal Pre-proof

78(2), 367-373.

Shi, X.M., Li, Z.X., Chen, W., Qiang, L.W., Xia, J.C., Chen, M., Zhu, L.Y., Alvarez, P.J.J., 2016. Fate of TiO2 nanoparticles entering sewage treatment plants and

bioaccumulation in fish in the receiving streams. Nanoimpact 3-4, 96-103.

Shin, N.R., Whon, T.W., Bae, J.W., 2015. Proteobacteria: microbial signature of

dysbiosis in gut microbiota. Trends in Biotechnology 33(9), 496-503.

Skírnisdóttir, S., Knobloch, S., Lauzon, H.L., Olafsdóttir, A., Steinthórsson, P.,

Bergsten, P., Marteinsson, V.U., 2021. Impact of onboard chitosan treatment of

whole cod (Gadus morhua) on the shelf life and spoilage bacteria of loins stored

superchilled under different atmospheres. Food Microbiology 97, 103723.

Song, H., Yu, Z.L., Yang, M.J., Zhang, T., Wang, H.Y., 2018. Analysis of microbial

abundance and community composition in esophagus and intestinal tract of wild

veined rapa whelk (Rapana venosa) by 16S rRNA gene sequencing. Journal of

General and Applied Microbiology 64(4), 158-166.

Sood, N., Pradhan, P.K., Verma, D.K., Gupta, S., Ravindra., Dev, A.K., Yadav, M.K.,

Swaminathan, T.R., Rathore, G., 2019. Epitheliocystis in rohu Labeo rohita

(Hamilton, 1822) is caused by novel Chlamydiales. Aquaculture 505, 539-543.

Tasnim, N., Abulizi, N., Pither, J., Hart, M.M., Gibson, D.L., 2017. Linking the Gut

Microbial Ecosystem with the Environment: Does Gut Health Depend on Where

We Live? Frontiers in Microbiology 8, 1935.

Taylor-Brown, A., Polkinghorne, A., 2017. New and emerging chlamydial infections

of creatures great and small. New Microbes and New Infections 18, 28-33.

Tejera, D., Limongi, G., Bertullo, M., Cancela, M., 2016. Ralstonia pickettii

bacteremia in hemodialysis patients: a report of two cases. Revista Brasileira de

terapia intensiva 28, 195-198.

Vezzulli, L., Stagnaro, L., Grande, C., Tassistro, G., Canesi, L., Pruzzo, C., 2018.

Comparative 16SrDNA Gene-Based Microbiota Profiles of the Pacific Oyster (Crassostrea gigas) and the Mediterranean Mussel (Mytilus galloprovincialis)

from a Shellfish Farm (Ligurian Sea, Italy). Microbial Ecology 75(2), 495-504.

Wan, Y., Wang, F.L., Yuan, J.H., Li, J., Jiang, D.D., Zhang, J.J., Li, H., Wang, R.Y.,

Journal Pre-proof

Journal Pre-proof

Tang, J., Huang, T., Zheng, J.S., Sinclair, A.J., Mann, J., Li, D., 2019. Effects of

dietary fat on gut microbiota and faecal metabolites, and their relationship with

cardiometabolic risk factors: a 6-month randomised controlled-feeding trial. Gut

68(8), 1417-1429.

Wang, Y., Sha, W.L., Zhang, C., Li, J.Y., Wang, C., Liu, C.C., Chen, J.F., Zhang, W.L .,

Song, Y.H., Wang, R.J., Gao, P.K., 2022. Toxic effect of triphenyl phosphate

(TPHP) on Cyprinus carpio and the intestinal microbial community response.

Chemosphere 299, 134463.

Watts, A.J.R., Lewis, C., Goodhead, R.M., Beckett, S.J., Moger, J., Tyler, C.R.,

Galloway, T.S., 2014. Uptake and Retention of Microplastics by the Shore Crab

Carcinus maenas. Environmental Science & Technology 48(15), 8823-8830.

Wu, F.S., Tang, K.H., Yuan, M., Shi, X.C., Shakeela, Q., Zhang, X.H., 2015. Studies

on bacterial pathogens isolated from diseased torafugu (Takifugu rubripes)

cultured in marine industrial recirculation aquaculture system in Shandong

Province, China. Aquaculture Research 46(3), 736-744.

Wu, H.L., Yang, C.Y., Hao, R.J., Liao, Y.S., Wang, Q.H.,Deng, Y.W., 2022. Lipidomic

insights into the immune response and pearl formation in transplanted pearl

oyster Pinctada fucata martensii. Frontiers in Immunology 13, 1018423.

Xia, J.Z., Lu, L., Jin, C.Y., Wang, S.Y., Zhou, J.C., Ni, Y.C., Fu, Z.W., Jin, Y.X., 2018.

Effects of short term lead exposure on gut microbiota and hepatic metabolism in

adult zebrafish. Comparative Biochemistry and Physiology C-Toxicology &

Pharmacology 209, 1-8.

Xie, S.L., Zhou, A.G., Wei, T.L., Li, S.Y., Yang, B., Xu, G.H., Zou, J.X., 2021.

Nanoplastics Induce More Serious Microbiota Dysbiosis and Inflammation in the

Gut of Adult Zebrafish than Microplastics. Bulletin of Environmental

Contamination and Toxicology 107(4), 640-650.

Xu, K.H., Zhang, Y.D., Huang, Y.M., Wang, J., 2021. Toxicological effects of

microplastics and phenanthrene to zebrafish (Danio rerio). Science of the Total

Environment 757, 143730.

Yan, J., Wang, D.G., Li, K., Chen, Q., Lai, W.Q., Tian, L., Lin, B.C.,Tan Y. Z., Liu,

Journal Pre-proof

Journal Pre-proof

X.H., Xi, Z.G., 2020. Toxic effects of the food additives titanium dioxide and

silica on the murine intestinal tract: Mechanisms related to intestinal barrier

dysfunction involved by gut microbiota. Environmental Toxicology and

Pharmacology 80, 103485.

Yang, C.Y., Wang, Q.H., Hao, R.J., Liao, Y.S., Du, X.D., Deng, Y.W., 2017. Effects of

replacing microalgae with an artificial diet on pearl production traits and

mineralization‐related gene expression in pearl oyster Pinctada fucata martensii.

Aquaculture Research 48, 5331–5337.

Yang, C.Y., Hao, R.J., He, C.Z., Deng, Y.W., Wang, Q.H., 2022. Cloning and

functional characterization of PmΔ5FAD in pearl oyster Pinctada fucata

martensii. Aquaculture Reports 23, 101036.

Yang, C.Y., Yang, J.M., Hao, R.J., Du, X.D., Deng, Y.W., 2020. Molecular

characterization of OSR1 in Pinctada fucata martensii and association of allelic

variants with growth traits. Aquaculture 516, 734617.

Yang, C.Y., Zeng, Y.T., Liao, Y.S., Deng, Y.W., Du, X.D., Wang, Q.H., 2021.

Integrated GC-MS- and LC-MS-Based Untargeted Metabolomics Studies of the

Effect of Vitamin D3 on Pearl Production Traits in Pearl Oyster Pinctada fucata

martensii. Frontiers in Molecular Biosciences 8, 614404.

Zha, Y.H., Eiler, A., Johansson, F., Svanb?ck, R., 2018. Effects of predation stress and

food ration on perch gut microbiota. Microbiome 6, 1-12.

Zhang, X.F., Yu, Q.R., Zhao, Y,H., Shi, Z,Y., Zhou, L., Li, E., Xie, J., 2022. Effect of

High Density-polyethylene on Growth Performance, Antioxidant Capacity,

Immune Functions and Intestinal Microbiota of Oreochromis Niloticus. Asian

Journal of Ecotoxicology17(6), 301-314 (in Chinese).

Zhang, M.L., Sun, Y.H., Chen, K., Yu, N., Zhou, Z.G., Chen, L.Q., Du, Z.Y., Li, E.C.,

2014. Characterization of the intestinal microbiota in Pacific white shrimp,

Litopenaeus vannamei, fed diets with different lipid sources. Aquaculture 434,

449-455.

Zhang, P., Lu, G.H., Sun, Y., Yan, Z.H., Dang, T.J., Liu, J.C., 2022. Metagenomic

analysis explores the interaction of aged microplastics and roxithromycin on gut

Journal Pre-proof

Journal Pre-proof

microbiota and antibiotic resistance genes of Carassius auratus. Journal of

Hazardous Materials 425, 127773.

Zhang, T.X., Zhang, Y., Xu, J.Y., Yan, Z.G., Sun, Q.H., Huang, Y., Wang, S.P., Li, S.,

Sun, B.B., 2021. Toxic effects of ammonia on the intestine of the Asian clam

(Corbicula fluminea). Environmental Pollution 2021, 117617.

Zhang, Y.M., Xie, S.W., Guo, T.Y., Liu, Z.L., Fang, H.H., Zheng, L., Xie, J.J., Tian,

L.X., Liu, Y.J., Jin, N., 2020. High dietary starch inclusion impairs growth and

antioxidant status, and alters liver organization and intestinal microbiota in

largemouth bass Micropterus salmoides. Aquaculture Nutrition 26(5),

1806-1821.

Zheng, Z., Hao, R.J., Yang, C.Y., Jiao, Y., Wang, Q.H., Huang, R.L., Liao, Y.S., Jian,

J.B., Ming, Y., Yin, L.X., He, W.M., Wang, Z.M., Li, C.Y., He, Q., Chen, K.,

Deng, Y.W., Du, X.D., 2022. Genome-wide association study analysis to resolve

the key regulatory mechanism of biomineralization in Pinctada fucata martensii.

Molecular Ecology Resources 23(3), 680-693.

Zheng, Z., Liao, Y.S., Ye, J.M., Yang, C.Y., Adzigbli, L., Wang, Q.H., Du, X.D., Deng,

Y.W., 2021. Microbiota diversity in pearl oyster Pinctada fucata martensii

intestine and its aquaculture environment. Frontiers in Marine Science 8,

655698.

Zhou, Y.F., Gui, L., Wei, W.B., Xu, E.G., Zhou, W.Z., Sokolova, I.M., Li, M.Y., Wang,

Y.J., 2023. Low particle concentrations of nanoplastics impair the gut health of

medaka. Aquatic Toxicology 256, 106422.

Journal Pre-proof

Journal Pre-proof

Table 1 Forward and reverse primer sequences used for quantitative real-time PCR

analyses

Gene name Gene function Primer sequence (5′ to 3′)

5′ -AMP activated protein kinase

(AMPK)

Energy

metabolism

F:

AAAGCCTGGATGTGGTT

GG

A:

TTGCTGGAAAAATCTGCG A

Pyruvate kinase (PK)

Energy

metabolism

F:

CCAAGGGGTAGACATGG

TGT

A:

CTCCCCTAGCAACCATCA

CA

Succinyl-CoA synthetase (SCS)-1

Energy

metabolism

F:

CAGTGGAGGGAGAGGTA

AAGG A:

TCATTTTTTCCGCAGTGG C

Succinyl-CoA synthetase (SCS)-2

Energy

metabolism

F:

GAACTTGGTGCTGGAACT

GG

A:

CTACCAGAGGCTCGAGT

GAC

Succinyl-CoA synthetase (SCS)-3

Energy

metabolism

F:

AGAGACTCACATGACGG

CAAG

Journal Pre-proof

Journal Pre-proof

A:

CAGCAGGCTTGTCATTTG

GT

GAPDH Reference gene

F:

CACTCGCCAAGATAATC

AACG

A:

CCATTCCTGTCAACTTCC

CAT

Journal Pre-proof

Journal Pre-proof

Fig.1 Gut microbiome alterations induced by different group. (A) Venn analysis at

ASVs level, (B) Principal coordinates analysis. Different letters mean a significant

difference between differenr groups (p < 0.05).

Journal Pre-proof

Journal Pre-proof

Fig.2 Effect of different groups on Alpha diversity in pearl oyster. \"*\" indicates

significant differences between different groups, respectively. (*: p < 0.05； **: p <

0.01； ***: p < 0.001； ****: p < 0.0001.)

Journal Pre-proof

Journal Pre-proof

Fig.3 Composition of intestinal bacterial community of pearl oysters at phylum

taxonomic level. (A) Only the top 30 most abundant (Based on relative abundance)

bacterial phyla was shown. (B) Significance of differences among three

groups.Different lower case letters mean a significant difference between different

groups (p < 0.05).

Journal Pre-proof

Journal Pre-proof

Fig.4 Composition of intestinal bacterial community of pearl oysters at genera

taxonomic level. (A) Only the top 30 most abundant (Based on relative abundance)

bacterial genera was shown. (B) Significance of differences among three groups.

Different lower case letters mean a significant difference between different groups (p

< 0.05).

Journal Pre-proof

Journal Pre-proof

Fig.5 LEfSe analysis of different groups of gut microbiome biomarkers Bar chart (A)

showing the LDA scores of bacterial taxa (LDA score >4, p < 0.05), and cladogram.

(B) showing the phylogenetic relationships of bacterial taxa revealed by LEfSe.

Journal Pre-proof

Journal Pre-proof

Fig.6 Functional annotation and relative abundance of pearl oysters intestinal

microbiota using Kyoto Encyclopedia of Gene and Genomes (KEGG) analysis at

level 2 and level 3. A, C for level 2, B, D for level 3.

Journal Pre-proof

Journal Pre-proof

Fig.7 Relative expression levels of genes 5′ -AMPactivated protein kinase (AMPK;

A), Pyruvate kinase (PK; B), Succinyl-CoA synthetase (SCS-1, C; SCS-2, D; SCS-3,

E).

Journal Pre-proof

Journal Pre-proof

Graphical abstract

Journal Pre-proof

Journal Pre-proof

Highlights

1. TiO2 nanoparticles increased the susceptibility of pearl oysters to potential

pathogenic bacteria infections.

2. TiO2 nanoparticles affected the abundance of microbial communities in the gut of

pearl oysters.

3. 7-days recovery did not allow the affected microbial communities and functions to

return to full normalcy.