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Impact of chronic exposure to trichlorfon on intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome in common carp (Cyprinus carpio L.)
Journal Pre-proof Impact of chronic exposure to trichlorfon on intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome in common carp (Cyprinus carpio L.) Xulu Chang, Xianfeng Wang, Junchang Feng, Xi Su, Junping Liang, Hui Li, Jianxin Zhang PII:
S0269-7491(19)33863-1
DOI:
https://doi.org/10.1016/j.envpol.2019.113846
Reference:
ENPO 113846
To appear in:
Environmental Pollution
Received Date: 17 July 2019 Revised Date:
30 November 2019
Accepted Date: 16 December 2019
Please cite this article as: Chang, X., Wang, X., Feng, J., Su, X., Liang, J., Li, H., Zhang, J., Impact of chronic exposure to trichlorfon on intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome in common carp (Cyprinus carpio L.), Environmental Pollution (2020), doi: https:// doi.org/10.1016/j.envpol.2019.113846. 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. © 2019 Published by Elsevier Ltd.
1
Impact of chronic exposure to trichlorfon on intestinal barrier,
2
oxidative stress, inflammatory response and intestinal microbiome in
3
common carp (Cyprinus carpio L.)
4 a,1
, Xianfeng Wang a,1, Junchang Feng a, Xi Su b, Junping
5
Xulu Chang
6
Liang a, Hui Li a, Jianxin Zhang a,*
7 8
a
9
China
College of Fisheries, Henan Normal University, Xinxiang, 453007, PR
10
b
11
Medical University, Xinxiang, 453007, PR China
12
1
13
* Corresponding author: Jianxin Zhang
14
Email: [email protected]
15
Tel.: +86-373-3326563
16
Fax: +86-373-3326563
17
Address: College of Fisheries, Henan Normal University, Xinxiang,
18
453007, PR China
19 20 21 22
Henan Mental Hospital, The Second Affiliated Hospital of Xinxiang
These authors contributed equally to this study.
23
Abstract
24
Trichlorfon is an organic phosphorus pesticide used to control
25
different parasitic infections in aquaculture. The repeated, excessive use
26
of trichlorfon can result in environmental pollution, thus affecting human
27
health. This study aimed to determine the effects of different
28
concentrations of trichlorfon (0, 0.1, 0.5 and 1.0 mg/L) on the intestinal
29
barrier,
30
microbiome of common carp. Trichlorfon exposure significantly reduced
31
the height of intestinal villus and decreased the expression levels of tight
32
junction genes, such as claudin-2, occludin and ZO-1, in common carp.
33
Moreover, the activities of antioxidant enzymes, such as CAT, SOD and
34
GSH-Px, exhibited a decreasing trend with increasing trichlorfon
35
concentrations, while the contents of MDA and ROS elevated in the
36
intestinal tissues of common carp. The mRNA and protein levels of
37
pro-inflammatory cytokines TNF-α and IL-1β were significantly
38
upregulated by trichlorfon exposure. The level of anti-inflammatory
39
cytokine TGF-β was remarkably higher in 1.0 mg/L trichlorfon treatment
40
group compared to control group. In addition, the results demonstrated
41
that trichlorfon exposure could affect the microbiota community
42
composition and decreased the community diversity in the gut of
43
common carp. Notably, the proportions of some probiotic bacteria,
44
namely, Lactobacillus, Bifidobacterium and Akkermansia, were observed
oxidative
stress,
inflammatory
response
and
intestinal
45
to be reduced after trichlorfon exposure. In summary, the findings of this
46
study indicate that exposure to different concentrations of trichlorfon can
47
damage intestinal barrier, induce intestinal oxidative damage, trigger
48
inflammatory reaction and alter gut microbiota structure in common carp.
49 50
Key words: Trichlorfon; Cyprinus carpio; intestinal barrier; oxidative
51
stress; inflammatory response; intestinal microbiome
52 53
1. Introduction
54
Organophosphorus pesticides (OPs) have been used as insecticides,
55
acaricides and anthelmintic compounds in agriculture, industry, and
56
households for long periods of time (Lu et al., 2018; Ma et al., 2018).
57
Trichlorfon is an organophosphate insecticide used for controlling
58
different parasitic infections in fish and other aquatic animals (Ma et al.,
59
2019; Woo et al., 2018). The doses of trichlorfon for ectoparasite
60
eradication are varying from 0.1 to 1.0 mg/L (Chang et al., 2006), but
61
large amounts are often used in aquaculture and agriculture farming
62
systems. Based on the guidelines of Environmental Protection Agency,
63
the environmental concentrations of trichlorfon in underground water and
64
surface waters are approximately 0.00027 and 0.179 mg/L, respectively
65
Error! Reference source not found..
66
67
Excessive application of trichlorfon in aquaculture can contribute to
68
severe environmental pollution. Previous studies have indicated that
69
trichlorfon can cause different types of toxicity, such as hepatotoxicity,
70
hematotoxicity and neurotoxicity, to many freshwater fish species
71
(Chandrasekara and Pathiratne, 2005; Ma et al., 2019; Sinha et al., 2010;
72
Woo et al., 2018; Xu et al., 2009; Yonar et al., 2015). It is worth noting
73
that humans have suffered from carcinogenicity, mutagenicity and
74
reproductive toxicity following accidental or occupational exposure to
75
trichlorfon residues Error! Reference source not found.. Thus, the
76
overuse of trichlorfon in aquaculture has raised considerable concern
77
about its influence on environmental and public health Error! Reference
78
source not found.; Woo et al., 2018).
79 80
The intestine is the primary site for digestion, nutrient acquisition
81
and toxin exposure, due to its broad surface areas and physiological
82
characteristics (Sun et al., 2018; Vismaya and Rajini, 2014). Thus, a
83
complete intestinal structure and functionality is beneficial to the host's
84
health (Suo et al., 2017). It has been demonstrated that OPs can alter the
85
structure and function of intestines, and thereby affecting host health
86
(Lecoeur et al., 2006; Vismaya and Rajini, 2014). However, there is lack
87
of information available on the changes in the intestinal health of fish
88
after trichlorfon exposure.
89 90
The fish gut is inhabited by a wide variety of microbial communities,
91
and a stable intestinal microbiota is a key to a healthy host by regulating
92
numerous physiological functions, such as pathogen resistance, nutrient
93
digestion, energy metabolism, and immune modulation (Jin et al., 2017b).
94
The correlation between several diseases and intestinal microbiota
95
composition changes has been reported (Lange et al., 2016). Numerous
96
factors, such as developmental stage, environmental factors (e.g. water
97
salinity and temperature), geographic habitat location, fish species and
98
age, can modulate the composition of gut microbiota (Li et al., 2018;
99
Zhag et al., 2016). Recent works have suggested that chronic exposure to
100
pesticides can alter the composition of intestinal microbiota (Evariste et
101
al., 2019; Kan et al., 2015). However, the impact of trichlorfon exposure
102
on the intestinal microbiota of aquatic organisms remains largely
103
unknown.
104 105
Common carp is the most commonly grown freshwater fish in China,
106
which can serve as a bio-indicator for assessing the status of
107
environmental pollution (Yeşilbudak and Erdem, 2014). The general aim
108
of this research was to understand the impact of different concentrations
109
of trichlorfon on intestinal health and microbiome in common carp. To
110
achieve this goal, the intestinal morphology, mRNA levels of occludin,
111
claudin-2, zonula occludens-1 (ZO-1),
112
(IL-1β), transforming growth factor-β (TGF-β) and tumour necrosis
113
factor-α (TNF-α) , activities of catalase (CAT), glutathione peroxidase
114
(GSH-Px) and superoxide dismutase (SOD), the contents of glutathione
115
malondialdehyde (MDA) and reactive oxygen species (ROS) and, as well
116
as composition and diversity of intestinal microbiota were investigated in
117
common carp exposed to different concentrations of trichlorfon. It is
118
hoped that the findings will offer novel insights into the toxicological
119
effects of trichlorfon on freshwater fish.
the levels of interleukin 1β
120 121
2. Material and methods
122
2.1. Reagents and fish
123
Trichlorfon (>90% purity) was supplied by Shanghai Biochemical
124
Reagent (Shanghai, China), while 5-month-old healthy common carp
125
(mean weight, 58.6 ± 1.6 g) were obtained from a farmland in the
126
Freshwater Aquaculture Institute of Henan Province, China. Before
127
starting the experiments, all fish were acclimated with a controlled diet
128
(containing 20 % fish meal, 20 % flour, 20 % soybean meal, 19 % casein,
129
10 % CMC-Na, 5 % cottonseed meal, 3 % soybean oil, 1.5 % CaH2PO3,
130
1 % mineral mixture, 0.2 % methylcellulose, 0.2 % salt and 0.1 %
131
vitamin mixture; Tongwei, Henan Province, China) twice daily (at 08:30
132
and 17:30) for 2 weeks.
133 134
2.2 Experimental design
135
After 2 weeks of acclimation, 360 common carp were divided into
136
four groups. The concentration of trichlorfon treatment in each group was
137
0, 0.1, 0.5 and 1.0 mg/L, corresponding to 0, 1/500, 1/100 and 1/50 of
138
72-h LC50 data (51.25 mg/L trichlorfon, according to our acute toxicity
139
experiment) for common carp, respectively. Approximately 1/3 of the
140
tank water was changed daily, and the common carp were fed twice per
141
day (at 08:30 and 17:30) for 4 consecutive weeks. The fish in each group
142
were weighed on days 0 and 28, and the weight gain was then calculated
143
for all the four groups. Throughout the entire experiment, the rearing
144
water temperatures were ranged from 25.0 to 27.0 °C, with 5.0-6.0 mg/L
145
dissolved oxygen, pH 7.1-7.4 and < 0.01 mg/L amino nitrogen. The
146
concentrations of trichlorfon were assessed twice weekly using
147
ultra-high-performance liquid chromatography combined with mass
148
spectrophotometry Error! Reference source not found.. The measured
149
trichlorfon concentrations for control, 0.1, 0.5 and 1.0 mg/L trichlorfon
150
treatment groups were 0, 72.28 ± 15.98, 409.34 ± 35.96 and 828.96 ±
151
42.34 µg /L, respectively.
152 153
2.3 Intestinal tissue sampling
154
After completing trichlorfon treatment, the fish were selected and
155
euthanized with 10 mg/L tricaine methanesulfonate. Intestinal tissue was
156
isolated from each fish, and then washed with physiological saline.
157
Samples of intestinal content were aseptically scraped with glass slides,
158
collected into autoclaved tubes, and transferred into a liquid nitrogen tank,
159
then stored at -80 °C for microbial DNA extraction. Meanwhile, the
160
tissue samples were quickly removed, placed in liquid nitrogen, and
161
stored at -80 °C for RNA extraction. A portion of the midgut was fixed in
162
Bouin's fluid for histomorphological analysis. The remaining intestinal
163
tissue was kept at -80 °C for enzyme activity evaluation.
164 165
2.4 Morphological analysis
166
The Bouin's fixed tissues were dehydrated and paraffin-embedded,
167
followed by sectioning at 6-µm thickness and staining with haematoxylin
168
and eosin (HE). The microstructure of the intestinal tissue was analyzed
169
using a light microscope (Nikon Eclipse E400).
170 171
2.5 Assessment of antioxidant enzyme activities, MDA contents and ROS
172
levels
173
Intestinal tissue sample was homogenized (1:9, wt/vol) with ice-cold
174
physiological saline, and then centrifuged at 3000 rpm for 10 minutes.
175
The activities of CAT, GSH-Px, and SOD, as well as the contents of
176
MDA and ROS were examined using commercial test kits (Jian Cheng
177
Bioengineering Institute, Nanjing, China). The procedures were
178
conducted by following the instructions of the kits.
179 180
2.6 Cytokines assays
181
The intestinal protein levels of IL-1β, TGF-β and TNF-α in common
182
carp were measured by ELISA method as described previously Error!
183
Reference source not found..
184 185
2.7 Real-time PCR assay
186
The transcriptional levels of the target genes were evaluated using
187
the real-time PCR method. Total RNA was extracted from the intestinal
188
tissues using a TRIzon Reagent RNA kit (Invitrogen) by following the
189
manufacturer's protocol. Subsequently, PrimeScript RT reagent kit with
190
gDNA eraser (Perfect Real Time, Takara, Japan) was applied for cDNA
191
synthesis. The corresponding primer sequences are described in Table S1.
192 193
Real-time PCR mixture (20 µl) containing 10 µl of 2×SYBR Premix
194
Ex Taq (TaKaRa, Japan), 0.4 µl of ROX reference dye II, 0.4 µl of each
195
primer (10 mM) and 1 µl of diluted cDNA template was prepared in a
196
96-well plate. The reaction program was set at 95 °C for 30 s, followed
197
by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. Common carp β-actin was
198
selected as an internal standard for normalizing the relative mRNA levels
199
of target genes via the ∆∆Ct method (Pfaffl, 2001).
200 201
2.8 Intestinal microbiota analysis
202
The intestinal microbial DNA was isolated by a QIAamp DNA Stool
203
Mini kit (Qiagen Inc., Hilden, Germany) as per the manufacturer's
204
protocol. V3-V4 hypervariable region of the 16S rRNA was selected for
205
the PCR amplification of the extracted DNA. The universal primers 338F
206
(ACTCCTACGGGAGGCAGCA)
207
(GGACTACHVGGGTWTCTAAT) were employed to amplify this
208
region. The PCR volume and reaction program were set as previously
209
reported for common carp (Chang et al., 2019). PCR product purification
210
was carried out by an AxyPrep DNA gel extraction kit (Axygen,
211
Hangzhou, China) before sequencing on an Illumina MiSeq system at
212
Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Finally,
213
the raw sequence reads were deposited into the NCBI’s Sequence Read
214
Archive database (accession number: PRJNA553584).
and
806R
215 216
The raw FASTQ file reads were assessed and quality-filtered as
217
previously described for common carp (Chang et al., 2019). Species
218
richness and taxonomic diversity were analyzed using Mothur version
219
1.31.2 (Schloss et al., 2009). Alpha diversity was estimated by Chao1,
220
Simpson's diversity and Good’s coverage indices. Beta diversity was
221
assessed using the unweighted UniFrac-based Principal Coordinates
222
Analysis (PCoA). To asses if statistically significant differences occurred
223
in microbial composition among the 4 groups, permutational analysis of
224
variance (PERMANOVA) analysis were completed at OUT level.
225 226
2.9 Statistical analyses
227
All data were analyzed by SPSS software version 22.0 (IBM Corp.,
228
Chicago, IL, USA). When a significant difference was resulted from
229
one-way analysis of variance (ANOVA), the difference between groups
230
was further evaluated by Duncan’s multiple comparisons. The results
231
were presented as mean values ± standard error of the mean (SEM).
232 233
3 Results
234
3.1 Effects of trichlorfon on weight gain in common carp
235
The effects of different trichlorfon concentrations on the weight of
236
common carp are shown in Tables S2. After 28 days of feeding, the mean
237
weights of common carp were 23.7 ± 1.6, 20.9 ± 2.2, 17.9 ± 1.8 and 15.5
238
± 1.6 g for 0 (control), 0.1, 0.5 and 1.0 mg/L trichlorfon treatment groups,
239
respectively. No significant difference (P = 0.292) in weight gain was
240
found between 0 and 0.1 mg/L trichlorfon exposure groups. There was a
241
significant difference in weight gain between the common carp exposed
242
to 0 mg/L trichlorfon and those exposed to 0.5 and 1.0 mg/L trichlorfon
243
after 28 days of feeding (P = 0.023, P = 0.002). These results indicate that
244
the high concentrations of trichlorfon could inhibit the growth of common
245
carp.
246 247
3.2 Effects of trichlorfon on the intestinal histomorphology of common
248
carp
249
The effects of trichlorfon on intestinal histomorphology were
250
examined by HE staining under light microscopy (Fig. 1). Intestinal villus
251
length and muscular layer thickness are important indicators of intestinal
252
health in fish. In this study, a typical healthy intestine was noted in
253
control group. The most notable histomorphology change in the
254
trichlorfon-treated groups was the shortening of villus length compared to
255
control group (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). The values of muscle
256
thickness between the four groups were not significantly different (P =
257
0.198, P = 0.132, P = 0.098).
258 259
3.3 Effects of trichlorfon on the transcriptional levels of tight junction
260
genes
261
The impacts of trichlorfon on the transcriptional levels of the three
262
intestinal mucosa tight junction genes, such as claudin-2, occludin and
263
ZO-1, are presented in Fig. 2. The results showed that low-dose (0.1 mg/L)
264
trichlorfon treatment exerted no significant effect on the mRNA levels of
265
occludin and claudin-2 (P = 0.063, P = 0.180). However, the
266
transcriptional levels of occludin and claudin-2 genes were markedly
267
reduced in 0.5 and 1.0 mg/L trichlorfon treatment group compared to
268
those in control group (P = 0.003, P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). In
269
addition, the mRNA level of ZO-1 gene was remarkably downregulated
270
in response to increasing trichlorfon concentrations (P ≤ 0.001, P ≤ 0.001,
271
P ≤ 0.001).
272 273
3.4 Effects of trichlorfon on the intestinal oxidative stress
274
The effects of trichlorfon on intestinal oxidative stress are shown in
275
Fig. 3. Notably, SOD activity was significantly decreased in the intestinal
276
tissue of common carp treated with different trichlorfon doses compared
277
to control common carp (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). Similarly,
278
CAT activity in the intestinal tissue of common carp displayed a
279
decreasing trend with increasing trichlorfon concentrations, and the
280
difference was statistically significant (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001).
281
The activities of GSH-Px were significantly lower in 0.5 and 1 mg/L
282
trichlorfon treatment groups than in control group (P = 0.006, P = 0.001),
283
but no statistical significance was reached for 0.1 mg/L treatment group
284
(P = 0.124). Similarly, the concentrations of MDA did not differ
285
significantly different between 0.1 mg/L treatment group and control
286
group (P = 0.173). However, with increasing trichlorfon doses, the MDA
287
content continued to increase significantly (P = 0.016, P = 0.006).
288
Moreover, the levels of ROS were markedly increased in the intestinal
289
tissues of common carp treated with different trichlorfon doses compared
290
to the control group (P = 0.004, P = 0.002, P≤0.001).
291 292 293
3.5 Effects of trichlorfon on the cytokines The effects of trichlorfon on the expression levels of the three
294
cytokine-related genes (i.e. IL-1β, TGF-β and TNF-α) are shown in Fig. 4.
295
The expression levels of pro-inflammatory cytokine IL-1β were
296
remarkably upregulated in the three trichlorfon treatment groups (P =
297
0.005, P ≤ 0.001, P ≤ 0.001). No significant difference was found for the
298
mRNA levels of pro-inflammatory cytokine TNF-α between control and
299
0.1 mg/L trichlorfon treatment groups (P = 0.621). However, its
300
expression levels were markedly elevated in 0.5 and 1.0 mg/L trichlorfon
301
treatment groups when compared to control group (P ≤ 0.001, P ≤ 0.001).
302
Furthermore, the mRNA level of anti-inflammatory cytokine TGF-β in
303
1.0 mg/L trichlorfon treatment group was significantly lower than that in
304
control group (P = 0.001). The effects of trichlorfon on the protein
305
levels of the three cytokines are presented in Fig. S1, and the results are
306
consistent with the above mRNA expression data.
307 308 309
3.6 Intestinal microbiota analysis After filtering out the low-quality sequence reads, a total of 610,495
310
raw reads (ranging from 33,938 to 68,259 per sample) were obtained
311
from the 12 sequenced samples. All matching tags were delineated into
312
OTUs. The similarity of the different tags was approximately 97%, and
313
the total number of OTUs was 336 at this similarity cut-off value. The
314
rarefaction curves appeared to reach the saturation plateau (Fig. S2),
315
indicating that the analysis has covered most of the microbial diversity.
316 317
The alpha diversity of intestinal microbiota was determined using
318
Chao 1 and Simpson indices for all the four groups. As shown in Fig. 5A,
319
the results of Chao 1 index displayed a decreasing richness tendency with
320
increasing trichlorfon concentrations, and a significant difference was
321
observed between 1.0 mg/L trichlorfon treatment and control group (P=
322
0.04). Moreover, the data of Simpson index (Fig. 5B) exhibited an
323
increasing trend with the increase of trichlorfon concentrations.
324
Compared with control group, the values of Simpson index in 0.5 and 1.0
325
mg/L trichlorfon treatment groups were remarkably increased (P = 0.012,
326
P ≤ 0.001). These findings suggest that the diversity of intestinal
327
microbial community is reduced in common carp following trichlorfon
328
exposure. Based on the PCoA analysis of unweighted UniFrac distances,
329
the microbial communities of gut samples in 0, 0.1, 0.5 and 1.0
330
mg/L trichlorfon exposure groups were categorized into four clear groups,
331
except that one sample was overlapped between 0.1 and 0.5 mg/L
332
trichlorfon exposure groups (Fig. 6). PERMANOVA analysis result
333
indicated that gut microbiota structure was significantly affected by
334
trichlorfon treatment (F = 4.284, P = 0.002 ). These findings indicate that
335
chronic exposure to trichlorfon may alter the gut microbiota structure of
336
common carp.
337 338
A total of 22 phyla were detected in all the fish samples. The most
339
abundant phyla found in the four groups were Fusobacteria,
340
Proteobacteria and Bacteroidetes (Fig. 7A). As shown in Fig. S3A,
341
exposure to trichlorfon caused a significant decrease in Fusobacteria
342
(control: 59.83%, T1: 55.18%, T2: 51.90% and T3: 43.99%; P = 0.043)
343
and a considerable increase in Bacteroidetes (control: 15.60%, T1:
344
18.89%, T2: 21.28% and T3: 24.99%). In addition, a total of 220 genera
345
were detected in the 12 samples. The most dominant genera in the
346
intestinal samples of common carp were Cetobacterium, Aeromonas and
347
Bacteroides (Fig. 7B). The results of Kruskal-Wallis test showed that
348
trichlorfon exposure altered the gut microbiota composition of common
349
carp at the genus level (Fig. S3B). Furthermore, the abundance levels of
350
some probiotics (e.g. Lactobacillus, Bifidobacterium and Akkermansia)
351
appeared to be decreased in common carp treated with trichlorfon (Table
352
S3).
353
354
4. Discussion
355
Trichlorfon is one of the most commonly applied OPs in
356
aquacultural field. However, its large-scale application has caused severe
357
environmental pollution. It has been demonstrated that trichlorfon
358
triggers several physiological alterations in fish (Lu et al., 2018). To the
359
best of our knowledge, this study is the first to reveal that trichlorfon
360
exposure disrupts intestinal barrier, triggers intestinal oxidative stress,
361
stimulates inflammatory response, and alters the composition and
362
structure of intestinal microbiota in common carp.
363 364
A recent study has demonstrated that some environmental pollutants
365
can be deposited into aquatic animals through direct water uptake or
366
ingestion of contaminated materials (Ding et al., 2019). Therefore, the
367
intestine is proposed to be a target organ of OPs, and its morphology can
368
serve as an important indicator for assessing the effects of environmental
369
pollutants (Ding et al., 2019; Tinkov et al., 2018). Several studies have
370
reported that pesticides can alter the structures of the intestine (Pinton et
371
al., 2009; Sun et al., 2018). The findings of histological observation and
372
haematoxylin staining revealed that the common carp exhibited
373
significantly decreased villus height after four weeks of trichlorfon
374
exposure. This proves that trichlorfon exposure tends to alter the structure
375
of the intestine, which may affect the status of nutrient absorption in
376
common carp, resulting in poor growth performance.
377 378
Tight junction proteins, such as the transmembrane proteins claudins
379
and occludin as well as the cytosolic protein ZO-1 are an important part
380
of the intestinal physical barrier (Yu et al., 2019). Abnormal expression
381
levels of tight junction proteins can affect the function of physical
382
intestinal barrier and regulate the change in gut permeability (Ding et al.,
383
2019). In mammals, the influence of pesticides on the expression levels
384
of tight junction proteins varies across different studies (Condette et al.,
385
2014; Sun et al., 2018). However, there has been very little research
386
investigating the effects of pesticides on the transcriptional levels of tight
387
junction proteins in fish. Our findings demonstrated that the mRNA
388
levels of claudin-2, occludin and ZO-1 were decreased in common carp
389
after trichlorfon exposure. These data indicate that trichlorfon exposure
390
can disrupt the intestinal physical barrier in common carp, leading to an
391
increase in intestinal permeability. Such phenomenon may aggravate
392
trichlorfon to pass through the intestinal barrier into the body of common
393
carp, and cause more serious damage to the host.
394 395
Oxidative stress can serve as an essential biomarker for determining
396
the impact of pesticide exposure Error! Reference source not found..
397
ROS are generated as byproducts of normal cell metabolism, and their
398
low levels can maintain biological functions and cellular homeostasis,
399
while their high levels can cause oxidative damages to cells Error!
400
Reference source not found.. In this work, the intestinal levels of ROS
401
in common carp were increased after trichlorfon exposure. Hence, our
402
results indicated that chronic exposure to trichlorfon could cause
403
oxidative damage in the intestine of common carp.
404 405
MDA is an end-product of lipid peroxidation, and the elevated MDA
406
contents are useful markers of oxidative stress induced by free radicals
407
(Valavanidis et al., 2006). It was observed that trichlorfon exposure
408
increased the concentrations of MDA in the gut of common carp.
409
Antioxidant enzyme plays major roles in protecting cells from oxidative
410
stress by removing free radicals generated during metabolic reactions
411
and/or activated by immunostimulants (Jin et al., 2019; Reyes-Becerril et
412
al., 2019). In our experiment, the levels of CAT, GSH-Px and SOD were
413
analyzed. Notably, the common carp treated with trichlorfon displayed
414
reduced activities of CAT, GSH-Px and SOD compared to the untreated
415
common carp, indicating that the antioxidant system is suppressed after
416
exposure of trichlorfon. Consistently, several studies have demonstrated
417
that trichlorfon exposure can reduce the activities of antioxidant enzymes
418
(Coelho et al., 2011; Lu et al., 2018). Taken together, the decreased CAT,
419
GSH-Px and SOD activities as well as the increased MDA levels could
420
explain the intestinal oxidative stress of common carp after trichlorfon
421
exposure.
422 423
There seem to be a close connection between inflammation and
424
oxidative stress (Sun et al., 2018). Inflammatory responses and immune
425
system activation in the gut usually result from substantial gut injury in
426
animals (Suo et al., 2017). OPs have been shown to cause inflammation
427
in the gut of rats (Sun et al., 2018). However, much less is known about
428
the impact of gut inflammation in fish. Cytokines, such as IL-1β, TNF-α
429
and TGF-β, play vital roles in mediating inflammatory response and
430
immune function in animals (Standen et al., 2016). Our findings indicated
431
that the transcriptional and protein levels of pro-inflammatory IL-1β and
432
TNF-α were remarkably elevated in trichlorfon treatment groups. While,
433
the expression levels of anti-inflammatory cytokine TGF-β was markedly
434
reduced in 1.0 mg/L trichlorfon treatment group compared to control
435
group. These data indicate that trichlorfon exposure triggers inflammation
436
and disrupts intestinal immune function in common carp. Therefore, we
437
assume that trichlorfon exposure can weaken the host's defence against
438
diseases.
439 440
Recently, intestinal microbiota have been found to be associated
441
with host health through the regulation of multiple physiological
442
functions, including pathogen resistance, immune regulation and mineral
443
metabolism maturation (Jin et al., 2017b). Among multiple factors that
444
affect the changes in gut microbiota, growing evidence has indicated that
445
pesticides can lead to significant alterations in intestinal microbiota
446
communities and dysbiosis (Evariste et al., 2019; Jin et al., 2017a).
447
However, limited studies have investigated the toxic effects of trichlorfon
448
on intestinal microbiota in fish. Our findings revealed that trichlorfon
449
profoundly affected the composition and diversity of intestinal microbiota
450
in common carp, which were supported by PCoA analysis and reduced
451
microbiome alpha diversity. Combined with the histological results, we
452
speculated that trichlorfon-induced intestinal damage might trigger a
453
severe deterioration of the intestinal microenvironment, which in turn
454
accelerates the changes in intestinal microbiota. Previous studies have
455
shown that imbalance of gut microbiome caused by environmental
456
pollutants could lead to disorders of nutrient absorption, energy
457
metabolism, and immune function (Evariste et al., 2019; Liu et al., 2017;
458
Meng et al., 2018). Thus, our findings highlight the hazards of chronic
459
trichlorfon exposure in affecting intestinal microbes that might cause
460
health problems in common carp.
461 462
In
the
present
study,
Fusobacteria,
Proteobacteria
and
463
Bacteroidetes were identified as the most dominant phyla of gut bacteria
464
in common carp. These findings are consistent with previous findings
465
showing that Fusobacteria, Proteobacteria and Bacteroidetes are
466
commonly distributed in common carp (Chang et al., 2019). An
467
over-representation of Fusobacteria was observed in the gut of common
468
carp, and this phylum is also the dominant member in many other fishes,
469
including omnivorous zebrafish Error! Reference source not found.,
470
bluegill Error! Reference source not found. and grass carp Error!
471
Reference source not found.. Our findings indicated that trichlorfon
472
exposure significantly altered the composition of gut microbiota at
473
phylum level. Specifically, the relative abundances of Fusobacteria
474
reduced with increasing trichlorfon concentrations. The relative
475
abundance levels of control and 0.1, 0.5 and 1.0 mg/L trichlorfon
476
treatment groups were 59.83%, 55.18%, 51.90% and 43.99%,
477
respectively, suggesting that trichlorfon could inhibit the growth of
478
Fusobacteria. Nearly all Fusobacteria identfied from the intestinal
479
samples of common carp belonged to the genus Cetobacterium. Previous
480
studies have reported that Cetobacterium is the most dominant microbiota
481
in the intestine of fish (Chang et al., 2019; Li et al., 2015; Ni et al., 2012),
482
and this bacterium has been proven to produce vitamin B12 (Tsuchiya et
483
al.,
484
Cetobacterium were reduced in high-dose trichlorfon treatment group.
485
Combined with the growth performance result, it is speculated that
486
trichlorfon-induced alteration in gut microbial community structure may
2008).
Our
results
demonstrated
that
the
abundances
of
487
affect the nutrient absorption status of common carp and inhibit the
488
growth of common carp.
489 490
Probiotics, such as Lactobacillus and Bifidobacterium, have been
491
commonly used in aquacultural field (Newaj-Fyzul et al., 2014; Wang et
492
al., 2019). The gut microbiota may serve as a vital source of probiotics for
493
fish (Verschuere et al., 2000). Previous research has demonstrated that
494
Bifidobacterium
495
microbiota, thus maintaining immune homeostasis in the host (Liu et al.,
496
2016). The ability to successfully colonize the intestine is regarded as a
497
basic requirement for probiotic strains, and such strains should be
498
originated preferably from host gut microbiota (Wu et al., 2018). Our
499
findings revealed that the relative abundances of Lactobacillus and
500
Bifidobacterium were reduced in the fish guts exposed to trichlorfon.
501
These results suggest that trichlorfon exposure could inhibit the
502
colonization of some potential probiotics in the gut of common carp.
503
Akkermansia is a group oh intestinal anaerobic bacteria distributed
504
throughout the fish intestine, including common carp, which has been
505
recognized as a new functional microorganism with probiotic features
506
(Chang et al., 2018; Gomez-Gallego et al., 2016; Meng et al., 2018).
507
Previous studies have indicated that Akkermansia can protect the gut
508
barrier (Belzer and de Vos, 2012; Derrien et al., 2017). In this work, the
and
Lactobacillus
positively
regulate
intestinal
509
relative abundance of Akkermansia was reduced in the fish guts exposed
510
to trichlorfon, suggesting that the functional barrier of the gut can be
511
disrupted by trichlorfon exposure .
512 513
In summary, the findings of this study reveal that chronic exposure
514
to trichlorfon can lead to intestinal barrier function damage, intestinal
515
oxidative damage, inflammatory response, immunity activation, and gut
516
microbial community alterations in common carp.
517 518
Acknowledgements
519
This work was supported by the National Natural Science
520
Foundation of China (31700446 and 31902361), the Key Technology
521
Research Project of Henan Province (182102110235 and 192102310138),
522
and the Doctoral Foundation of Henan Normal University (qd16157).
523 524
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751
Figure caption
752
Fig. 1 Haematoxylin-eosin staining in intestinal sections from common
753
carp. (A) Control; (B) 0.1; (C) 0.5 and (D) 1.0 mg/L trichlorfon treatment
754
groups. VH=villus height; MT=muscle thickness. Scale bar=200 µm.
755
Effects of trichlorfon on villus height (E) and muscle thickness (G) in
756
common carp. * Stands for significant differences among treatments
757
compared with the control using one-way analysis of variance (ANOVA)
758
followed by by Duncan’s multiple comparisons (P < 0.05).
759 760
Fig. 2. Effects of trichlorfon exposure on the mRNA levels of tight
761
junction genes in the gut of common carp after 4 weeks of trichlorfon
762
exposure. Values are given as the mean ± SEM (n = 3 replicate tanks. For
763
the mRNA expression analysis, six fish from each tank were sampled).
764
The mRNA expression level values were normalized to β-actin and
765
expressed as a ratio of the control. * Stands for significant differences
766
among treatments compared with the control using one-way analysis of
767
variance (ANOVA) followed by by Duncan’s multiple comparisons (P <
768
0.05).
769 770
Fig. 3. Glutathione peroxidase (GSH-Px) (A), superoxide dismutase
771
(SOD), and catalase (CAT) activity and malondialdehyde (MDA)
772
concentration in the gut of common carp after exposure to trichlorfon.
773
Data are presented as the mean ± SE (n=18 fish/treatment). * Stands for
774
significant differences among treatments compared with the control using
775
one-way analysis of variance (ANOVA) followed by by Duncan’s
776
multiple comparisons (P < 0.05).
777 778
Fig. 4. Effects of trichlorfon exposure on the mRNA levels of
779
cytokine-related genes in the intestine of common carp. Values are given
780
as the mean ± SEM (n = 3 replicate tanks. For the mRNA expression
781
analysis, six fish from each tank were sampled). The mRNA expression
782
level values were normalized to those of β-actin and expressed as a ratio
783
of the control. * Stands for significant differences among treatments
784
compared with the control using one-way analysis of variance (ANOVA)
785
followed by by Duncan’s multiple comparisons (P < 0.05).
786 787
Fig. 5. Significant differences between the four groups in terms of alpha
788
diversity of the intestinal microbial communities in common carp. (A)
789
Bacterial community richness (measured by the Chao index). (B)
790
Bacterial community diversity (measured by the Simpson index).
791
significant differences (*: P < 0.05; **: P < 0.01) was analysed using
792
one-way analysis of variance (ANOVA).
793 794
Fig. 6. Principal coordinates analysis of the unweighted UniFrac scores of
795
the microbial communities. Principal components (PCs) 1 and 2 explain
796
42.3% and 19.9% of the variance, respectively.
797 798
Fig. 7. Bacterial composition of the different communities at the phylum
799
level (A) and genus level (B). Taxa with abundances <1% are included in
800
“others”.
801
802 803
Figures
804 805 806 807 808 809
Fig.1
810 811
Fig.2
812
813 814 815
Fig.3
816 817
Fig.4
818
819 820 821 822
Fig.5
823 824
Fig.6
825
826 827 828
Fig.7
Highlights: Trichlorfon exposure reduced the height of intestinal villus and decreased the expression levels of tight junction genes. Trichlorfon exposure increased the levels of ROS and MDA and decreased the antioxidant enzyme activity. Trichlorfon exposure affected the intestinal microbiota community composition of common carp.
Chang Xulu: Conceptualization, Methodology, Investigation. Wang Xianfeng: Methodology, Validation, Writing - Original Draft. Feng Junchang: Formal analysis: Su Xi: Investigation. Li Hui: Resources. Liang Junping: Visualization, Supervision. Zhang Jianxin: Project administration, Writing - Review & Editing.
Conflict of interest statement The authors declare no conflicts of interest.
S0269-7491(19)33863-1
DOI:
https://doi.org/10.1016/j.envpol.2019.113846
Reference:
ENPO 113846
To appear in:
Environmental Pollution
Received Date: 17 July 2019 Revised Date:
30 November 2019
Accepted Date: 16 December 2019
Please cite this article as: Chang, X., Wang, X., Feng, J., Su, X., Liang, J., Li, H., Zhang, J., Impact of chronic exposure to trichlorfon on intestinal barrier, oxidative stress, inflammatory response and intestinal microbiome in common carp (Cyprinus carpio L.), Environmental Pollution (2020), doi: https:// doi.org/10.1016/j.envpol.2019.113846. 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. © 2019 Published by Elsevier Ltd.
1
Impact of chronic exposure to trichlorfon on intestinal barrier,
2
oxidative stress, inflammatory response and intestinal microbiome in
3
common carp (Cyprinus carpio L.)
4 a,1
, Xianfeng Wang a,1, Junchang Feng a, Xi Su b, Junping
5
Xulu Chang
6
Liang a, Hui Li a, Jianxin Zhang a,*
7 8
a
9
China
College of Fisheries, Henan Normal University, Xinxiang, 453007, PR
10
b
11
Medical University, Xinxiang, 453007, PR China
12
1
13
* Corresponding author: Jianxin Zhang
14
Email: [email protected]
15
Tel.: +86-373-3326563
16
Fax: +86-373-3326563
17
Address: College of Fisheries, Henan Normal University, Xinxiang,
18
453007, PR China
19 20 21 22
Henan Mental Hospital, The Second Affiliated Hospital of Xinxiang
These authors contributed equally to this study.
23
Abstract
24
Trichlorfon is an organic phosphorus pesticide used to control
25
different parasitic infections in aquaculture. The repeated, excessive use
26
of trichlorfon can result in environmental pollution, thus affecting human
27
health. This study aimed to determine the effects of different
28
concentrations of trichlorfon (0, 0.1, 0.5 and 1.0 mg/L) on the intestinal
29
barrier,
30
microbiome of common carp. Trichlorfon exposure significantly reduced
31
the height of intestinal villus and decreased the expression levels of tight
32
junction genes, such as claudin-2, occludin and ZO-1, in common carp.
33
Moreover, the activities of antioxidant enzymes, such as CAT, SOD and
34
GSH-Px, exhibited a decreasing trend with increasing trichlorfon
35
concentrations, while the contents of MDA and ROS elevated in the
36
intestinal tissues of common carp. The mRNA and protein levels of
37
pro-inflammatory cytokines TNF-α and IL-1β were significantly
38
upregulated by trichlorfon exposure. The level of anti-inflammatory
39
cytokine TGF-β was remarkably higher in 1.0 mg/L trichlorfon treatment
40
group compared to control group. In addition, the results demonstrated
41
that trichlorfon exposure could affect the microbiota community
42
composition and decreased the community diversity in the gut of
43
common carp. Notably, the proportions of some probiotic bacteria,
44
namely, Lactobacillus, Bifidobacterium and Akkermansia, were observed
oxidative
stress,
inflammatory
response
and
intestinal
45
to be reduced after trichlorfon exposure. In summary, the findings of this
46
study indicate that exposure to different concentrations of trichlorfon can
47
damage intestinal barrier, induce intestinal oxidative damage, trigger
48
inflammatory reaction and alter gut microbiota structure in common carp.
49 50
Key words: Trichlorfon; Cyprinus carpio; intestinal barrier; oxidative
51
stress; inflammatory response; intestinal microbiome
52 53
1. Introduction
54
Organophosphorus pesticides (OPs) have been used as insecticides,
55
acaricides and anthelmintic compounds in agriculture, industry, and
56
households for long periods of time (Lu et al., 2018; Ma et al., 2018).
57
Trichlorfon is an organophosphate insecticide used for controlling
58
different parasitic infections in fish and other aquatic animals (Ma et al.,
59
2019; Woo et al., 2018). The doses of trichlorfon for ectoparasite
60
eradication are varying from 0.1 to 1.0 mg/L (Chang et al., 2006), but
61
large amounts are often used in aquaculture and agriculture farming
62
systems. Based on the guidelines of Environmental Protection Agency,
63
the environmental concentrations of trichlorfon in underground water and
64
surface waters are approximately 0.00027 and 0.179 mg/L, respectively
65
Error! Reference source not found..
66
67
Excessive application of trichlorfon in aquaculture can contribute to
68
severe environmental pollution. Previous studies have indicated that
69
trichlorfon can cause different types of toxicity, such as hepatotoxicity,
70
hematotoxicity and neurotoxicity, to many freshwater fish species
71
(Chandrasekara and Pathiratne, 2005; Ma et al., 2019; Sinha et al., 2010;
72
Woo et al., 2018; Xu et al., 2009; Yonar et al., 2015). It is worth noting
73
that humans have suffered from carcinogenicity, mutagenicity and
74
reproductive toxicity following accidental or occupational exposure to
75
trichlorfon residues Error! Reference source not found.. Thus, the
76
overuse of trichlorfon in aquaculture has raised considerable concern
77
about its influence on environmental and public health Error! Reference
78
source not found.; Woo et al., 2018).
79 80
The intestine is the primary site for digestion, nutrient acquisition
81
and toxin exposure, due to its broad surface areas and physiological
82
characteristics (Sun et al., 2018; Vismaya and Rajini, 2014). Thus, a
83
complete intestinal structure and functionality is beneficial to the host's
84
health (Suo et al., 2017). It has been demonstrated that OPs can alter the
85
structure and function of intestines, and thereby affecting host health
86
(Lecoeur et al., 2006; Vismaya and Rajini, 2014). However, there is lack
87
of information available on the changes in the intestinal health of fish
88
after trichlorfon exposure.
89 90
The fish gut is inhabited by a wide variety of microbial communities,
91
and a stable intestinal microbiota is a key to a healthy host by regulating
92
numerous physiological functions, such as pathogen resistance, nutrient
93
digestion, energy metabolism, and immune modulation (Jin et al., 2017b).
94
The correlation between several diseases and intestinal microbiota
95
composition changes has been reported (Lange et al., 2016). Numerous
96
factors, such as developmental stage, environmental factors (e.g. water
97
salinity and temperature), geographic habitat location, fish species and
98
age, can modulate the composition of gut microbiota (Li et al., 2018;
99
Zhag et al., 2016). Recent works have suggested that chronic exposure to
100
pesticides can alter the composition of intestinal microbiota (Evariste et
101
al., 2019; Kan et al., 2015). However, the impact of trichlorfon exposure
102
on the intestinal microbiota of aquatic organisms remains largely
103
unknown.
104 105
Common carp is the most commonly grown freshwater fish in China,
106
which can serve as a bio-indicator for assessing the status of
107
environmental pollution (Yeşilbudak and Erdem, 2014). The general aim
108
of this research was to understand the impact of different concentrations
109
of trichlorfon on intestinal health and microbiome in common carp. To
110
achieve this goal, the intestinal morphology, mRNA levels of occludin,
111
claudin-2, zonula occludens-1 (ZO-1),
112
(IL-1β), transforming growth factor-β (TGF-β) and tumour necrosis
113
factor-α (TNF-α) , activities of catalase (CAT), glutathione peroxidase
114
(GSH-Px) and superoxide dismutase (SOD), the contents of glutathione
115
malondialdehyde (MDA) and reactive oxygen species (ROS) and, as well
116
as composition and diversity of intestinal microbiota were investigated in
117
common carp exposed to different concentrations of trichlorfon. It is
118
hoped that the findings will offer novel insights into the toxicological
119
effects of trichlorfon on freshwater fish.
the levels of interleukin 1β
120 121
2. Material and methods
122
2.1. Reagents and fish
123
Trichlorfon (>90% purity) was supplied by Shanghai Biochemical
124
Reagent (Shanghai, China), while 5-month-old healthy common carp
125
(mean weight, 58.6 ± 1.6 g) were obtained from a farmland in the
126
Freshwater Aquaculture Institute of Henan Province, China. Before
127
starting the experiments, all fish were acclimated with a controlled diet
128
(containing 20 % fish meal, 20 % flour, 20 % soybean meal, 19 % casein,
129
10 % CMC-Na, 5 % cottonseed meal, 3 % soybean oil, 1.5 % CaH2PO3,
130
1 % mineral mixture, 0.2 % methylcellulose, 0.2 % salt and 0.1 %
131
vitamin mixture; Tongwei, Henan Province, China) twice daily (at 08:30
132
and 17:30) for 2 weeks.
133 134
2.2 Experimental design
135
After 2 weeks of acclimation, 360 common carp were divided into
136
four groups. The concentration of trichlorfon treatment in each group was
137
0, 0.1, 0.5 and 1.0 mg/L, corresponding to 0, 1/500, 1/100 and 1/50 of
138
72-h LC50 data (51.25 mg/L trichlorfon, according to our acute toxicity
139
experiment) for common carp, respectively. Approximately 1/3 of the
140
tank water was changed daily, and the common carp were fed twice per
141
day (at 08:30 and 17:30) for 4 consecutive weeks. The fish in each group
142
were weighed on days 0 and 28, and the weight gain was then calculated
143
for all the four groups. Throughout the entire experiment, the rearing
144
water temperatures were ranged from 25.0 to 27.0 °C, with 5.0-6.0 mg/L
145
dissolved oxygen, pH 7.1-7.4 and < 0.01 mg/L amino nitrogen. The
146
concentrations of trichlorfon were assessed twice weekly using
147
ultra-high-performance liquid chromatography combined with mass
148
spectrophotometry Error! Reference source not found.. The measured
149
trichlorfon concentrations for control, 0.1, 0.5 and 1.0 mg/L trichlorfon
150
treatment groups were 0, 72.28 ± 15.98, 409.34 ± 35.96 and 828.96 ±
151
42.34 µg /L, respectively.
152 153
2.3 Intestinal tissue sampling
154
After completing trichlorfon treatment, the fish were selected and
155
euthanized with 10 mg/L tricaine methanesulfonate. Intestinal tissue was
156
isolated from each fish, and then washed with physiological saline.
157
Samples of intestinal content were aseptically scraped with glass slides,
158
collected into autoclaved tubes, and transferred into a liquid nitrogen tank,
159
then stored at -80 °C for microbial DNA extraction. Meanwhile, the
160
tissue samples were quickly removed, placed in liquid nitrogen, and
161
stored at -80 °C for RNA extraction. A portion of the midgut was fixed in
162
Bouin's fluid for histomorphological analysis. The remaining intestinal
163
tissue was kept at -80 °C for enzyme activity evaluation.
164 165
2.4 Morphological analysis
166
The Bouin's fixed tissues were dehydrated and paraffin-embedded,
167
followed by sectioning at 6-µm thickness and staining with haematoxylin
168
and eosin (HE). The microstructure of the intestinal tissue was analyzed
169
using a light microscope (Nikon Eclipse E400).
170 171
2.5 Assessment of antioxidant enzyme activities, MDA contents and ROS
172
levels
173
Intestinal tissue sample was homogenized (1:9, wt/vol) with ice-cold
174
physiological saline, and then centrifuged at 3000 rpm for 10 minutes.
175
The activities of CAT, GSH-Px, and SOD, as well as the contents of
176
MDA and ROS were examined using commercial test kits (Jian Cheng
177
Bioengineering Institute, Nanjing, China). The procedures were
178
conducted by following the instructions of the kits.
179 180
2.6 Cytokines assays
181
The intestinal protein levels of IL-1β, TGF-β and TNF-α in common
182
carp were measured by ELISA method as described previously Error!
183
Reference source not found..
184 185
2.7 Real-time PCR assay
186
The transcriptional levels of the target genes were evaluated using
187
the real-time PCR method. Total RNA was extracted from the intestinal
188
tissues using a TRIzon Reagent RNA kit (Invitrogen) by following the
189
manufacturer's protocol. Subsequently, PrimeScript RT reagent kit with
190
gDNA eraser (Perfect Real Time, Takara, Japan) was applied for cDNA
191
synthesis. The corresponding primer sequences are described in Table S1.
192 193
Real-time PCR mixture (20 µl) containing 10 µl of 2×SYBR Premix
194
Ex Taq (TaKaRa, Japan), 0.4 µl of ROX reference dye II, 0.4 µl of each
195
primer (10 mM) and 1 µl of diluted cDNA template was prepared in a
196
96-well plate. The reaction program was set at 95 °C for 30 s, followed
197
by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. Common carp β-actin was
198
selected as an internal standard for normalizing the relative mRNA levels
199
of target genes via the ∆∆Ct method (Pfaffl, 2001).
200 201
2.8 Intestinal microbiota analysis
202
The intestinal microbial DNA was isolated by a QIAamp DNA Stool
203
Mini kit (Qiagen Inc., Hilden, Germany) as per the manufacturer's
204
protocol. V3-V4 hypervariable region of the 16S rRNA was selected for
205
the PCR amplification of the extracted DNA. The universal primers 338F
206
(ACTCCTACGGGAGGCAGCA)
207
(GGACTACHVGGGTWTCTAAT) were employed to amplify this
208
region. The PCR volume and reaction program were set as previously
209
reported for common carp (Chang et al., 2019). PCR product purification
210
was carried out by an AxyPrep DNA gel extraction kit (Axygen,
211
Hangzhou, China) before sequencing on an Illumina MiSeq system at
212
Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Finally,
213
the raw sequence reads were deposited into the NCBI’s Sequence Read
214
Archive database (accession number: PRJNA553584).
and
806R
215 216
The raw FASTQ file reads were assessed and quality-filtered as
217
previously described for common carp (Chang et al., 2019). Species
218
richness and taxonomic diversity were analyzed using Mothur version
219
1.31.2 (Schloss et al., 2009). Alpha diversity was estimated by Chao1,
220
Simpson's diversity and Good’s coverage indices. Beta diversity was
221
assessed using the unweighted UniFrac-based Principal Coordinates
222
Analysis (PCoA). To asses if statistically significant differences occurred
223
in microbial composition among the 4 groups, permutational analysis of
224
variance (PERMANOVA) analysis were completed at OUT level.
225 226
2.9 Statistical analyses
227
All data were analyzed by SPSS software version 22.0 (IBM Corp.,
228
Chicago, IL, USA). When a significant difference was resulted from
229
one-way analysis of variance (ANOVA), the difference between groups
230
was further evaluated by Duncan’s multiple comparisons. The results
231
were presented as mean values ± standard error of the mean (SEM).
232 233
3 Results
234
3.1 Effects of trichlorfon on weight gain in common carp
235
The effects of different trichlorfon concentrations on the weight of
236
common carp are shown in Tables S2. After 28 days of feeding, the mean
237
weights of common carp were 23.7 ± 1.6, 20.9 ± 2.2, 17.9 ± 1.8 and 15.5
238
± 1.6 g for 0 (control), 0.1, 0.5 and 1.0 mg/L trichlorfon treatment groups,
239
respectively. No significant difference (P = 0.292) in weight gain was
240
found between 0 and 0.1 mg/L trichlorfon exposure groups. There was a
241
significant difference in weight gain between the common carp exposed
242
to 0 mg/L trichlorfon and those exposed to 0.5 and 1.0 mg/L trichlorfon
243
after 28 days of feeding (P = 0.023, P = 0.002). These results indicate that
244
the high concentrations of trichlorfon could inhibit the growth of common
245
carp.
246 247
3.2 Effects of trichlorfon on the intestinal histomorphology of common
248
carp
249
The effects of trichlorfon on intestinal histomorphology were
250
examined by HE staining under light microscopy (Fig. 1). Intestinal villus
251
length and muscular layer thickness are important indicators of intestinal
252
health in fish. In this study, a typical healthy intestine was noted in
253
control group. The most notable histomorphology change in the
254
trichlorfon-treated groups was the shortening of villus length compared to
255
control group (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). The values of muscle
256
thickness between the four groups were not significantly different (P =
257
0.198, P = 0.132, P = 0.098).
258 259
3.3 Effects of trichlorfon on the transcriptional levels of tight junction
260
genes
261
The impacts of trichlorfon on the transcriptional levels of the three
262
intestinal mucosa tight junction genes, such as claudin-2, occludin and
263
ZO-1, are presented in Fig. 2. The results showed that low-dose (0.1 mg/L)
264
trichlorfon treatment exerted no significant effect on the mRNA levels of
265
occludin and claudin-2 (P = 0.063, P = 0.180). However, the
266
transcriptional levels of occludin and claudin-2 genes were markedly
267
reduced in 0.5 and 1.0 mg/L trichlorfon treatment group compared to
268
those in control group (P = 0.003, P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). In
269
addition, the mRNA level of ZO-1 gene was remarkably downregulated
270
in response to increasing trichlorfon concentrations (P ≤ 0.001, P ≤ 0.001,
271
P ≤ 0.001).
272 273
3.4 Effects of trichlorfon on the intestinal oxidative stress
274
The effects of trichlorfon on intestinal oxidative stress are shown in
275
Fig. 3. Notably, SOD activity was significantly decreased in the intestinal
276
tissue of common carp treated with different trichlorfon doses compared
277
to control common carp (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001). Similarly,
278
CAT activity in the intestinal tissue of common carp displayed a
279
decreasing trend with increasing trichlorfon concentrations, and the
280
difference was statistically significant (P ≤ 0.001, P ≤ 0.001, P ≤ 0.001).
281
The activities of GSH-Px were significantly lower in 0.5 and 1 mg/L
282
trichlorfon treatment groups than in control group (P = 0.006, P = 0.001),
283
but no statistical significance was reached for 0.1 mg/L treatment group
284
(P = 0.124). Similarly, the concentrations of MDA did not differ
285
significantly different between 0.1 mg/L treatment group and control
286
group (P = 0.173). However, with increasing trichlorfon doses, the MDA
287
content continued to increase significantly (P = 0.016, P = 0.006).
288
Moreover, the levels of ROS were markedly increased in the intestinal
289
tissues of common carp treated with different trichlorfon doses compared
290
to the control group (P = 0.004, P = 0.002, P≤0.001).
291 292 293
3.5 Effects of trichlorfon on the cytokines The effects of trichlorfon on the expression levels of the three
294
cytokine-related genes (i.e. IL-1β, TGF-β and TNF-α) are shown in Fig. 4.
295
The expression levels of pro-inflammatory cytokine IL-1β were
296
remarkably upregulated in the three trichlorfon treatment groups (P =
297
0.005, P ≤ 0.001, P ≤ 0.001). No significant difference was found for the
298
mRNA levels of pro-inflammatory cytokine TNF-α between control and
299
0.1 mg/L trichlorfon treatment groups (P = 0.621). However, its
300
expression levels were markedly elevated in 0.5 and 1.0 mg/L trichlorfon
301
treatment groups when compared to control group (P ≤ 0.001, P ≤ 0.001).
302
Furthermore, the mRNA level of anti-inflammatory cytokine TGF-β in
303
1.0 mg/L trichlorfon treatment group was significantly lower than that in
304
control group (P = 0.001). The effects of trichlorfon on the protein
305
levels of the three cytokines are presented in Fig. S1, and the results are
306
consistent with the above mRNA expression data.
307 308 309
3.6 Intestinal microbiota analysis After filtering out the low-quality sequence reads, a total of 610,495
310
raw reads (ranging from 33,938 to 68,259 per sample) were obtained
311
from the 12 sequenced samples. All matching tags were delineated into
312
OTUs. The similarity of the different tags was approximately 97%, and
313
the total number of OTUs was 336 at this similarity cut-off value. The
314
rarefaction curves appeared to reach the saturation plateau (Fig. S2),
315
indicating that the analysis has covered most of the microbial diversity.
316 317
The alpha diversity of intestinal microbiota was determined using
318
Chao 1 and Simpson indices for all the four groups. As shown in Fig. 5A,
319
the results of Chao 1 index displayed a decreasing richness tendency with
320
increasing trichlorfon concentrations, and a significant difference was
321
observed between 1.0 mg/L trichlorfon treatment and control group (P=
322
0.04). Moreover, the data of Simpson index (Fig. 5B) exhibited an
323
increasing trend with the increase of trichlorfon concentrations.
324
Compared with control group, the values of Simpson index in 0.5 and 1.0
325
mg/L trichlorfon treatment groups were remarkably increased (P = 0.012,
326
P ≤ 0.001). These findings suggest that the diversity of intestinal
327
microbial community is reduced in common carp following trichlorfon
328
exposure. Based on the PCoA analysis of unweighted UniFrac distances,
329
the microbial communities of gut samples in 0, 0.1, 0.5 and 1.0
330
mg/L trichlorfon exposure groups were categorized into four clear groups,
331
except that one sample was overlapped between 0.1 and 0.5 mg/L
332
trichlorfon exposure groups (Fig. 6). PERMANOVA analysis result
333
indicated that gut microbiota structure was significantly affected by
334
trichlorfon treatment (F = 4.284, P = 0.002 ). These findings indicate that
335
chronic exposure to trichlorfon may alter the gut microbiota structure of
336
common carp.
337 338
A total of 22 phyla were detected in all the fish samples. The most
339
abundant phyla found in the four groups were Fusobacteria,
340
Proteobacteria and Bacteroidetes (Fig. 7A). As shown in Fig. S3A,
341
exposure to trichlorfon caused a significant decrease in Fusobacteria
342
(control: 59.83%, T1: 55.18%, T2: 51.90% and T3: 43.99%; P = 0.043)
343
and a considerable increase in Bacteroidetes (control: 15.60%, T1:
344
18.89%, T2: 21.28% and T3: 24.99%). In addition, a total of 220 genera
345
were detected in the 12 samples. The most dominant genera in the
346
intestinal samples of common carp were Cetobacterium, Aeromonas and
347
Bacteroides (Fig. 7B). The results of Kruskal-Wallis test showed that
348
trichlorfon exposure altered the gut microbiota composition of common
349
carp at the genus level (Fig. S3B). Furthermore, the abundance levels of
350
some probiotics (e.g. Lactobacillus, Bifidobacterium and Akkermansia)
351
appeared to be decreased in common carp treated with trichlorfon (Table
352
S3).
353
354
4. Discussion
355
Trichlorfon is one of the most commonly applied OPs in
356
aquacultural field. However, its large-scale application has caused severe
357
environmental pollution. It has been demonstrated that trichlorfon
358
triggers several physiological alterations in fish (Lu et al., 2018). To the
359
best of our knowledge, this study is the first to reveal that trichlorfon
360
exposure disrupts intestinal barrier, triggers intestinal oxidative stress,
361
stimulates inflammatory response, and alters the composition and
362
structure of intestinal microbiota in common carp.
363 364
A recent study has demonstrated that some environmental pollutants
365
can be deposited into aquatic animals through direct water uptake or
366
ingestion of contaminated materials (Ding et al., 2019). Therefore, the
367
intestine is proposed to be a target organ of OPs, and its morphology can
368
serve as an important indicator for assessing the effects of environmental
369
pollutants (Ding et al., 2019; Tinkov et al., 2018). Several studies have
370
reported that pesticides can alter the structures of the intestine (Pinton et
371
al., 2009; Sun et al., 2018). The findings of histological observation and
372
haematoxylin staining revealed that the common carp exhibited
373
significantly decreased villus height after four weeks of trichlorfon
374
exposure. This proves that trichlorfon exposure tends to alter the structure
375
of the intestine, which may affect the status of nutrient absorption in
376
common carp, resulting in poor growth performance.
377 378
Tight junction proteins, such as the transmembrane proteins claudins
379
and occludin as well as the cytosolic protein ZO-1 are an important part
380
of the intestinal physical barrier (Yu et al., 2019). Abnormal expression
381
levels of tight junction proteins can affect the function of physical
382
intestinal barrier and regulate the change in gut permeability (Ding et al.,
383
2019). In mammals, the influence of pesticides on the expression levels
384
of tight junction proteins varies across different studies (Condette et al.,
385
2014; Sun et al., 2018). However, there has been very little research
386
investigating the effects of pesticides on the transcriptional levels of tight
387
junction proteins in fish. Our findings demonstrated that the mRNA
388
levels of claudin-2, occludin and ZO-1 were decreased in common carp
389
after trichlorfon exposure. These data indicate that trichlorfon exposure
390
can disrupt the intestinal physical barrier in common carp, leading to an
391
increase in intestinal permeability. Such phenomenon may aggravate
392
trichlorfon to pass through the intestinal barrier into the body of common
393
carp, and cause more serious damage to the host.
394 395
Oxidative stress can serve as an essential biomarker for determining
396
the impact of pesticide exposure Error! Reference source not found..
397
ROS are generated as byproducts of normal cell metabolism, and their
398
low levels can maintain biological functions and cellular homeostasis,
399
while their high levels can cause oxidative damages to cells Error!
400
Reference source not found.. In this work, the intestinal levels of ROS
401
in common carp were increased after trichlorfon exposure. Hence, our
402
results indicated that chronic exposure to trichlorfon could cause
403
oxidative damage in the intestine of common carp.
404 405
MDA is an end-product of lipid peroxidation, and the elevated MDA
406
contents are useful markers of oxidative stress induced by free radicals
407
(Valavanidis et al., 2006). It was observed that trichlorfon exposure
408
increased the concentrations of MDA in the gut of common carp.
409
Antioxidant enzyme plays major roles in protecting cells from oxidative
410
stress by removing free radicals generated during metabolic reactions
411
and/or activated by immunostimulants (Jin et al., 2019; Reyes-Becerril et
412
al., 2019). In our experiment, the levels of CAT, GSH-Px and SOD were
413
analyzed. Notably, the common carp treated with trichlorfon displayed
414
reduced activities of CAT, GSH-Px and SOD compared to the untreated
415
common carp, indicating that the antioxidant system is suppressed after
416
exposure of trichlorfon. Consistently, several studies have demonstrated
417
that trichlorfon exposure can reduce the activities of antioxidant enzymes
418
(Coelho et al., 2011; Lu et al., 2018). Taken together, the decreased CAT,
419
GSH-Px and SOD activities as well as the increased MDA levels could
420
explain the intestinal oxidative stress of common carp after trichlorfon
421
exposure.
422 423
There seem to be a close connection between inflammation and
424
oxidative stress (Sun et al., 2018). Inflammatory responses and immune
425
system activation in the gut usually result from substantial gut injury in
426
animals (Suo et al., 2017). OPs have been shown to cause inflammation
427
in the gut of rats (Sun et al., 2018). However, much less is known about
428
the impact of gut inflammation in fish. Cytokines, such as IL-1β, TNF-α
429
and TGF-β, play vital roles in mediating inflammatory response and
430
immune function in animals (Standen et al., 2016). Our findings indicated
431
that the transcriptional and protein levels of pro-inflammatory IL-1β and
432
TNF-α were remarkably elevated in trichlorfon treatment groups. While,
433
the expression levels of anti-inflammatory cytokine TGF-β was markedly
434
reduced in 1.0 mg/L trichlorfon treatment group compared to control
435
group. These data indicate that trichlorfon exposure triggers inflammation
436
and disrupts intestinal immune function in common carp. Therefore, we
437
assume that trichlorfon exposure can weaken the host's defence against
438
diseases.
439 440
Recently, intestinal microbiota have been found to be associated
441
with host health through the regulation of multiple physiological
442
functions, including pathogen resistance, immune regulation and mineral
443
metabolism maturation (Jin et al., 2017b). Among multiple factors that
444
affect the changes in gut microbiota, growing evidence has indicated that
445
pesticides can lead to significant alterations in intestinal microbiota
446
communities and dysbiosis (Evariste et al., 2019; Jin et al., 2017a).
447
However, limited studies have investigated the toxic effects of trichlorfon
448
on intestinal microbiota in fish. Our findings revealed that trichlorfon
449
profoundly affected the composition and diversity of intestinal microbiota
450
in common carp, which were supported by PCoA analysis and reduced
451
microbiome alpha diversity. Combined with the histological results, we
452
speculated that trichlorfon-induced intestinal damage might trigger a
453
severe deterioration of the intestinal microenvironment, which in turn
454
accelerates the changes in intestinal microbiota. Previous studies have
455
shown that imbalance of gut microbiome caused by environmental
456
pollutants could lead to disorders of nutrient absorption, energy
457
metabolism, and immune function (Evariste et al., 2019; Liu et al., 2017;
458
Meng et al., 2018). Thus, our findings highlight the hazards of chronic
459
trichlorfon exposure in affecting intestinal microbes that might cause
460
health problems in common carp.
461 462
In
the
present
study,
Fusobacteria,
Proteobacteria
and
463
Bacteroidetes were identified as the most dominant phyla of gut bacteria
464
in common carp. These findings are consistent with previous findings
465
showing that Fusobacteria, Proteobacteria and Bacteroidetes are
466
commonly distributed in common carp (Chang et al., 2019). An
467
over-representation of Fusobacteria was observed in the gut of common
468
carp, and this phylum is also the dominant member in many other fishes,
469
including omnivorous zebrafish Error! Reference source not found.,
470
bluegill Error! Reference source not found. and grass carp Error!
471
Reference source not found.. Our findings indicated that trichlorfon
472
exposure significantly altered the composition of gut microbiota at
473
phylum level. Specifically, the relative abundances of Fusobacteria
474
reduced with increasing trichlorfon concentrations. The relative
475
abundance levels of control and 0.1, 0.5 and 1.0 mg/L trichlorfon
476
treatment groups were 59.83%, 55.18%, 51.90% and 43.99%,
477
respectively, suggesting that trichlorfon could inhibit the growth of
478
Fusobacteria. Nearly all Fusobacteria identfied from the intestinal
479
samples of common carp belonged to the genus Cetobacterium. Previous
480
studies have reported that Cetobacterium is the most dominant microbiota
481
in the intestine of fish (Chang et al., 2019; Li et al., 2015; Ni et al., 2012),
482
and this bacterium has been proven to produce vitamin B12 (Tsuchiya et
483
al.,
484
Cetobacterium were reduced in high-dose trichlorfon treatment group.
485
Combined with the growth performance result, it is speculated that
486
trichlorfon-induced alteration in gut microbial community structure may
2008).
Our
results
demonstrated
that
the
abundances
of
487
affect the nutrient absorption status of common carp and inhibit the
488
growth of common carp.
489 490
Probiotics, such as Lactobacillus and Bifidobacterium, have been
491
commonly used in aquacultural field (Newaj-Fyzul et al., 2014; Wang et
492
al., 2019). The gut microbiota may serve as a vital source of probiotics for
493
fish (Verschuere et al., 2000). Previous research has demonstrated that
494
Bifidobacterium
495
microbiota, thus maintaining immune homeostasis in the host (Liu et al.,
496
2016). The ability to successfully colonize the intestine is regarded as a
497
basic requirement for probiotic strains, and such strains should be
498
originated preferably from host gut microbiota (Wu et al., 2018). Our
499
findings revealed that the relative abundances of Lactobacillus and
500
Bifidobacterium were reduced in the fish guts exposed to trichlorfon.
501
These results suggest that trichlorfon exposure could inhibit the
502
colonization of some potential probiotics in the gut of common carp.
503
Akkermansia is a group oh intestinal anaerobic bacteria distributed
504
throughout the fish intestine, including common carp, which has been
505
recognized as a new functional microorganism with probiotic features
506
(Chang et al., 2018; Gomez-Gallego et al., 2016; Meng et al., 2018).
507
Previous studies have indicated that Akkermansia can protect the gut
508
barrier (Belzer and de Vos, 2012; Derrien et al., 2017). In this work, the
and
Lactobacillus
positively
regulate
intestinal
509
relative abundance of Akkermansia was reduced in the fish guts exposed
510
to trichlorfon, suggesting that the functional barrier of the gut can be
511
disrupted by trichlorfon exposure .
512 513
In summary, the findings of this study reveal that chronic exposure
514
to trichlorfon can lead to intestinal barrier function damage, intestinal
515
oxidative damage, inflammatory response, immunity activation, and gut
516
microbial community alterations in common carp.
517 518
Acknowledgements
519
This work was supported by the National Natural Science
520
Foundation of China (31700446 and 31902361), the Key Technology
521
Research Project of Henan Province (182102110235 and 192102310138),
522
and the Doctoral Foundation of Henan Normal University (qd16157).
523 524
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751
Figure caption
752
Fig. 1 Haematoxylin-eosin staining in intestinal sections from common
753
carp. (A) Control; (B) 0.1; (C) 0.5 and (D) 1.0 mg/L trichlorfon treatment
754
groups. VH=villus height; MT=muscle thickness. Scale bar=200 µm.
755
Effects of trichlorfon on villus height (E) and muscle thickness (G) in
756
common carp. * Stands for significant differences among treatments
757
compared with the control using one-way analysis of variance (ANOVA)
758
followed by by Duncan’s multiple comparisons (P < 0.05).
759 760
Fig. 2. Effects of trichlorfon exposure on the mRNA levels of tight
761
junction genes in the gut of common carp after 4 weeks of trichlorfon
762
exposure. Values are given as the mean ± SEM (n = 3 replicate tanks. For
763
the mRNA expression analysis, six fish from each tank were sampled).
764
The mRNA expression level values were normalized to β-actin and
765
expressed as a ratio of the control. * Stands for significant differences
766
among treatments compared with the control using one-way analysis of
767
variance (ANOVA) followed by by Duncan’s multiple comparisons (P <
768
0.05).
769 770
Fig. 3. Glutathione peroxidase (GSH-Px) (A), superoxide dismutase
771
(SOD), and catalase (CAT) activity and malondialdehyde (MDA)
772
concentration in the gut of common carp after exposure to trichlorfon.
773
Data are presented as the mean ± SE (n=18 fish/treatment). * Stands for
774
significant differences among treatments compared with the control using
775
one-way analysis of variance (ANOVA) followed by by Duncan’s
776
multiple comparisons (P < 0.05).
777 778
Fig. 4. Effects of trichlorfon exposure on the mRNA levels of
779
cytokine-related genes in the intestine of common carp. Values are given
780
as the mean ± SEM (n = 3 replicate tanks. For the mRNA expression
781
analysis, six fish from each tank were sampled). The mRNA expression
782
level values were normalized to those of β-actin and expressed as a ratio
783
of the control. * Stands for significant differences among treatments
784
compared with the control using one-way analysis of variance (ANOVA)
785
followed by by Duncan’s multiple comparisons (P < 0.05).
786 787
Fig. 5. Significant differences between the four groups in terms of alpha
788
diversity of the intestinal microbial communities in common carp. (A)
789
Bacterial community richness (measured by the Chao index). (B)
790
Bacterial community diversity (measured by the Simpson index).
791
significant differences (*: P < 0.05; **: P < 0.01) was analysed using
792
one-way analysis of variance (ANOVA).
793 794
Fig. 6. Principal coordinates analysis of the unweighted UniFrac scores of
795
the microbial communities. Principal components (PCs) 1 and 2 explain
796
42.3% and 19.9% of the variance, respectively.
797 798
Fig. 7. Bacterial composition of the different communities at the phylum
799
level (A) and genus level (B). Taxa with abundances <1% are included in
800
“others”.
801
802 803
Figures
804 805 806 807 808 809
Fig.1
810 811
Fig.2
812
813 814 815
Fig.3
816 817
Fig.4
818
819 820 821 822
Fig.5
823 824
Fig.6
825
826 827 828
Fig.7
Highlights: Trichlorfon exposure reduced the height of intestinal villus and decreased the expression levels of tight junction genes. Trichlorfon exposure increased the levels of ROS and MDA and decreased the antioxidant enzyme activity. Trichlorfon exposure affected the intestinal microbiota community composition of common carp.
Chang Xulu: Conceptualization, Methodology, Investigation. Wang Xianfeng: Methodology, Validation, Writing - Original Draft. Feng Junchang: Formal analysis: Su Xi: Investigation. Li Hui: Resources. Liang Junping: Visualization, Supervision. Zhang Jianxin: Project administration, Writing - Review & Editing.
Conflict of interest statement The authors declare no conflicts of interest.