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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.
http://dx.doi.org/10.1016/j.fsi.2016.03.018
1050-4648/ 2016 Elsevier Ltd. All rights reserved.
B. Liu
B. Liu
et al.
et /al.Fish
/ Fish
& Shellsh
& Shellsh
Immunology
Immunology
52 (2016)
52 (2016)
31 317e324
317e324
31
[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.
2.1. Fish
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
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
B. Liu
B. Liu
et al.
et /al.Fish
/ Fish
& Shellsh
& Shellsh
Immunology
Immunology
52 (2016)
52 (2016)
32 317e324
317e324
32
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
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)
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).
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