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Fish & Shellsh Immunology 52 (2016) 317e324

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Fish & Shellsh Immunology


j o u r n a l h o m e p a g e : w w w.el s e vi e r. c o m / l o c a t e / f s i

Full length article

The effect of hyperthermia on liver histology, oxidative stress and


disease resistance of the Wuchang bream, Megalobrama amblycephala
Bo Liu a, Pao Xu a, Paul B. Brown b, Jun Xie a, *, Xianping Ge a, **
, Linghong Miao a,
Qunlan Zhou a, Mingchun Ren a, Liangkun Pan a
a
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese
Academy of Fishery Sciences, Wuxi 214081, PR China
b
Department of Forestry and Natural Resources, Purdue University, West Lafayette, 47907, Indiana, USA

a r t i c l e i n f o a b s t r a c t
Article history: This study aimed to investigate the effects of hyperthermia on serum hormones, hepatic oxidization
Received 18 December 2015
indices, hepatic heat shock protein (HSP60, 70, and 90) mRNA expression levels and liver cell ultra-
Received in revised form
structure in Megalobrama amblycephala before and after high temperature stress. Fish were exposed to
5 March 2016
Accepted 11 March 2016 the optimal temperature (25 1 C) or high temperature (32 1 C) and then challenged with
Available online 22 March 2016 Aero- monas hydrophila. The results showed that hyperthermic stress signicantly increased serum
adreno- corticotropic hormone (ACTH) at 0.5 and 2 d, serum cortisol (COR) at 0.5, 14, and 21 d and
Keywords: serum 3,5,30 - triiodothyronine (T3) at 1, 14, and 21 d after stress. Additionally, hyperthermia led to
High temperature oxidative stress, as evidenced by a signicant decrease in the hepatic anti-superoxide anion free
Oxidative stress radical concentration (ASAFER) at 1, 2, 7, and 21 d and in hepatic superoxide dismutase (SOD) activity at
Liver histology 1, 2, 14 and 21 d after stress; however, hepatic malondialdehyde content (MDA) increased at 1, 2, and 7
HSPs gene expression d after stress. Moreover, the expression of HSP60 at 1 d, HSP70 at 1 and 2 d, and HSP90 at 0.25, 0.5, 1
Megalobrama amblycephala
and 2 d after stress was higher in the stress group compared with the control group. The histological
results clearly showed that hyperthermia resulted in fat and glycogen accumulation and structural
alterations of the hepatocytes, mitochondria, and nuclei. The cumulative mortality increased in the high
temperature stress group at 1 d after acute stress and at 2 and 7 d after chronic stress compared with
the control group. Overall, 1 d or 2 d after hyperthermia stress damaged the hepatic ultrastructure and
impaired mitochondrial bioenergetics. Dysfunction of the mitochondria subsequently mediated
oxidative stress and improved HSP expression modulated the cellular anti-stress response, which in
turn led to reduced efcacy of the immune system and increased mortality from Aeromonas hydrophila
infection in Megalobrama amblycephala.
2016 Elsevier Ltd. All rights reserved.

1. Introduction integrity, and result in protein carbonylation and cellular aggrega-


tion or fragmentation [8].
Temperature is an important ecological factor. Temperatures To cope with these injuries, sh possess an efcient innate im-
above the optimum affect growth performance and physiological mune and antioxidant defense system to protect themselves
functions of sh, and they increase concentrations of harmful against hyperthermia stress and maintain biochemical, molecular
reactive oxygen species (ROS) [1e4]. Cellular ROS cause damage and physiological homeostasis. Antioxidant enzymes such as su-
to DNA, a general disturbance of the cellular redox balance [5], peroxide dismutase (SOD) and antisuperoxide anion free radical
and increase susceptibility to infection [6,7]. In addition, ROS, (ASAFR) directly detoxify harmful ROS and other compounds
especially HO , lead to a chain reaction and severe injury to involved in ROS generation [9]. SOD is a well-known antioxidative
plasma mem- branes, cause abnormal cellular function and loss enzyme that converts superoxide to hydrogen peroxide and oxy-
of membrane gen, and catalase catalyzes the decomposition of hydrogen
peroxide into water and oxygen to remove the free radical of
oxy- gen and reduce lipid peroxidation damage [10,11]. In
* Corresponding author. Present address: No.9 Shanshui East Road, FFRC CAFS,
Wuxi 214081, PR China.
addition to hyperthermia stress, the elevation of ROS is known to
** Corresponding author. increase heat shock factor [12] and heat shock protein (HSP)
E-mail addresses: liub@ffrc.cn (B. Liu), Xiej@ffrc.cn (J. Xie), gexp@ffrc.cn (X. Ge). expression levels

http://dx.doi.org/10.1016/j.fsi.2016.03.018
1050-4648/ 2016 Elsevier Ltd. All rights reserved.
B. Liu
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[13], which prevent protein aggregation, assist in refolding any was the control group (commercial diet with a water
misfolded proteins, and maintain the integrity of the temperature of
mitochondrial membrane [14e16]. Thus, the relationships 25 1 C) for Aeromonas hydrophila infection, and the other groups
between oxidative stress, antioxidants and HSPs play an
important role in sh survival during exposure to elevated
temperatures [17].
Previous studies have shown that histological examination of
the liver could provide an index of the general condition of the
sh [18,19]. Mitochondria are considered to be the major source of
ROS production [20,21]. Moreover, ROS are also produced by the
microsomal systems of the endoplasmic reticulum [22]. An un-
derstanding of the organelle ultrastructure could provide addi-
tional information regarding sh health and the metabolic
condition. However, to our knowledge, the effects of
hyperthermia- induced oxidative stress on the hepatic
ultrastructure of Mega- lobrama amblycephala remain poorly
studied.
Megalobrama amblycephala, also known as the blunt snout or
Wuchang bream, is one of the principal species in Chinese fresh-
water culture systems. Production of this species in China reached
approximately 0.73 million tons in 2013 [23]. The optimal tem-
perature for the growth of M. amblycephala is 25e28 C, but they
commonly experience water temperatures of more than 32 C;
chronically elevated temperatures above 30 C for 7e15 days or
more in summer are common. Hyperthermia may increase
M. amblycephala vulnerability to opportunistic bacterial pathogens
and result in signicant economic losses [24]. However, informa-
tion regarding the physiological mechanisms, oxidative stress and
pathogen invasion of M. amblycephala during increasing tempera-
tures is not well understood. Therefore, we hypothesized that
high temperature would affect the immune ability of the sh
and in- crease the prevalence of bacterial infection. To test our
hypothesis, the sh were exposed to the optimal temperature
(25 1 C) or high temperature (32 1 C), and then challenged
with bacteria. To compare the efciency of their oxidative
system and thermal resistance ability, we examined serum
hormones, hepatic oxi- dization indices, hepatic HSP mRNA
expression levels and liver cell ultrastructure during exposure.
Using these experiments, we ex- pected to gain an
understanding of the relationship of high temperature-induced
oxidative stress and disease resistance in sh challenged with
bacteria and high-temperature stress. The results would provide
guidance for understanding high-temperature stress and
immune defense and the potential adverse effects of this
condition on sh.

2. Materials and methods

2.1. Fish

We obtained 360 healthy M. amblycephala of a similar size


(mean weight: 77.04 2.18 g) from the Freshwater Fisheries
Research Center, Chinese Academy of Fishery Sciences, China. The
sh were placed in an indoor recirculating system containing 24
round berglass tanks (4820 700 mm, N 15 sh/tank) and
acclimated for 20 d. The experimental facilities consisted of a
thermo-regulated recirculating system and mechanical ltration
units with recycled water at a rate of 3 L min 1. All sh were
starved
for 24 h before the experiments. After acclimation, the sh in the
12 tanks were randomly divided into two groups (6 tanks per
group) for acute or chronic high temperature stress experiments:
control group (CT group, commercial diet with a water
temperature of
25 1 C), high temperature stress group (HTS group, commercial
diet with a water temperature of 32 1 C). The sh in the
remaining 12 tanks were randomly divided into four groups (3
tanks per group) for the pathogenic infection experiment. One
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31
were exposed to three temperature stresses (commercial 25 1 C for 30 d of acclimation. The water temperature in the CT
a water temperature of 32 1 C) placed in water at 32 1 C on d control group was subsequently maintained at 25 1 C. The sh
1, in the HTS group were subjected to an acute heat shock and
2 and 7 and then challenged with Aeromonas hydrophila. main- tained at 32 1 C for 24 h. The water temperature of the 25
Daily removal of 5e6% water from the tanks was performed
1 C system was quickly raised to 32 1 C within 3 h.
to remove leftover feed and excreta, and replenishment of the
During the stress experiment, the water temperature was
same volume and temperature of freshwater was provided in each
monitored using a data logger, and feed was withheld. Dissolved
group. During the experimental period, the water temperature in
oxygen was not less than 6.0 mg/L, and ammonia nitrogen was
the each tank was monitored using data logger and adjusted
lower than 0.05 mg/L. Minimal human interference prevented the
using ther- mostatic water heaters (XiLong XL-888, range 20e34
sh from experiencing additional stress. Six samples (1 sh per
C).
replicate tank) were taken prior to the heat stress and at 0.5 d and
An optimum extruded commercial diet with a proximate
1 d after challenge.
composition of 32.16% crude protein and 6.17% lipid (dry matter
basis) was obtained from Wuxi Tongwei Feed Co. Ltd., China. Dur-
2.2.2. Chronic high temperature stress
ing acclimation, the sh were hand-fed to apparent satiation three
After the acute high temperature stress, the CT control and HTS
times daily (8:00e8:30, 12:00e12:30, and 16:00e16:30).
group were maintained in the same water temperature of 25 1
During the experimental period, the water temperature for the
control and high temperature stress groups was maintained at 25 C or 32 1 C for 21 d, respectively. During the chronic high
tem- perature stress stage, the sh were hand-fed to apparent
1 C and
satiation once daily (8:00e8:30). The water temperature was
32 1 C, respectively. The water quality was monitored weekly monitored using a data logger; dissolved oxygen was not less
to ensure that the conditions remained within ranges acceptable than 6.0 mg/L, and ammonia nitrogen was lower than 0.05 mg/L.
for sh growth; the dissolved oxygen concentration of the water Six samples (1 sh per replicate tank) were collected on d 2, 7, 14
was and 21 of the experiment.
>6 mg/L, the ammonia-nitrogen concentration was <0.05 mg/L,
the photoperiod was 12-h light and 12-h dark, and the pH 2.2.3. Pathogen infection
remained between 7.60 and 7.80 throughout the study. Four groups (3 tanks per group) were assessed in the pathogen
infection experiment: the control group with a water temperature
2.2. Challenge experiments
of 25 C, and three high temperature stress groups with a water
2.2.1. Acute high temperature stress temperature of 32 C. Before application of the stress, and at 1 d
According to the procedure described by Fast et al. [25], there (acute stress point), 2 d and 7 d (one of chronic stress points) after
were two experimental groups: the control (CT) group and the stress, the sh were challenged with the bacterial septicemia
stressed (HTS) group (6 replicate tanks in each group). The CT and pathogen A. hydrophila. Thirty sh (3 tanks/group, N 10 sh/tank)
HTS groups were maintained in the same water temperature of at each time point were used in the infection experiment. Ac-
cording to the method described by Liu et al. [26], the strain of
A. hydrophi1a was activated using agar medium and then puried.
The colony was selected and incubated in a tube with nutritious follows: (1) 50 -TGCTGTCTACTGCTGAAGCCGTTGT-30 and 50
broth for 18e24 h. The number of bacteria was then counted -CCATCACT- CAGTTTCGGCAGGTTT-30 for HSP60 cDNA;
under a microscope. A. hydrophi1a was diluted in sterile normal
(2) 50 -
saline,
and the nal concentration was set at 5 107 cells mL 1. The
bacterial suspension (0.5 mL per 50 g body weight) was injected
into the abdominal cavity of the sh. After A. hydrophi1a
infection, the sh in the control group with a water temperature of
25 C or in the treatment groups with a water temperature of
32 C were exposed to the same water temperature for 5 days.
Fish mortality was checked at 0 h, 12 h, 24 h, 48 h, 72 h and 120 h
after challenge.

2.3. Serum and liver collection

At the end of the acclimation trial, the sh were starved for 48


h to evacuate the alimentary tract contents prior to sampling,
and serum and liver samples of 6 sh (1 sh per replicate tank) in
the CT or HTS group were obtained. To eliminate the change
in sh metabolism level after feeding on serum and liver
parameters, the CT and HTS group were sampled concomitantly at
each stress point. The serum and liver samples of 6 sh (1 per
replicate tank) in each group were collected at 0.5 d, 1 d, 2 d, 7 d,
14 d and 21 d after stress challenge. At each sampling point, the
sh were rapidly netted and then anesthetized with 150 mg/L MS-
222. We collected serum from the caudal vein and stored the
samples in a refrigerator at 4 C for
1e2 h. The serum was then centrifuged at 3000 g (4 C) for
10 min, and the supernatant was removed and stored at 20 C
for subsequent serum hormone measurements. In addition, part of
the liver in each sh was removed, frozen in liquid nitrogen, and
stored at 80 C until further analysis for subsequent assays of
oxidative parameters and HSP gene expressions. Part of the liver
from each sh was xed with 2.5% buffered glutaraldehyde for
observation of the liver ultrastructure.

2.4. Serum and liver measurements

2.4.1. Serum ACTH, COR and T3


measurements
The levels of adrenocorticotropic hormone (ACTH), cortisol
(COR) and 3,5,30 -triiodothyronine (T3) were measured using
the automatic chemiluminescence immunoassay analyzer
MAGLUMI
1000 (Shenzhen, China) with assay kits purchased from Shenzhen
New Industries Biomedical Engineering Co., Ltd, China, according to
a previously described method [27,28].

2.4.2. Hepatic ASAFR, SOD and MDA measurements


Hepatic samples were homogenized in ice-cold phosphate
buffer (1:10 dilution) (phosphate-buffered saline: 0.064 M, pH
7.4). The homogenate was then centrifuged for 10 min (4 C, 4000
g), and aliquots of the supernatant were used to quantify the
hepatic anti-superoxide anion free radical concentration
(ASAFER), super- oxide dismutase activity (SOD) and
malondialdehyde content (MDA). Hepatic ASAFR activity, SOD
activity and MDA content were measured using the xanthine
oxidase method [29], xanthine oxi- dase method [30] and
barbituric acid colorimetry [31], respectively. We measured the
hepatic protein content using the Folin method with bovine
serum albumin as a standard. Chemical assay kits were purchased
from the Nanjing Jiancheng Bioengineering Institute of China.

2.4.3. Real-time PCR for hepatic HSP60, HSP70 and HSP90


We used the M. amblycephala cDNA sequences in GenBank to
design the primers for HSP60 (accession No. KC521465), HSP70
(accession no. EU884290.2), HSP90 (accession no. KC521466) and
beta-actin (accession no. AY170122.2). The primers were as
CGACGCCAACGGAATCCTAAAT-3 and 50 actin) were obtained from each sample. We measured the
- CTTTGCTCAGTCTGCCCTTGT-3 0
for HSP70 cDNA; (3) 50 standard equation and correlation coefcient by constructing a
-TGCGGGA- CAACTCCACCAT-3 0
and 50 standard curve using a serial dilution of cDNA: HSP60: Y
0.310x10.65,
-TCCAATGAGAACCCAGAGGAAAGC-3 for HSP90 cDNA; and (4) 50
0
R2 0.991; HSP70: Y 0.361x13.38, R2 0.995; HSP90:
-TCTGCTATGTGGCTCTTGACTTCG-30 and 50 - Y 0.314x10.29, R2 0.996; Beta-actin: Y
CCTCTGGGCACCTGAACCTCT-30 for beta-actin cDNA. All primers 2
0.304x9.817, R 0.990; where Y is the logarithm of the
were synthesized by Shanghai Biocolor, BioScience & Technology starting template to
Company, China. The PCR products were 100e150 bp long. base 10, and x is the Ct value. The relative expression level of the
Total RNA was extracted from 50 to 100 mg liver tissue using
gene could be calculated using the double-standard curve method
TRIzol reagent (Dalian Takara Co. Ltd., China). In general, the [33].
puri- ed RNA had an OD260/OD280 ratio of 1.8e2.0. RNA samples
were treated with RQ1 RNase-Free DNase (Dalian Takara Co.
Limited, China) to avoid genomic DNA amplication. We
generated cDNA from 500 ng DNase-treated RNA using the 2.5. Transmission electron microscopy of liver
ExScript RT-PCR Kit (Dalian Takara Co. Ltd., China). The reverse
transcription PCR re- action solution consisted of 500 ng RNA, 2 mL Liver samples for electron microscopy observation were xed in
5 Buffer, 0.5 mL dT- 2.5% glutaraldehyde for 24 h, post-xed in 1% osmium tetroxide
AP Primer (50 mM), 0.25 mL ExScript RTase (200 U mL 1), and (OsO4) for 1 h, and stored at 4 C. Sections were embedded in
DEPC H2O up to a nal volume of 10 mL. The reaction conditions epoxy resin Epon812, cut into 70-mm thick slices with an RMC
were as follows: 42 C for 40 min, 90 C for 2 min, and 4 C PowerTome XL microtome, stained with uranyl acetate and lead
thereafter. citrate, and
examined under a Hitachi H-7650 transmission electron micro-
We used real-time quantitative PCR to determine the mRNA
levels with a SYBR Green one uorescence kit according to a pre- scope (Hitachi, Tokyo, Japan).
viously described method [32]. Real-time quantitative PCR was
performed using a Mini Opticon Real-Time Detector (Bio-Rad, USA).
The uorescent quantitative PCR reaction solution consisted of 2.6. Data statistics and analysis
12.5 mL SYBR premix Ex TaqTM (2 ), 0.5 mL PCR Forward Primer
(10 mM), 0.5 mL PCR Reverse Primer (10 mM), 2.0 mL RT reaction mix All results were expressed as the mean standard error of the
(cDNA solution), and 9.5 mL dH2O. The reaction conditions were as mean (XSEM). Data were analyzed by analysis of variance
follows: 95 C for 10 s, followed by 45 cycles consisting of 95 C (ANOVA). If ANOVA indicated signicant differences between
for treatments, Duncan's multiple range test was used to separate
5 s, 62 C for 15 s, 72 C for 10 s, plate reading, and a nal step treatment effects using SPSS, version 11.5. Letters indicate signi-
at cant differences (P < 0.05) compared with pre-stress levels ac-
72 C for 3 min. After the program nished, the Ct values of cording to Turkey's b test. Asterisks indicate signicant differences
the target genes (three HSPs) and a chosen reference gene (beta- (P < 0.05) between the CT and HST groups at the same sampling
time according to the t-test.
Adrenocorticotropic hormone (pg/mL)
3. Results 700 25
(A)
600 32
a*
ab
3.1. Effects of acute and chronic high temperature stress on 500 ab* ab
bc bc
cumulative mortality 400
c c
300
The sh in the CT group before stress and the sh in the HTS 200
group at 1 d after acute stress and at 2 and 7 d after chronic stress 100
were challenged with A. hydrophila, and the cumulative mortality 0
was calculated for 120 h (Fig. 1). The maximum cumulative mor-
tality occurred between 24 and 48 h after A. hydrophila infection. 0h 0.25d 0.5d 1d 2d 7d 14d 21d
Before stress Days after stress
At (B)

48 h after bacterial challenge, the cumulative mortality at 1 d 500

Serum cortisol content(ng/mL)


after acute stress and at 2 and 7 d after chronic stress in the HTS 400
groups was 100%, while the cumulative mortality in the CT a*

group was
b*
63.3%. In addition, the cumulative mortality improved in the HST
group at 1 d after acute stress and at 2 and 7 d after chronic stress
300 bcd* abc cd bcd abc
compared with the CT group (P < 0.05, Fig. 1). abc bc abc
a
cd ab
200
bc
3.2. Effects of acute and chronic high temperature stress on serum 100
c d

hormones
0

0h 0.25d 0.5d 1d 2d 7d 14d 21d


The effects of acute and chronic temperature stress on serum (C) Before stress Days after stress
ACTH, COR and T3 in M. amblycephala are shown in Fig. 2. Serum
Serum triiodothyronine T3 (ng/mL)

ACTH signicantly increased in the HTS group at 0.5 and 1 d after


10 25
acute stress and at 2 and 7 d after chronic stress compared with 32
the pre-stress level (P < 0.05, Fig. 2A). In addition, serum ACTH 8
was
signicantly elevated in the HTS group compared with the CT
group
abc a
at 0.5 d after acute stress and at 2 d after chronic stress (P < 0.05, 6
ab ab*
ab* a*
c
Fig. 2A). 4 c
The serum COR level increased dramatically in the CT group at
2
0.5 d after acute stress and at 7 d after chronic stress compared
with the pre-stress level (P < 0.05, Fig. 2B), while the serum COR 0
level 0h 0.25d 0.5d 1d 2d 7d 14d 21d
improved in the HTS group at 7, 14 and 21 d after chronic stress Before stress Days after stress

compared with the pre-stress level (P < 0.05, Fig. 2B). Serum COR
signicantly increased in the HTS group at 0.5 d after acute stress Fig. 2. Effects of acute and chronic stress on serum ACTH (A), COR (B), and T3 (C) in
M. amblycephala. Note: Data are expressed as the mean SEM (n 9). Letters indicate
and at 14, 21 d after chronic stress compared with the CT group
signicant differences (P < 0.05) in different dosage groups for each sampling point
(P < 0.05, Fig. 2B). using Turkey's b test. Asterisks indicate signicant differences (P < 0.05) between
The serum T3 level increased in the CT group at 0.5 d after values obtained pre-stress versus post-stress using a t-test.
acute stress and at 7 d after chronic stress compared with the
pre-stress levels (P < 0.05, Fig. 2C), while the serum T3 level
signicantly improved in the HTS group at 0.5 and 1 d after acute 3.3. Effects of acute and chronic high temperature stress on hepatic
stress and at 7, anti-oxidization enzymes
14 and 21 d after chronic stress compared with the pre-stress
level (P < 0.05, Fig. 2C). The comparison between groups before We examined the effects of acute and chronic high temperature
and after stress showed that serum T3 was signicantly enhanced stress on hepatic anti-oxidization enzymes in sh, and the results
in the HTS group at 1 d after acute stress and at 14 and 21 d after are shown in Fig. 3. There were no signicant differences in the
chronic stress compared with the CT group (P < 0.05, Fig. 2C). liver ASAFER concentration, SOD activity or MDA concentration in
the CT group at the time of stress exposure compared with the
pre-stress level (P > 0.05, Fig. 3).
25
160 32 for 1d The liver ASAFER concentration signicantly decreased in the
140 32 for 2d HTS group at 0.5 and 1 d after acute stress, and at 2, 7, 14 and 21 d
32 for 7d after chronic stress compared with the pre-stress level (P < 0.05,
Cumulative mortality (%)

120
* * * Fig. 3A). In addition, the hepatic ASAFER concentration in the HTS
100 group was signicantly reduced compared with that in the CT
* group at 1 d after acute stress and at 2, 7 and 21 d after chronic
80
stress (P < 0.05, Fig. 3A).
60 Liver SOD activity was signicantly reduced in the HTS group at
14 and 21 d after chronic stress compared with the pre-stress
40
level (P < 0.05, Fig. 3B). Moreover, hepatic SOD activity in the HTS
20 group was signicantly lower than that in the CT group at 1 d
0
after acute stress and at 2, 14 and 21 d after chronic stress (P <
0.05, Fig. 3B).
0h 12h 24h 48h 96h 120h The liver MDA concentration increased signicantly in the HTS
group at 1 d after acute stress and at 2 and 7 d after chronic stress
Fig. 1. Effects of high temperature stress on the cumulative mortality after
A. hydrophi1a infection of M. amblycephala. Note: Asterisks indicate signicant differ- compared with the pre-stress level (P < 0.05, Fig. 3C). Furthermore,
ences (P < 0.05) between values for the high temperature group and those for the the hepatic MDA concentration in the HTS group was signicantly
control group. higher than that in the CT group at 1 d after acute stress and at 2
and
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Relative HSP60 mRNA (arbitrary units


(A) (A)
1 25
a* 32
500 25 0.8
anti-superoxide anion free radical

450 32
a
400 a 0.6 b
bc
350 b*
b b* 0.4
300 b* bcd
b* cd
250 b d
0.2 d
d
200
150 0

100 0h 0.25d 0.5d 1d 2d 7d 14d 21d


50 Before stress Days after stress
(B)
0 0h 0.25d 0.5d 1d 2d 7d 14d 21d

Relative HSP70 mRNA (arbitrary units


Before stress Days after stress
1

(B) 180 0.8


a* a*
Superoxide dismutase activitie

150
0.6
120 a
a
a 0.4 b
a* a* b
90 a
b b
60 0.2 a
b* b* ab b ab ab
b b a
ab ab
0
(U/mg protein)

30 0h 0.25d 0.5d 1d 2d 7d 14d 21d


0 (C) Before stress Days after stress

Relative HSP90 mRNA (arbitrary units


0h 0.25d 0.5d 1d 2d 7d 14d 1.6
21d
Before stress Days after stress 1.4
a*
1.2
1 ab*
(C) 6
ab
Malondialdehyde contentl

a*
a*

a* b* b*
4 0.8 bc
b bc
b
0.6
b b b
0.4 c
2
0.2
(nmol/mg protein)

0
0h 0.25d 0.5d 1d 2d 7d 14d 21d
0 Before stress Days after stress
0h 0.25d 0.5d 1d 2d 7d 14d 21d
Before stress Days after stress Fig. 4. Effects of acute and chronic stress on liver HSP60 (A), HSP70 (B), and HSP90 (C)
in M. amblycephala. Note: Data are expressed as means SEM (n 9). Legends are the
Fig. 3. Effects of acute and chronic stress on liver ASAFR (A), SOD (B), and MDA (C) in same as described in Fig. 2.
M. amblycephala. Note: Data are expressed as means SEM (n 9). Legends are the
same as described in Fig. 2.

CT group at 0.25, 0.5 and 1.0 d after acute stress and at 2 d after
chronic stress (P < 0.05, Fig. 4C).
7 d after chronic stress (P < 0.05, Fig. 3C).

3.4. Effects of acute and chronic high temperature stress on hepatic


HSP gene expression
3.5. Effects of high temperature stress on hepatic cell ultrastructure
The effects of acute and chronic high temperature stress on
hepatic HSP gene expression in sh are shown in Fig. 4. The mRNA The hepatic cell ultrastructure of the sh at 48 h after high
expression level of liver HSP60 increased signicantly in the HTS temperature stress is shown in Fig. 5. Livers of sh that were
group at 1 d after acute stress compared with the pre-stress level acclimated to 25 C exhibited a normal ultrastructure. Each hepa-
(P < 0.05, Fig. 4A). In addition, liver HSP60 mRNA levels in the HTS tocyte had a large, round nucleus that was centrally located
group were signicantly higher than those in the CT group at 1 d within a moderate and homogeneous cytoplasm (Fig. 5A, 5B). The
after acute stress (P < 0.05, Fig. 4A). nucleus was ovoid and contained a prominent nucleolus (Fig.
Liver HSP70 mRNA expression signicantly increased in the CT 5A). The rough endoplasmic reticulum (Fig. 5A) and smooth
group at 2 and 14 d after chronic stress compared with the pre- endoplasmic reticulum (Fig. 5A, B) were well developed and often
stress level (P < 0.05, Fig. 4B). Liver HSP70 mRNA expression adjacent to the nucleus and mitochondria. Hepatocytes
signicantly increased in the HTS group at 1 d after acute stress displayed dark, slender mitochondria with well-developed cristae
and at 2 d after chronic stress (P < 0.05, Fig. 4B). In addition, the and matrix (Fig. 5A, 5B), and they contained very few lipid
mRNA levels of liver HSP70 were signicantly elevated in the droplets (Fig. 5B).
HTS group compared with the CT group at 1 d after acute stress However, a number of abnormalities were observed in the
and at 2 d after chronic stress (P < 0.05, Fig. 4B). livers of sh examined at 48 h after high temperature
Liver HSP90 mRNA expression signicantly increased in the CT exposure. The hepatocytes exhibited many large electron-dense
group at 0.25 and 0.5 d after acute stress and at 7, 14 and 21 d fat droplets (Fig. 5C, 5D), some of which were even larger than
after chronic stress compared with the pre-stress level (P < 0.05, the nucleus (Fig. 5C, 5D). These extensive intracellular lipid
Fig. 4C). Liver HSP90 mRNA expression remarkably increased in droplets resulted in displacement of the nucleus to the cell
the HTS group at 0.25, 0.5 and 1 d after acute stress and at 2 and margin as well as a loss of cytoplasm (Fig. 5C, 5D). The nucleus
21 d after chronic stress (P < 0.05, Fig. 4C). Furthermore, the was atrophic and presented as a polygon (Fig. 5D). Many
mRNA levels of liver HSP90 were signicantly higher in the HTS mitochondria crowded together and exhibited an irregular
group than in the arrangement, loss of cristae and matrix and structural damage to
the outer and the inner membrane (Fig. 5D).
Fig. 5. Effects of high temperature stress on the hepatic cell ultrastructure of M. amblycephalaina. Note: The 25 C control group for 2 d: Fig. A, 25 C( 2500), showing a large,
round nucleus surrounded by a moderate and homogeneous cytoplasm (see arrow); Fig. B, 25 C( 3000), displaying a clear and normal ultrastructure (see arrow). The group
exposed to 32 C high temperature stress for 2 d: Fig. C, 32 C for 2 d ( 1000), resulting in extensive intracellular lipid droplets (see arrow); Fig. D, 32 C for 2 d ( 1200),
resulting in atrophy (see arrow); C-Chromatin; M-mitochondria; N-nucleus; NM-nuclear membrane; RER-rough endoplasmic reticulum; SER-smooth endoplasmic reticulum; L-
lysosome; G-
glycogen; F-fat.

4. Discussion serum ACTH and, subsequently, the COR level [36e38]. Cortisol is
considered a key response to stress in sh and, in more general
Wuchang bream has an optimum growing temperature be- terms, to an animal's welfare [37,38]. In the present study, serum
tween 25 C and 28 C. This study provides the rst data showing ACTH was signicantly elevated in the HTS group compared with
oxidative stress damage resulting from a change in temperature the CT group at 0.5 d after acute stress and at 2 d after chronic
from 25 to 32 C, as well as the effect of bacterial infection. stress. Serum COR signicantly increased in the HTS group at 0.5 d
Previous researchers have shown that temperatures above or after acute stress and at 14 and 21 d after chronic stress compared
below their thermal optima can affect physiological functions, with the CT group. Similar results have been reported for the same
adaptive and innate immunity and result in an increased species [32] as well as for the Atlantic cod [39] in response to heat
susceptibility to infection and even death [6,7,34,35]. Consistent stress.
with these studies, we also observed an increase in mortality after T3 also plays an important role in the hypothalamic-pituitary
A. hydrophila chal- lenge at 1 d after acute stress and 2 and 7 d axis and affects some features of the immune system [40,41]. Liu
after chronic stress compared with the CT group. et al. [42] demonstrated that cold water stress increased concen-
The increased temperature inuenced the serum trations of serum T3 and T4. Deane et al. [43] reported that serum
concentrations of ACTH, COR and T3. If the sh are exposed to concentrations of T3 and T4 decreased signicantly after vibriosis
chronic stress during culturing activities, the hypothalamus- infection. Similarly, our studies indicated that serum T3
pituitary-inter-renal axis of the sh will be continuously signicantly increased in the HTS group at 1 d after acute stress
stimulated to cause an increase in and at 14 and
21 d after chronic stress compared with the CT group. It is worth However, high temperature stress is often related to rapid
mentioning that the elevated levels of cortisol and T3 hormones
are generally thought to have a negative effect on the immune
system, increasing susceptibility to disease [40,41,44,45]. In the
present study, we also found that the high levels of serum cortisol
and T3 of sh were related to high mortality of the sh infected
with bacteria and exposed to a high temperature.
Under high temperature stress conditions, the increase in
tem- perature inuences oxidative stress parameters such as
SOD, ASAFER and MDA. For example, Vinagre et al. [46] found high
MDA levels and catalase activities in the juvenile seabass
exposed to temperatures outside their thermal optimum.
Madeira et al. [47] reported that lipid peroxidation, catalase
activity and glutathione S-transferase activity increased as the
temperature became closer to the Critical Thermal Maximum. In
the present study, similar patterns of the reduction of ASAFER
concentrations and SOD ac- tivities were observed at 1, 2, and 21
d after 32 C high temperature stress, and an improvement of the
MDA concentrations was iden- tied at 1, 2, and 7 d after 32 C
high temperature stress. This phenomenon was due to the
capacity of high temperature to inactivate and damage
antioxidant enzymes [48], reduce antioxi- dant defenses and
disturb physiological homeostasis, leading to lipid peroxidation
injury [49,50].
ROS production and the accumulation of denatured proteins
under high temperature stress may subsequently trigger HSP
expression [13]. In the present study, the 32 C high temperature
stress increased liver HSP60 mRNA levels in the HTS group at 1 d
after acute stress, liver HSP70 mRNA levels at 1 d after acute stress
and at 2 d after chronic stress, and liver HSP90 mRNA levels at
0.25,
0.5 and 1 d after acute stress and at 2 d after chronic stress
compared with the control group. These ndings indicated
that HSP expression levels were up-regulated by higher
temperature. Similar results have been obtained for aquatic
animals such as grass carp [51], sea lamprey [52] and rainbow
trout [53].
The elevation of HSPs might be used to refold and reassemble
denatured proteins [54,55], or to modulate the redox status of the
cytosol via reactions between their cysteine groups and cyto-
chrome c [13,56], which contributed to modulate cellular anti-
stress responses and to play key roles in protecting organisms
against heat stress. The up-regulation of HSPs at 1 d or 2 d after
exposure to 32 C high temperature stress might be evidence of
the presence of toxic ROS accumulation or lipid peroxidation.
As previously mentioned, an imbalance between lipid peroxides
and the antioxidant system may result in cell dysfunction and the
production of lipid peroxides and free radicals, leading to cell
damage [57]. In the present study, the hyperthermia-induced
tissue damage further conrmed the hepatic ultrastructure of
the Wuchang bream. At 2 d after 32 C high temperature
exposure, the hepatocytes of the sh exhibited many large
electron-dense fat droplets, and the nucleus was atrophic and
presented as a polygon. The endoplasmic reticulum was poorly
developed and had lost most of its ribosomes. Many
mitochondria crowded together and exhibited an irregular
arrangement, loss of cristae and matrix and structural damage to
the outer and the inner membrane. This observation
suggested that distinct differences in mitochondrial structure
distinguished the high temperature group and the control group.
Based on these results, we considered the liver alterations
observed in the sh to be hepatic lesions due to the high temper-
ature. Damage to the mitochondria contribute to the production of
ROS, which is released into the cytosol and causes oxidative
stress [20,21,58]. In the present study, the increase in MDA at 1 d
or 2 d after 32 C high temperature exposure showed that
mitochondria alteration may be an important contributor to the
leakage of ROS and subsequent oxidative damage.
changes in gene expression followed by the synthesis of proteins hyperthermia stress result in damage to the hepatic ultrastructure
involved in adaptation [59]. It should be noted that HSP60, 70 and and impaired mitochondrial bioenergetics. The dysfunction of the
90 expression levels and lipid peroxidation (MDA concentration) in mitochondria subsequently mediated oxidative stress and the
the liver exhibited the same trends and peaked at 1 d or 2 d after improvement in HSP expression modulated the cellular anti-stress
stress and then decreased back to baseline levels. The chronic response, which in turn reduced the efcacy of the immune
stress of 21 d high temperature did not affect the HSP60, 70 system and increased mortality of A. hydrophila-infected
or 90 expression levels, or the MDA concentration. It is possible Wuchang bream.
that sh can gradually adapt to chronic stress and establish a
new physio- logical homeostasis to protect themselves against Acknowledgements
heat stress. It is worth mentioning that a signicant difference
was detected in serum ACTH, COR and T3 and hepatic ASAFER, This work was supported by the National Nonprot Institute
SOD, MDA and HSP expression at some sampling times. In the Research Grant of Freshwater Fisheries Research Center, Chinese
present study, sh in the stage of acute stress were not fed; Academy of Fishery Sciences (2014A08XK02); the National Tech-
however, they were hand-fed to apparent satiation once daily nology System for Conventional Freshwater Fish Industries, the
during the chronic stress stage. Different physiologies and Modern Agriculture Industrial Technology System (CARS-46); the
biochemical parameters after feeding in sh likely underlie the National Natural Science Foundation of China (31572662) and the
peak and valley values. This ndings may provide an Three New Projects of Fishery in Jiangsu Province (D2013-5).
explanation for the self-metabolism of the sh for nutrient
acquisition. However, this hypothesis requires further study. References

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