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Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus
Accepted Manuscript Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus Yafei Duan, Jiasong Zhang, Hongbiao Dong, Yun Wang, Qingsong Liu, Hua Li PII:
S1050-4648(15)30277-1
DOI:
10.1016/j.fsi.2015.12.018
Reference:
YFSIM 3739
To appear in:
Fish and Shellfish Immunology
Received Date: 16 October 2015 Revised Date:
6 December 2015
Accepted Date: 12 December 2015
Please cite this article as: Duan Y, Zhang J, Dong H, Wang Y, Liu Q, Li H, Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2015.12.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT 1
Effect of desiccation and resubmersion on the oxidative stress response of the
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kuruma shrimp Marsupenaeus japonicus
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Yafei Duan, Jiasong Zhang , Hongbiao Dong, Yun Wang, Qingsong Liu, Hua Li
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Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of
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Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
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Guangzhou 510300, PR China
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ABSTRACT In the present study, the oxidative stress response in hepatopancreas of
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Marsupenaeus japonicas to desiccation stress and resubmersed in seawater were studied, such as
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respiratory burst, ROS production (·O2-), activities of antioxidant enzymes (CAT, GPx, SOD, POD and
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GST) and oxidative damage to lipid and protein (indexed by contents of MDA). The duration of
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desiccation significantly influenced shrimp survival, and the mortality rates were 37.5% and 87.5%
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after desiccation 5 h and 10 h, respectively. After desiccation stress 3 h, the respiratory burst, ROS
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production, and the activity of SOD and CAT were up-regulated significantly. The activity of GPx and
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POD, and the content of MDA decreased significantly at 0.5 h and 1 h, and then increased significantly
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at 3 h. But GST activity was no significant change after desiccation. During the resubmersion period,
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most of the antioxidant enzymes activities could recover to the control level at 24 h, but a small
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quantity of the oxidative stress still existed in tissues. HE staining showed that desiccation stress
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induced damage symptoms in hepatopancreas of M. japonicus. These results revealed that desiccation
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influenced the antioxidative status and caused oxidative stress and tissue damage via confusion of
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antioxidant enzymes in M. japonicas, but the oxidative stress could be eliminated within a certain range
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* Corresponding author. Tel: +86 20 84451349; fax: +86 20 84451442. E-mail address: [email protected] (J.S. Zhang).
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after the shrimps were resubmersed in seawater. Keywords: Marsupenaeus japonicus, Desiccation, Resubmersion, Antioxidants, Oxidative stress
24 1. Introduction
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The kuruma shrimp Marsupenaeus japonicus is an economically important shrimp species widely
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cultured in Taiwan and southeastern Asia, and the shrimp productions increase every year [1-2]. Due to
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its multiple merits, such as good reproductive performance, fast growth rate, and the capability of being
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transported live without water, the culture areas of which expands rapidly. In the shrimp-grade culture
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system, live shrimps are removed from the water and exposure in the air for transport between ponds
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[3]. However, shrimps usually suffer from desiccation during transportation without water. In addition,
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the habits of M. japonicus hidden in the sand, may lead to it exposed to the desiccation stress during the
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process of the water exchange and ponds leakage. Previous studies have demonstrated that desiccation
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is one of the more severe stressors to crustaceans, which can cause serious metabolic and respiratory
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disturbances [4-8]. Therefore, better understanding of the adaptation mechanism of the kuruma shrimp
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will be beneficial to the health management and varieties breeding in shrimp aquaculture.
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Stress factors, including pathogen infection [9], acute salinity or pH changes [10], temperature stress
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[11], hypoxia [12], and desiccation [3,13] have been shown to induce reactive oxygen species (ROS)
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generation in aquatic animals. ROS, such as hydrogen peroxide (H2O2), superoxide anion (·O2-) and
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hydroxyl free radical (·OH), are thought to be involved in cancer, aging and various inflammatory
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disorders [13,14]. In addition, these ROSs can kill harmful pathogens like bacteria and virus efficiently
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and also play an important role in immune signal transduction [15,16]. However, the mass
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accumulation of ROS in animals will cause serious cell damage, resulting in various diseases [17]. To
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ACCEPTED MANUSCRIPT protect themselves against damages of ROS, aerobic organisms have developed a set of antioxidant
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defense systems, including antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT),
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glutathione peroxidase (GPx), peroxidase (POD) and glutathione S-transferase (GST) [18,19], for
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protecting cells against oxidative stress and preventing or repairing oxidative damage.
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However, little is known about the effects of desiccation on shrimp physiology and the role of
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antioxidants in M. japonicus exposed to desiccation is uncertain. Whether the antioxidant enzymes of
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M. japonicus play important roles in the resistance to desiccation stress worth for studying, and the
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screening of evaluation index for desiccation is signality. Therefore, the aim of this study was to: (1)
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investigate the mortalities of M. japonicus under desiccation exposure; (2) evaluate the effects of
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desiccation on the ultrastructural changes of the cells in hepatopancreas of M. japonicus; (3) analyze
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the effects of desiccation and resubmersion on the respiratory burst, ROS production and antioxidant
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enzyme changes in hepatopancreas of M. japonicus. These results will be essential to better understand
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the roles of antioxidant defense systems in immune response of M. japonicus.
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2. Materials and methods
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2.1. Animal materials
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Healthy adult M. japonicus, averaging weight 10.65 ± 0.34 g, were randomly collected from a
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commercial farm in Zhongshan, China, and did not distinguish male and female. They were cultured in
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filtered aerated seawater (salinity 16‰, pH 8.5) at 25 ± 0.5 ℃ for 7 days before processing. The
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shrimps were fed daily with a ration of 5% of body weight, and one-thirds of the water in each group
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was renewed once daily.
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2.2. Experimental design of desiccation and resubmersion
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The desiccation experiments was conducted in the closed without wind laboratory and the room
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group and the desiccation group. The shrimps were examined health status and with no signs of
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infection before starting the experiment. For the control group, shrimps were cultured in filtered
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aerated seawater as described above. For the desiccation group, shrimps were performed individually in
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the dissect plates without water at 25 ℃, and wet gauze was used in the plates to maintain air humidity
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between 70% and 75%. There were three replicates for each group. Hepatopancreas of six shrimps
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from each treatment (the control group and the desiccation group) were randomly sampled at 0, 0.5, 1
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and 3 h post- desiccation, respectively.
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After desiccation as described above, the rest of live shrimps were used for the resubmersion
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experiments, which were divided into four groups, the control group, the group Ⅰ, group Ⅱ and
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group Ⅲ. For the control group, shrimps were cultured in filtered aerated seawater as described above.
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For the group Ⅰ, Ⅱ and Ⅲ, after desiccation 0.5, 1 and 3 h respectively as described above, shrimps
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were resubmersed for 24 h in aerated seawater and fed at 25 ℃. There were three replicates for each
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group. Hepatopancreas of six shrimps from each treatment (the control group, group Ⅰ, group Ⅱ and
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group Ⅲ) were randomly sampled at 3, 6, 12 and 24 h post-resubmersion, respectively.
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2.3. HE stain of the hepatopancreas
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Hepatopancreas of shrimps from each treatment (the desiccation group and the control group) were
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randomly sampled at 3 h post-desiccation, respectively, and stored in 4% paraformaldehyde for 2 h.
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After rinsed with flow water 8 h, the tissues were dehydrated in series of ethanol (70%, 80%, 90% and
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100%), transparented with xylene, embedded in paraffin and cut in an microtome (Leica, RM2016,
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Germany) at 4 µm thickness. After HE dye, stained sections were examined and photographed under an
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inverted phase-contrast microscope (Hitachi, Japan).
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2.4. Sample preparation Hepatopancreas were homogenized by adding sterile 0.9% saline solution to prepare 10% (W:V)
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homogenates. Homogenates were centrifuged at 3500 rpm for 10 min at 4 ℃. After removing
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precipitates, supernatants were immediately used for antioxidant enzyme activity analyses, which were
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carried out using a spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Assays were run in
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three replicate samples.
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2.5. Determination of respiratory burst and ROS production
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Respiratory bursts in the hepatopancreas of M. japonicus were determined respectively according to
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the 2’, 7’-DCFH-DA method. Hepatopancreas cells were collected from homogenates as described
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above, which centrifuged at 3500 rpm for 10 min at 4 ℃. 200 µL hepatopancreas cells were incubated
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with 2 µL dichlorofluorescein diacetate (DCFH-DA) for 30 min in darkness, and diluted with 200 µL
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Modified Alsevier's Solution (MAS) to a final concentration of 1 × 106 cells/mL. Samples were then
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analyzed by the flow cytometry analysis. Two light-scattering parameters (forward scatter and side
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scatter) were used to define a gate that excluded debris and aggregates from all fluorescence analyses.
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From each sample, 10000 cells were analyzed for the two fluorescent signals. The percentage of cells
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and mean fluorescence values were calculated for the data.
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ROS production, such as ·O2- generation capacity in the hepatopancreas of M. japonicus were
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determined with the ·O2- kits respectively (Jiancheng, Ltd, Nanjing, China) and read on a
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spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Total protein concentration in tissue
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homogenates was measured by Coomassie brilliant blue protein assay kit (Jiancheng, Ltd, Nanjing,
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China) following the manufacturer’s protocol.
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2.6. Determination of antioxidant enzymes
ACCEPTED MANUSCRIPT Hepatopancreas tissue extracts were used for immune enzyme activities analysis, such as CAT, GPx,
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SOD, POD and GST and the content analysis of malondialdehyde (MDA). The activities of CAT, GPx,
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SOD, POD and GST and the content of MDA in the hepatopancreas of M. japonicus were determined
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with the CAT, GPx, SOD, POD, GST and MDA kits respectively (Jiancheng, Ltd, Nanjing, China) and
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read on a spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Total protein concentration in
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tissue homogenates was measured as described above. Activities of all the enzymes were measured at
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37 ℃ following the protocol of the manufacturer.
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CAT was determined by measuring the degradation rate of H2O2 at 412 nm. One unit of CAT activity
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was defined as the amount of enzyme per mg tissue protein every second needed to reduce 1 µmol
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H2O2 under the assay condition. GPx activity was monitored by following the decrease of GSH at 412
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nm using H2O2 as substrate. One unit of GPx activity was defined as the amount of enzyme per mg
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tissue protein every minute deduction of enzymatic reaction reduce 1 µmol/L GSH in the reaction
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system. SOD activity was measured using xanthine/xanthine oxidase as the superoxide generator and
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hydroxylamine as the detector. One unit of SOD activity was defined as the SOD amount of per mg
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tissue protein to inhibit the rate of xanthine reduction by 50% in 1 mL reaction solution. POD activity
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was measured by catalyzing reaction of H2O2 at 420 nm. One unit of POD activity was defined as the
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amount of enzyme per mg tissue protein every minute catalyzes 1 µg substrate. GST was determined
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by monitoring the conjugation of reduced glutathione with 1-chloro-2, 4-dinitrobenzene (CDNB) at
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412 nm. One unit of GST activity was defined as the amount of enzyme per mg tissue protein every
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minute deduction of enzymatic reaction reduce 1 µmol/L GSH at 37 ℃.
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2.7. Statistical analysis
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The value of each variable was expressed as mean ± SD. Statistical analysis was performed using
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Tukey’s post hoc test for multiple comparison analysis. Significance was set at P < 0.05.
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3. Results
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3.1. Symptoms of M. japonicus after desiccation stress
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The cumulative survival of M. japonicus after desiccation stress was shown in Fig. 1. There were no
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obvious stress symptoms in the early stage (0-2 h) after M. japonicus exposed to desiccation stress. As
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the time progressed, shrimps of the desiccation group began to show stress symptoms at 3 h and
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gradually died at 5 h (mortality rate was 37.5%). After desiccation 10 h, shrimps showed serious
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symptoms, such as occasional jump, lessen breath, slowness gill cavity motion, and abundantly died
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(mortality rate was 87.5%). However, there were no shrimp died in the control group.
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The hepatopancreas tissues of M. japonicus in the desiccation group and the control group stained
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with HE dye were shown in Fig. 2. Compared with the control group (Fig. 2A, 2B), the hepatopancreas
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of M. japonicus in the desiccation group showed damage morbidity symptoms, including liver tubules
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arranged disorder, boundaries blurred, and the lumen also largen. In addition, the epithelial and
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connective tissue between liver tubule injured, and cell nucleus pyknosised or disappeared (Fig. 2C,
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2D).
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(Figure 1)
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(Figure 2)
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3.2. Respiratory burst and ROS production of M. japonicus after desiccation and resubmersion
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The changes of respiratory burst in hepatopancreas of M. japonicus after desiccation and
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resubmersion was shown in Fig. 3. After shrimps exposed to desiccation, respiratory burst activity in
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hepatopancreas increased gradually and reached a peak level at 3 h (5.44-fold of the control group, P <
ACCEPTED MANUSCRIPT 0.05) (Fig. 3A). In the resubmersion period, respiratory burst activity of Group Ⅰ decreased to
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1.65-fold of the control group (P < 0.05) at 3 h, then recovered to the control level at 6-24 h.
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Respiratory burst activity of Group Ⅱ decreased significantly at 3 h, then decreased gradually while it
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still higher than that of the control group (P < 0.05). Respiratory burst activity of Group Ⅲ increased
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significantly at 3 h and reached the highest at 6 h (6.82-fold of the control group, P < 0.05), then
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decreased gradually at 12-24 h and higher than that of the control group (P < 0.05) (Fig. 3B).
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(Figure 3)
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·O2- generation capacity changes in hepatopancreas of M. japonicus after desiccation and
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resubmersion was shown in Fig. 4. After shrimps exposed to desiccation, ·O2- generation capacity in
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hepatopancreas increased significantly at 0.5 h and reached to 15.43-fold of the control group at 3 h (P
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< 0.05) (Fig. 4A). In the resubmersion period, ·O2- generation capacity in Group Ⅰ was significantly
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higher than the control (P < 0.05), and recovered to the control level at 6 h. ·O2- generation capacity in
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Group Ⅱ decreased gradually to the control level at 12 h (P < 0.05). ·O2- generation capacity in Group
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Ⅲ decreased significantly, but still significantly higher than the control after resubmersion 24 h (Fig.
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4B).
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(Figure 4)
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3.3. Antioxidant enzyme changes of M. japonicus after desiccation and resubmersion
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CAT activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 5. After shrimps exposed to desiccation, CAT activity in hepatopancreas increased significantly
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and reached the highest at 0.5 h (12.39-fold of the control group, P < 0.05), then decreased gradually at
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1 h and 3 h, but it still higher than that of the control group (P < 0.05) (Fig. 5A). In the resubmersion
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period, CAT activity of Group Ⅰ and Ⅱ both decreased gradually and recovered to the control level
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at 12 h. CAT activity of Group Ⅲ increased significantly at 3 h and reached a peak level at 6 h
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(3.82-fold of the control group, P < 0.05), then decreased gradually at 24 h (P < 0.05) (Fig. 5B).
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(Figure 5) GPx activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in Fig.
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6. After shrimps exposed to desiccation, GPx activity in hepatopancreas decreased significantly at 0.5 h
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and 1 h (0.44 and 0.64-fold of the control group respectively, P < 0.05), then increased significantly to
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the 1.65-fold of the control group (P < 0.05) (Fig. 6A). In the resubmersion period, GPx activity of
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Group Ⅰ and Ⅱ both increased significantly and reached to the maximum at 12 h (1.35 and
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1.69-fold of the control group respectively, P < 0.05), then recovered to the control level at 24 h. GPx
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activity of Group Ⅲ increased significantly to 2.16-fold of the control group at 3 h (P < 0.05), then
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decreased gradually and reached to the control level at 24 h (Fig. 6B).
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(Figure 6)
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SOD activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 7. After shrimps exposed to desiccation, SOD activity in hepatopancreas increased significantly at
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1 h and reached to 1.79-fold of the control group at 3 h (P < 0.05) (Fig. 7A). In the resubmersion period,
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there were no apparent changes of SOD activity in Group Ⅰ. As time progressed, SOD activity in
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Group Ⅱ decreased gradually to the control level at 12 h, but increased slightly and higher than the
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control level at 24 h (P < 0.05). SOD activity in Group Ⅲ increased significantly and reached to the
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highest at 6 h (2.82-fold of the control group, P < 0.05), then decreased gradually at 12 and 24 h (Fig.
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7B).
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(Figure 7)
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POD activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
ACCEPTED MANUSCRIPT Fig. 8. After shrimps exposed to desiccation, POD activity in hepatopancreas decreased significantly at
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0.5 and 1 h (0.44 and 0.46-fold of the control group, P < 0.05), then increased to the control level at 3 h
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(Fig. 8A). In the resubmersion period, POD activity of Group Ⅰ up-regulated significantly and
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reached to 1.32-fold of the control group at 24 h (P < 0.05). POD activity of Group Ⅱ increased
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significantly to the maximum at 6 h (1.58-fold of the control group, P < 0.05), then it decreased at 12 h
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and recovered to the control level at 24 h. POD activity of Group Ⅲ increased significantly at 12 h
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and reached to 1.67-fold of the control group at 24 h (P < 0.05) (Fig. 8B).
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(Figure 8)
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GST activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 9. After shrimps exposed to desiccation, GST activity in hepatopancreas were no significant
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changes throughout the whole experimental periods (P ＞ 0.05) (Fig. 9A). In the resubmersion period,
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GST activity of Group Ⅱ and Ⅲ both increased significantly at 3 h (1.32 and 1.46-fold of the
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control group respectively, P < 0.05), then decreased significantly at 6 h (P < 0.05) and recovered to
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the control level at 12 h (Fig. 9B).
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(Figure 9)
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3.4. MDA content changes of of M. japonicus after desiccation and resubmersion
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MDA content in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 10. After shrimps exposed to desiccation, MDA content in hepatopancreas decreased significantly
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to a lower level at 0.5 and 1 h, then induced significantly and higher than the control level at 3 h
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(1.29-fold of the control group, P < 0.05) (Fig. 10A). In the resubmersion period, MDA content in three
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groups (Group Ⅰ, Ⅱ and Ⅲ) induced significantly and reached to the maximum at 6 h (1.47, 1.68
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and 1.62-fold of the control group, P < 0.05), then it decreased at 12 h while it still significantly higher
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than that of the control group (P < 0.05), and recovered to the control level at 24 h (Fig. 10B).
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(Figure 10)
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4. Discussion Oxidative bursts, a rapid, transient, production of large amounts of ROS, are one of the important
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defense strategies of crustaceans against stress factors, including chemical, physical and biological
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stressors [20]. During desiccation stress condition, the combined effects of low dissolved oxygen, pH,
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salinity and temperature causes severely damage and triggers more complicated oxidative stress such as
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ROS production response to aquatic organisms [19]. Most cells have protective mechanisms to balance
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ROS production and avoid oxidative stress, namely antioxidants. The antioxidant system of aerobic
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organisms prevents the deleterious effects of ROS, playing a vital role in protecting cells against
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oxidative stress, preventing or repairing oxidative damage [21-23]. Previous studies have demonstrated
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that organism recovery a period of time after environment stresses could eliminate the metabolites in
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tissues and alleviate the pressure of the stresses [24]. Hepatopancreas was regarded as the metabolic
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center for ROS production and the most sensitive tissue response to external stress stimulation in
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crustacean [25,26]. Therefore, information on the antioxidant enzymes activity in hepatopancreas of
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shrimps after desiccation and resubmersion would be helpful to better understand its immunological
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function.
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The ·O2- is the first product released from a respiratory burst [20]. In the present study, ·O2- was
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significantly induced by desiccation. As an important endogenous antioxidant protein, SOD provides
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the first and most important line of ROS eliminated from the cells, which catalyzes the conversion
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of ·O2- to H2O2 and molecular oxygen, then H2O2 is transformed to water and oxygen via CAT or GPx
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[27,28]. In the present study, respiratory bursts and ·O2- generation capacity in hepatopancreas of M.
ACCEPTED MANUSCRIPT japonicus increased significantly after desiccation, indicating that desiccation affects the oxidative
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status of M. japonicus. Following the elevation of respiratory bursts, SOD activity in hepatopancreas of
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M. japonicus increased significantly after desiccation 1 h and 3 h. Similar patterns exhibited in several
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aquatic animals under limited O2 conditions. For example, SOD activity increased significantly in
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Litopenaeus vannamei [3], Paralomis granulosa [29] and Chlamys farreri [30] after them under
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desiccation. In addition, the muscles of L. vannamei showed a higher SOD activity in response to
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hypoxia than under controlled conditions, which all revealed that SOD activity could be due to a
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transient systemic response to the injury inflicted during desiccation that would be expected to generate
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increased systemic levels of ROS for avoiding harmful effects caused by desiccation [31]. After
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resubmersed in aerated seawater for 24 h, respiratory bursts and SOD activity in hepatopancreas of M.
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japonicus still higher than the control, indicating that the oxidative stress caused by desiccation not
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enough to be alleviated in resubmersion 24 h.
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GPx is generally thought to be more important than CAT as a H2O2-removing system because GPx
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have a higher degree of affinity for H2O2 than CAT [9]. GPx can eliminate H2O2 effectively even if the
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concentration of H2O2 is very low, whereas CAT can not. In addition, GPx are distributed widely in cell,
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but CAT only located in the peroxisome, which causes it to function only when the concentration of
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H2O2 is high in cell and H2O2 diffuses into the peroxisome [32,33]. In the present study, following the
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elevation of respiratory bursts and SOD activity, the CAT activity of hepatopancreas significantly
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increased after desiccation 0.5 h, then decreased gradually at 1 h and 3 h, but still higher than the
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control, suggesting that the enhancement of CAT activity is associated with increasing protection to
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diminish the harm from a H2O2 induced by desiccation. But GPx activity dropped to a low level
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significantly after desiccation 0.5 h and 1 h, similar results were found in the activity of POD, which
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ACCEPTED MANUSCRIPT might because of the shrimp’s cellular over-production of ROS inhibit the activity of GPx and POD,
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but induced the activity of CAT. As time progressed, GPx activity could recover to the normal after
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resubmersion 24 h, but the CAT activity of the desiccation 3 h group still higher than the control,
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indicating that there was still some remaining oxidative stress existing in tissues.
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GST have been demonstrated that it play roles in detoxification and protection from oxidative
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damage in aquatic organisms, such as pathogen challenge and environmental stress [34,35]. However,
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in this study, no difference were observed in GST activity when M. japonicus under desiccation
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condition. After resubmersion 3 h and 6 h, GST activity of the desiccation 1 h and 3 h groups increased
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significantly, implying that it might be involved in shrimp resistance to desiccation, but didn’t sensitive
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to the desiccation stress.
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MDA is the end product of lipid peroxidation, which can reflect the degree of lipid peroxidation and
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is recognized as an important indicator of oxidative damage to the cellular membrane [36,37]. In P.
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granulosa, lipid peroxidation levels of the hepatopancreas increased after subjected to air exposure [38].
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In our study, a significant decrease in MDA levels was observed in hepatopancreas of M. japonicus
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after desiccation 0.5 h and 1 h, then increased significantly at 3 h, and at last recovered to the normal
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during the resubmersion period, which indicated that the ROS of tissues could be eliminated by
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antioxidant enzymes at earlier periods, but the longtime of desiccation induced ROS formation
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excessive and intensified membranous lipid peroxidation, leading to cellular damage and oxidative
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stress [39,40].
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In conclusion, the present study documented changes of respiratory bursts, ·O2- generation capacity,
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antioxidant enzymes (CAT, GPx, SOD, POD and GST) activity and MDA contents in hepatopancreas
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of M. japonicus after desiccation stress and resubmersion. Antioxidant enzymes are modulated in
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serve as sensitive indicator of oxidative damage in monitoring desiccation stress. Oxidative stress can
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be eliminated within a certain stress range, such as desiccation 3 h, when the shrimp back to the normal
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habitat environments. These results will be essential to provide the valuable data about the
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transportation without water and the regulatory mechanisms of the resistance to desiccation in M.
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japonicus.
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Acknowledgments
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The authors were grateful to all the laboratory members for experimental material preparation and
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technical assistance. This study was supported by the earmarked fund for National Twelfth Five-year
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Science and Technology Support Program (2011BAD13B10), Guangdong Provincial Special Fund for
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Marine Fisheries Technology (A201501B15, A201508B05), and Special Scientific Research Funds for
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Central Non-profit Institutes, South China Sea Fisheries Research Institute, Chinese Academy of
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Fishery Sciences (2014TS15).
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Fig. 1. Cumulative survival of M. japonicus at different time intervals after desiccation stress treatment.
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Vertical bars represented the mean ± SD (N = 3).
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h. The control group: (A) ×100 magnification, (B) ×400 magnification. The desiccation group: (C)
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×100 magnification, (D) ×400 magnification. The letters in the figure indicated that: A (liver tubules),
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B (lumen), C (cell nucleus), D (connective tissue), E (epithelial tissue).
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Fig. 3 Respiratory burst activity in hepatopancreas of M. japonicus at different time intervals after
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desiccation stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3).
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Data without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 4. ·O2- generation capacity in hepatopancreas of M. japonicus at different time intervals after
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desiccation stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3).
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Data without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 5. CAT activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 7. SOD activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 8. POD activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 9. GST activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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ACCEPTED MANUSCRIPT ► Desiccation damaged the ultrastructure of hepatopancreas in Marsupenaeus japonicas. ► Desiccation induced the respiratory burst and ROS production in M. japonicas. ► Antioxidant enzyme activities were significantly affected by desiccation. ► Resubmersion could eliminate the oxidative
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stress within a certain range.
S1050-4648(15)30277-1
DOI:
10.1016/j.fsi.2015.12.018
Reference:
YFSIM 3739
To appear in:
Fish and Shellfish Immunology
Received Date: 16 October 2015 Revised Date:
6 December 2015
Accepted Date: 12 December 2015
Please cite this article as: Duan Y, Zhang J, Dong H, Wang Y, Liu Q, Li H, Effect of desiccation and resubmersion on the oxidative stress response of the kuruma shrimp Marsupenaeus japonicus, Fish and Shellfish Immunology (2016), doi: 10.1016/j.fsi.2015.12.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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Effect of desiccation and resubmersion on the oxidative stress response of the
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kuruma shrimp Marsupenaeus japonicus
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Yafei Duan, Jiasong Zhang , Hongbiao Dong, Yun Wang, Qingsong Liu, Hua Li
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Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of
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Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,
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Guangzhou 510300, PR China
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ABSTRACT In the present study, the oxidative stress response in hepatopancreas of
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Marsupenaeus japonicas to desiccation stress and resubmersed in seawater were studied, such as
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respiratory burst, ROS production (·O2-), activities of antioxidant enzymes (CAT, GPx, SOD, POD and
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GST) and oxidative damage to lipid and protein (indexed by contents of MDA). The duration of
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desiccation significantly influenced shrimp survival, and the mortality rates were 37.5% and 87.5%
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after desiccation 5 h and 10 h, respectively. After desiccation stress 3 h, the respiratory burst, ROS
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production, and the activity of SOD and CAT were up-regulated significantly. The activity of GPx and
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POD, and the content of MDA decreased significantly at 0.5 h and 1 h, and then increased significantly
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at 3 h. But GST activity was no significant change after desiccation. During the resubmersion period,
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most of the antioxidant enzymes activities could recover to the control level at 24 h, but a small
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quantity of the oxidative stress still existed in tissues. HE staining showed that desiccation stress
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induced damage symptoms in hepatopancreas of M. japonicus. These results revealed that desiccation
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influenced the antioxidative status and caused oxidative stress and tissue damage via confusion of
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antioxidant enzymes in M. japonicas, but the oxidative stress could be eliminated within a certain range
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* Corresponding author. Tel: +86 20 84451349; fax: +86 20 84451442. E-mail address: [email protected] (J.S. Zhang).
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after the shrimps were resubmersed in seawater. Keywords: Marsupenaeus japonicus, Desiccation, Resubmersion, Antioxidants, Oxidative stress
24 1. Introduction
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The kuruma shrimp Marsupenaeus japonicus is an economically important shrimp species widely
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cultured in Taiwan and southeastern Asia, and the shrimp productions increase every year [1-2]. Due to
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its multiple merits, such as good reproductive performance, fast growth rate, and the capability of being
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transported live without water, the culture areas of which expands rapidly. In the shrimp-grade culture
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system, live shrimps are removed from the water and exposure in the air for transport between ponds
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[3]. However, shrimps usually suffer from desiccation during transportation without water. In addition,
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the habits of M. japonicus hidden in the sand, may lead to it exposed to the desiccation stress during the
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process of the water exchange and ponds leakage. Previous studies have demonstrated that desiccation
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is one of the more severe stressors to crustaceans, which can cause serious metabolic and respiratory
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disturbances [4-8]. Therefore, better understanding of the adaptation mechanism of the kuruma shrimp
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will be beneficial to the health management and varieties breeding in shrimp aquaculture.
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Stress factors, including pathogen infection [9], acute salinity or pH changes [10], temperature stress
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[11], hypoxia [12], and desiccation [3,13] have been shown to induce reactive oxygen species (ROS)
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generation in aquatic animals. ROS, such as hydrogen peroxide (H2O2), superoxide anion (·O2-) and
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hydroxyl free radical (·OH), are thought to be involved in cancer, aging and various inflammatory
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disorders [13,14]. In addition, these ROSs can kill harmful pathogens like bacteria and virus efficiently
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and also play an important role in immune signal transduction [15,16]. However, the mass
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accumulation of ROS in animals will cause serious cell damage, resulting in various diseases [17]. To
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defense systems, including antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT),
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glutathione peroxidase (GPx), peroxidase (POD) and glutathione S-transferase (GST) [18,19], for
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protecting cells against oxidative stress and preventing or repairing oxidative damage.
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However, little is known about the effects of desiccation on shrimp physiology and the role of
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antioxidants in M. japonicus exposed to desiccation is uncertain. Whether the antioxidant enzymes of
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M. japonicus play important roles in the resistance to desiccation stress worth for studying, and the
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screening of evaluation index for desiccation is signality. Therefore, the aim of this study was to: (1)
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investigate the mortalities of M. japonicus under desiccation exposure; (2) evaluate the effects of
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desiccation on the ultrastructural changes of the cells in hepatopancreas of M. japonicus; (3) analyze
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the effects of desiccation and resubmersion on the respiratory burst, ROS production and antioxidant
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enzyme changes in hepatopancreas of M. japonicus. These results will be essential to better understand
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the roles of antioxidant defense systems in immune response of M. japonicus.
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2. Materials and methods
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2.1. Animal materials
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Healthy adult M. japonicus, averaging weight 10.65 ± 0.34 g, were randomly collected from a
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commercial farm in Zhongshan, China, and did not distinguish male and female. They were cultured in
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filtered aerated seawater (salinity 16‰, pH 8.5) at 25 ± 0.5 ℃ for 7 days before processing. The
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shrimps were fed daily with a ration of 5% of body weight, and one-thirds of the water in each group
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was renewed once daily.
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2.2. Experimental design of desiccation and resubmersion
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The desiccation experiments was conducted in the closed without wind laboratory and the room
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group and the desiccation group. The shrimps were examined health status and with no signs of
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infection before starting the experiment. For the control group, shrimps were cultured in filtered
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aerated seawater as described above. For the desiccation group, shrimps were performed individually in
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the dissect plates without water at 25 ℃, and wet gauze was used in the plates to maintain air humidity
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between 70% and 75%. There were three replicates for each group. Hepatopancreas of six shrimps
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from each treatment (the control group and the desiccation group) were randomly sampled at 0, 0.5, 1
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and 3 h post- desiccation, respectively.
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After desiccation as described above, the rest of live shrimps were used for the resubmersion
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experiments, which were divided into four groups, the control group, the group Ⅰ, group Ⅱ and
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group Ⅲ. For the control group, shrimps were cultured in filtered aerated seawater as described above.
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For the group Ⅰ, Ⅱ and Ⅲ, after desiccation 0.5, 1 and 3 h respectively as described above, shrimps
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were resubmersed for 24 h in aerated seawater and fed at 25 ℃. There were three replicates for each
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group. Hepatopancreas of six shrimps from each treatment (the control group, group Ⅰ, group Ⅱ and
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group Ⅲ) were randomly sampled at 3, 6, 12 and 24 h post-resubmersion, respectively.
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2.3. HE stain of the hepatopancreas
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Hepatopancreas of shrimps from each treatment (the desiccation group and the control group) were
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randomly sampled at 3 h post-desiccation, respectively, and stored in 4% paraformaldehyde for 2 h.
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After rinsed with flow water 8 h, the tissues were dehydrated in series of ethanol (70%, 80%, 90% and
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100%), transparented with xylene, embedded in paraffin and cut in an microtome (Leica, RM2016,
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Germany) at 4 µm thickness. After HE dye, stained sections were examined and photographed under an
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inverted phase-contrast microscope (Hitachi, Japan).
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2.4. Sample preparation Hepatopancreas were homogenized by adding sterile 0.9% saline solution to prepare 10% (W:V)
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homogenates. Homogenates were centrifuged at 3500 rpm for 10 min at 4 ℃. After removing
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precipitates, supernatants were immediately used for antioxidant enzyme activity analyses, which were
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carried out using a spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Assays were run in
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three replicate samples.
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2.5. Determination of respiratory burst and ROS production
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Respiratory bursts in the hepatopancreas of M. japonicus were determined respectively according to
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the 2’, 7’-DCFH-DA method. Hepatopancreas cells were collected from homogenates as described
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above, which centrifuged at 3500 rpm for 10 min at 4 ℃. 200 µL hepatopancreas cells were incubated
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with 2 µL dichlorofluorescein diacetate (DCFH-DA) for 30 min in darkness, and diluted with 200 µL
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Modified Alsevier's Solution (MAS) to a final concentration of 1 × 106 cells/mL. Samples were then
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analyzed by the flow cytometry analysis. Two light-scattering parameters (forward scatter and side
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scatter) were used to define a gate that excluded debris and aggregates from all fluorescence analyses.
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From each sample, 10000 cells were analyzed for the two fluorescent signals. The percentage of cells
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and mean fluorescence values were calculated for the data.
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ROS production, such as ·O2- generation capacity in the hepatopancreas of M. japonicus were
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determined with the ·O2- kits respectively (Jiancheng, Ltd, Nanjing, China) and read on a
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spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Total protein concentration in tissue
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homogenates was measured by Coomassie brilliant blue protein assay kit (Jiancheng, Ltd, Nanjing,
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China) following the manufacturer’s protocol.
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2.6. Determination of antioxidant enzymes
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SOD, POD and GST and the content analysis of malondialdehyde (MDA). The activities of CAT, GPx,
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SOD, POD and GST and the content of MDA in the hepatopancreas of M. japonicus were determined
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with the CAT, GPx, SOD, POD, GST and MDA kits respectively (Jiancheng, Ltd, Nanjing, China) and
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read on a spectrophotometer (TA-88, Sinsche Tech, Shenzhen, China). Total protein concentration in
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tissue homogenates was measured as described above. Activities of all the enzymes were measured at
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37 ℃ following the protocol of the manufacturer.
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CAT was determined by measuring the degradation rate of H2O2 at 412 nm. One unit of CAT activity
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was defined as the amount of enzyme per mg tissue protein every second needed to reduce 1 µmol
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H2O2 under the assay condition. GPx activity was monitored by following the decrease of GSH at 412
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nm using H2O2 as substrate. One unit of GPx activity was defined as the amount of enzyme per mg
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tissue protein every minute deduction of enzymatic reaction reduce 1 µmol/L GSH in the reaction
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system. SOD activity was measured using xanthine/xanthine oxidase as the superoxide generator and
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hydroxylamine as the detector. One unit of SOD activity was defined as the SOD amount of per mg
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tissue protein to inhibit the rate of xanthine reduction by 50% in 1 mL reaction solution. POD activity
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was measured by catalyzing reaction of H2O2 at 420 nm. One unit of POD activity was defined as the
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amount of enzyme per mg tissue protein every minute catalyzes 1 µg substrate. GST was determined
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by monitoring the conjugation of reduced glutathione with 1-chloro-2, 4-dinitrobenzene (CDNB) at
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412 nm. One unit of GST activity was defined as the amount of enzyme per mg tissue protein every
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minute deduction of enzymatic reaction reduce 1 µmol/L GSH at 37 ℃.
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2.7. Statistical analysis
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The value of each variable was expressed as mean ± SD. Statistical analysis was performed using
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Tukey’s post hoc test for multiple comparison analysis. Significance was set at P < 0.05.
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3. Results
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3.1. Symptoms of M. japonicus after desiccation stress
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The cumulative survival of M. japonicus after desiccation stress was shown in Fig. 1. There were no
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obvious stress symptoms in the early stage (0-2 h) after M. japonicus exposed to desiccation stress. As
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the time progressed, shrimps of the desiccation group began to show stress symptoms at 3 h and
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gradually died at 5 h (mortality rate was 37.5%). After desiccation 10 h, shrimps showed serious
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symptoms, such as occasional jump, lessen breath, slowness gill cavity motion, and abundantly died
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(mortality rate was 87.5%). However, there were no shrimp died in the control group.
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The hepatopancreas tissues of M. japonicus in the desiccation group and the control group stained
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with HE dye were shown in Fig. 2. Compared with the control group (Fig. 2A, 2B), the hepatopancreas
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of M. japonicus in the desiccation group showed damage morbidity symptoms, including liver tubules
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arranged disorder, boundaries blurred, and the lumen also largen. In addition, the epithelial and
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connective tissue between liver tubule injured, and cell nucleus pyknosised or disappeared (Fig. 2C,
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2D).
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(Figure 1)
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(Figure 2)
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3.2. Respiratory burst and ROS production of M. japonicus after desiccation and resubmersion
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The changes of respiratory burst in hepatopancreas of M. japonicus after desiccation and
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resubmersion was shown in Fig. 3. After shrimps exposed to desiccation, respiratory burst activity in
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hepatopancreas increased gradually and reached a peak level at 3 h (5.44-fold of the control group, P <
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1.65-fold of the control group (P < 0.05) at 3 h, then recovered to the control level at 6-24 h.
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Respiratory burst activity of Group Ⅱ decreased significantly at 3 h, then decreased gradually while it
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still higher than that of the control group (P < 0.05). Respiratory burst activity of Group Ⅲ increased
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significantly at 3 h and reached the highest at 6 h (6.82-fold of the control group, P < 0.05), then
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decreased gradually at 12-24 h and higher than that of the control group (P < 0.05) (Fig. 3B).
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(Figure 3)
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·O2- generation capacity changes in hepatopancreas of M. japonicus after desiccation and
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resubmersion was shown in Fig. 4. After shrimps exposed to desiccation, ·O2- generation capacity in
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hepatopancreas increased significantly at 0.5 h and reached to 15.43-fold of the control group at 3 h (P
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< 0.05) (Fig. 4A). In the resubmersion period, ·O2- generation capacity in Group Ⅰ was significantly
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higher than the control (P < 0.05), and recovered to the control level at 6 h. ·O2- generation capacity in
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Group Ⅱ decreased gradually to the control level at 12 h (P < 0.05). ·O2- generation capacity in Group
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Ⅲ decreased significantly, but still significantly higher than the control after resubmersion 24 h (Fig.
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4B).
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(Figure 4)
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3.3. Antioxidant enzyme changes of M. japonicus after desiccation and resubmersion
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CAT activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 5. After shrimps exposed to desiccation, CAT activity in hepatopancreas increased significantly
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and reached the highest at 0.5 h (12.39-fold of the control group, P < 0.05), then decreased gradually at
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1 h and 3 h, but it still higher than that of the control group (P < 0.05) (Fig. 5A). In the resubmersion
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period, CAT activity of Group Ⅰ and Ⅱ both decreased gradually and recovered to the control level
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at 12 h. CAT activity of Group Ⅲ increased significantly at 3 h and reached a peak level at 6 h
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(3.82-fold of the control group, P < 0.05), then decreased gradually at 24 h (P < 0.05) (Fig. 5B).
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(Figure 5) GPx activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in Fig.
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6. After shrimps exposed to desiccation, GPx activity in hepatopancreas decreased significantly at 0.5 h
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and 1 h (0.44 and 0.64-fold of the control group respectively, P < 0.05), then increased significantly to
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the 1.65-fold of the control group (P < 0.05) (Fig. 6A). In the resubmersion period, GPx activity of
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Group Ⅰ and Ⅱ both increased significantly and reached to the maximum at 12 h (1.35 and
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1.69-fold of the control group respectively, P < 0.05), then recovered to the control level at 24 h. GPx
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activity of Group Ⅲ increased significantly to 2.16-fold of the control group at 3 h (P < 0.05), then
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decreased gradually and reached to the control level at 24 h (Fig. 6B).
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(Figure 6)
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SOD activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 7. After shrimps exposed to desiccation, SOD activity in hepatopancreas increased significantly at
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1 h and reached to 1.79-fold of the control group at 3 h (P < 0.05) (Fig. 7A). In the resubmersion period,
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there were no apparent changes of SOD activity in Group Ⅰ. As time progressed, SOD activity in
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Group Ⅱ decreased gradually to the control level at 12 h, but increased slightly and higher than the
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control level at 24 h (P < 0.05). SOD activity in Group Ⅲ increased significantly and reached to the
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highest at 6 h (2.82-fold of the control group, P < 0.05), then decreased gradually at 12 and 24 h (Fig.
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7B).
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(Figure 7)
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POD activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
ACCEPTED MANUSCRIPT Fig. 8. After shrimps exposed to desiccation, POD activity in hepatopancreas decreased significantly at
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0.5 and 1 h (0.44 and 0.46-fold of the control group, P < 0.05), then increased to the control level at 3 h
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(Fig. 8A). In the resubmersion period, POD activity of Group Ⅰ up-regulated significantly and
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reached to 1.32-fold of the control group at 24 h (P < 0.05). POD activity of Group Ⅱ increased
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significantly to the maximum at 6 h (1.58-fold of the control group, P < 0.05), then it decreased at 12 h
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and recovered to the control level at 24 h. POD activity of Group Ⅲ increased significantly at 12 h
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and reached to 1.67-fold of the control group at 24 h (P < 0.05) (Fig. 8B).
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(Figure 8)
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GST activity in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 9. After shrimps exposed to desiccation, GST activity in hepatopancreas were no significant
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changes throughout the whole experimental periods (P ＞ 0.05) (Fig. 9A). In the resubmersion period,
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GST activity of Group Ⅱ and Ⅲ both increased significantly at 3 h (1.32 and 1.46-fold of the
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control group respectively, P < 0.05), then decreased significantly at 6 h (P < 0.05) and recovered to
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the control level at 12 h (Fig. 9B).
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(Figure 9)
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3.4. MDA content changes of of M. japonicus after desiccation and resubmersion
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MDA content in hepatopancreas of M. japonicus after desiccation and resubmersion was shown in
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Fig. 10. After shrimps exposed to desiccation, MDA content in hepatopancreas decreased significantly
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to a lower level at 0.5 and 1 h, then induced significantly and higher than the control level at 3 h
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(1.29-fold of the control group, P < 0.05) (Fig. 10A). In the resubmersion period, MDA content in three
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groups (Group Ⅰ, Ⅱ and Ⅲ) induced significantly and reached to the maximum at 6 h (1.47, 1.68
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and 1.62-fold of the control group, P < 0.05), then it decreased at 12 h while it still significantly higher
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than that of the control group (P < 0.05), and recovered to the control level at 24 h (Fig. 10B).
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(Figure 10)
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4. Discussion Oxidative bursts, a rapid, transient, production of large amounts of ROS, are one of the important
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defense strategies of crustaceans against stress factors, including chemical, physical and biological
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stressors [20]. During desiccation stress condition, the combined effects of low dissolved oxygen, pH,
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salinity and temperature causes severely damage and triggers more complicated oxidative stress such as
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ROS production response to aquatic organisms [19]. Most cells have protective mechanisms to balance
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ROS production and avoid oxidative stress, namely antioxidants. The antioxidant system of aerobic
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organisms prevents the deleterious effects of ROS, playing a vital role in protecting cells against
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oxidative stress, preventing or repairing oxidative damage [21-23]. Previous studies have demonstrated
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that organism recovery a period of time after environment stresses could eliminate the metabolites in
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tissues and alleviate the pressure of the stresses [24]. Hepatopancreas was regarded as the metabolic
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center for ROS production and the most sensitive tissue response to external stress stimulation in
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crustacean [25,26]. Therefore, information on the antioxidant enzymes activity in hepatopancreas of
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shrimps after desiccation and resubmersion would be helpful to better understand its immunological
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function.
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The ·O2- is the first product released from a respiratory burst [20]. In the present study, ·O2- was
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significantly induced by desiccation. As an important endogenous antioxidant protein, SOD provides
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the first and most important line of ROS eliminated from the cells, which catalyzes the conversion
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of ·O2- to H2O2 and molecular oxygen, then H2O2 is transformed to water and oxygen via CAT or GPx
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[27,28]. In the present study, respiratory bursts and ·O2- generation capacity in hepatopancreas of M.
ACCEPTED MANUSCRIPT japonicus increased significantly after desiccation, indicating that desiccation affects the oxidative
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status of M. japonicus. Following the elevation of respiratory bursts, SOD activity in hepatopancreas of
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M. japonicus increased significantly after desiccation 1 h and 3 h. Similar patterns exhibited in several
245
aquatic animals under limited O2 conditions. For example, SOD activity increased significantly in
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Litopenaeus vannamei [3], Paralomis granulosa [29] and Chlamys farreri [30] after them under
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desiccation. In addition, the muscles of L. vannamei showed a higher SOD activity in response to
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hypoxia than under controlled conditions, which all revealed that SOD activity could be due to a
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transient systemic response to the injury inflicted during desiccation that would be expected to generate
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increased systemic levels of ROS for avoiding harmful effects caused by desiccation [31]. After
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resubmersed in aerated seawater for 24 h, respiratory bursts and SOD activity in hepatopancreas of M.
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japonicus still higher than the control, indicating that the oxidative stress caused by desiccation not
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enough to be alleviated in resubmersion 24 h.
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GPx is generally thought to be more important than CAT as a H2O2-removing system because GPx
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have a higher degree of affinity for H2O2 than CAT [9]. GPx can eliminate H2O2 effectively even if the
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concentration of H2O2 is very low, whereas CAT can not. In addition, GPx are distributed widely in cell,
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but CAT only located in the peroxisome, which causes it to function only when the concentration of
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H2O2 is high in cell and H2O2 diffuses into the peroxisome [32,33]. In the present study, following the
259
elevation of respiratory bursts and SOD activity, the CAT activity of hepatopancreas significantly
260
increased after desiccation 0.5 h, then decreased gradually at 1 h and 3 h, but still higher than the
261
control, suggesting that the enhancement of CAT activity is associated with increasing protection to
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diminish the harm from a H2O2 induced by desiccation. But GPx activity dropped to a low level
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significantly after desiccation 0.5 h and 1 h, similar results were found in the activity of POD, which
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265
but induced the activity of CAT. As time progressed, GPx activity could recover to the normal after
266
resubmersion 24 h, but the CAT activity of the desiccation 3 h group still higher than the control,
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indicating that there was still some remaining oxidative stress existing in tissues.
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GST have been demonstrated that it play roles in detoxification and protection from oxidative
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damage in aquatic organisms, such as pathogen challenge and environmental stress [34,35]. However,
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in this study, no difference were observed in GST activity when M. japonicus under desiccation
271
condition. After resubmersion 3 h and 6 h, GST activity of the desiccation 1 h and 3 h groups increased
272
significantly, implying that it might be involved in shrimp resistance to desiccation, but didn’t sensitive
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to the desiccation stress.
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MDA is the end product of lipid peroxidation, which can reflect the degree of lipid peroxidation and
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is recognized as an important indicator of oxidative damage to the cellular membrane [36,37]. In P.
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granulosa, lipid peroxidation levels of the hepatopancreas increased after subjected to air exposure [38].
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In our study, a significant decrease in MDA levels was observed in hepatopancreas of M. japonicus
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after desiccation 0.5 h and 1 h, then increased significantly at 3 h, and at last recovered to the normal
279
during the resubmersion period, which indicated that the ROS of tissues could be eliminated by
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antioxidant enzymes at earlier periods, but the longtime of desiccation induced ROS formation
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excessive and intensified membranous lipid peroxidation, leading to cellular damage and oxidative
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stress [39,40].
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In conclusion, the present study documented changes of respiratory bursts, ·O2- generation capacity,
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antioxidant enzymes (CAT, GPx, SOD, POD and GST) activity and MDA contents in hepatopancreas
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of M. japonicus after desiccation stress and resubmersion. Antioxidant enzymes are modulated in
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287
serve as sensitive indicator of oxidative damage in monitoring desiccation stress. Oxidative stress can
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be eliminated within a certain stress range, such as desiccation 3 h, when the shrimp back to the normal
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habitat environments. These results will be essential to provide the valuable data about the
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transportation without water and the regulatory mechanisms of the resistance to desiccation in M.
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japonicus.
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Acknowledgments
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The authors were grateful to all the laboratory members for experimental material preparation and
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technical assistance. This study was supported by the earmarked fund for National Twelfth Five-year
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Science and Technology Support Program (2011BAD13B10), Guangdong Provincial Special Fund for
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Marine Fisheries Technology (A201501B15, A201508B05), and Special Scientific Research Funds for
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Central Non-profit Institutes, South China Sea Fisheries Research Institute, Chinese Academy of
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Fishery Sciences (2014TS15).
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Fig. 1. Cumulative survival of M. japonicus at different time intervals after desiccation stress treatment.
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Vertical bars represented the mean ± SD (N = 3).
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h. The control group: (A) ×100 magnification, (B) ×400 magnification. The desiccation group: (C)
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×100 magnification, (D) ×400 magnification. The letters in the figure indicated that: A (liver tubules),
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B (lumen), C (cell nucleus), D (connective tissue), E (epithelial tissue).
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Fig. 3 Respiratory burst activity in hepatopancreas of M. japonicus at different time intervals after
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desiccation stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3).
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Data without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 4. ·O2- generation capacity in hepatopancreas of M. japonicus at different time intervals after
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desiccation stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3).
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Data without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 5. CAT activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 7. SOD activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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Fig. 8. POD activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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Fig. 9. GST activity in hepatopancreas of M. japonicus at different time intervals after desiccation
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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treatments in the same exposure time.
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stress (A) and resubmersion (B) treatment. Vertical bars represented the mean ± SD (N = 3). Data
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without shared letters or indicated with asterisks were significantly different (P < 0.05) among
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ACCEPTED MANUSCRIPT ► Desiccation damaged the ultrastructure of hepatopancreas in Marsupenaeus japonicas. ► Desiccation induced the respiratory burst and ROS production in M. japonicas. ► Antioxidant enzyme activities were significantly affected by desiccation. ► Resubmersion could eliminate the oxidative
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stress within a certain range.