Effect of acute temperature change on antioxidant enzyme activities and lipid peroxidation in the ark shell Scapharca subcrenata (Lischke, 1869).
KEY WORDS: acute temperature change, Scapharca subcrenata, antioxidant enzymes, lipid peroxidation, hemolymph, high tolerance
The genus Scapharca (family Arcidae) contains 200 species (Morton 1960), with characteristic hooked beaks which are usually separated with a wide ligament between them, and the straight hinge line possesses numerous small teeth. Hemoglobin or myoglobin pigment is found in blood and tissue cells (Morton 1960). The ark shell Scapharca subcrenata is a bivalve mollusc that inhabits muddy sediments in shallow coastal waters of the northwestern Pacific Ocean and is a commercially important species in China, Japan, and Korea. Natural populations of this species have been decreasing quickly and dramatically in recent years, largely as a result of unsustainable exploitation and environmental impact (Xu et al. 2005a). Park et al. (1998) studied ark shell physiology and environmental factors of their habitat in relation to sustainable production of cultured ark shells. The reason why production has decreased remarkably, however, remains unclear. To ensure sustainable development, many S. subcrenata studies have focused on reproductive biology, artificial breeding, morphology, ultrastructure, and genetic responses (Xu et al. 2005b, Jiang et al. 2006, Li et al. 2007, Cho et al. 2009, Zhao et al. 2011, Wang et al. 2015), but few studies have investigated the environmental immunology of bivalve species. Bivalves are exposed to many seasonal and short-term environmental factors during the grow-out phase, including changes in water temperature, salinity, and food availability (Harding et al. 2004). In particular, temperature is the primary controlling factor affecting bivalve activities in ecological systems (Kinne 1963). Available evidence across various aquatic phyla, including annelids, sipunculids, molluscs (bivalves and gastropods), crustaceans, and fish, supports the view that impaired oxygen supply capacity in aquatic ectotherms is the primary thermal limitation at low and high temperatures (Portner 2002, Portner et al. 2004).
Recent studies emphasize that many diseases of aquatic animals caused by stress are accompanied by the generation of free oxygen radicals aimed at lipid peroxidation (Ahuja et al. 2001, Lantos et al. 2001). Organisms defend against free oxygen radicals by activating antioxidant defense systems, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), which eliminate the harmful effects of reactive oxygen species (ROS). Oxidative stress occurs when the balance between ROS generation and antioxidant activity is disturbed, which complicates underlying disease (Todorova et al. 2005). Malondialdehyde (MDA) is a final product of lipid peroxidation and is another commonly measured oxidative stress biomarker (Viarengo et al. 1990, Viarengo et al. 1991, Doyotte et al. 1997). It is not surprising that temperature plays an important role in the nature and extent of massive ark shell mortality (Cho et al. 2009), particularly during the acute temperature changes induced by storms and strong winds (Song et al. 2004).
No study has evaluated the antioxidant defense system and lipid peroxidation contents in the economically important ark shell Scapharca subcrenata during environmental stress. To our knowledge, this is the first study to investigate antioxidant enzyme activities in this ark shell species and will provide a basis for future disease control and efficient culture strategies. The aim of the present study was to assess the effect of acute temperature change (sudden transfers from 22[degrees]C to 17[degrees]C, 27[degrees]C, and 32[degrees]C for 72 h, followed by transfer back to 22[degrees]C for 6 h) on the lipid peroxidation profile, and SOD and GPX activities in cell-free hemolymph (CFH) and the hemocyte lysate (HL) of S. subcrenata.
MATERIALS AND METHODS
Specimens of Scapharca subcrenata (shell length, 3.8 [+ or -] 0.31 cm and shell height, 3.4 [+ or -] 0.60 cm) were collected from Zhuanghe, China (April 2009) and acclimatized to 22[degrees]C for 2 wk before exposure to the experimental temperatures. The ark shells (>500 specimens) were maintained in 300-1 tanks containing aerated seawater (salinity, 32[per thousand] [+ or -] 1 [per thousand], temperature, 22[degrees]C [+ or -] 0.5[degrees]C, and pH 7.8) that was renewed daily. The ark shells were fed a mixed microalgal diet of Nitzsehia closteztuma and Platymonas subeordiformis.
Exposure to Experimental Temperatures
After the 2-wk acclimation at 22[degrees]C, 315 ark shells were selected randomly and transferred in equal numbers into tanks with water temperatures of 17[degrees]C, 27[degrees]C, and 32[degrees]C for 72 h. Each treatment had three replicates. Hemolymph samples were collected at 0, 1, 6, 12, 24, 48, and 72 h. Then, the ark shells were transferred back to 22[degrees]C to recover, and hemolymph was collected at 1 and 6 h during recovery. Ark shells maintained at 22[degrees]C throughout the experiment were sampled at the same times as controls. During the acclimation, stress exposure, and remission periods, we assumed that seawater pH and salinity remained constant.
Three hemolymph pools were collected from three ark shells held at each experimental temperature (17[degrees]C, 27[degrees]C, and 32[degrees]C) and each sampling time point (0, 1, 6, 12, 24, 48, and 72 h during stress and 1 and 6 h during recovery). Hemolymph (~1,000 [micro]l/ark shell) was collected from the anterior adductor muscle with a 1-ml syringe fitted with a 25-gauge needle and stored temporarily in individual microcentrifuge tubes on ice.
The initial pooled hemolymph subsample was centrifuged at 780 g for 10 min. After centrifugation, the supernatant (CFH) and the pelleted hemocytes were stored separately at-80[degrees]C for later analyses.
Superoxide dismutase and GPX activities and MDA content were quantified in the CFH and HL. The hemocyte pellet was treated prior to analysis. Briefly, the pelleted hemocytes were resuspended in the same volume of distilled water. The cell suspension was hemolyzed using a hand-driven glass-Teflon homogenizer in an ice bath. The HL was divided into two aliquots; one aliquot was used to determine the hemoglobin concentration and erythrocyte GPX activity, and the second aliquot (0.4 ml) was added to a 1:1 ethanol: chloroform (v:v) mixture to precipitate the hemoglobin. The tubes were shaken for 5 min and centrifuged at 3,500 rpm for 10 min. The supernatant was used to determine SOD activity and MDA content.
Enzyme Assays and MDA Measurements
Superoxide dismutase activity was determined according to the method of Sun et al. (1988). Briefly, superoxide generators inhibit the reduction of nitroblue tetrazolium by the xanthine/ xanthine oxidase system. One unit of SOD activity was the amount of enzyme causing a 50% inhibition in the rate of nitroblue tetrazolium reduction. Glutathione peroxidase activity was assayed using a commercially available kit (Jiancheng Bioengineering Institute, Nanjing, China), and reduced glutathione was the substrate used to measure conjugation with 5,5'-dithiobis-(2-nitrobenzoic acid). Malondialdehyde levels were estimated using the thiobarbituric acid method of Asakawa and Matsushita (1980). Malondialdehyde values were calculated using the extinction coefficient of the MDA-thiobarbituric acid complex at 532 nm.
Hemoglobin concentration in the erythrocyte lysate was estimated using the cyanmethemoglobin method (Mahoney et al. 1993).
All values are expressed as mean [+ or -] SD. Two-way analysis of variance was performed to identify differences in antioxidant enzyme activities and MDA contents at different temperatures using SPSS 13.0 software (SPSS Inc., Chicago, IL). Tukey's multiple comparison test was used to identify differences among treatments (duration and temperature) A P value < 0.05 was considered significant.
Super oxide Dismutase Activity
Significant (P < 0.05) decreases in SOD activity were found in CFH of ark shells after 48 h exposure to 27[degrees]C and 32[degrees]C. No differences in SOD activity were detected among ark shells exposed to 17[degrees]C, 27[degrees]C, or 32[degrees]C before 24 h of stress. Superoxide dismutase activity returned to the initial values during the recovery period, and SOD activity in CFH of ark shells exposed to 27[degrees]C recovered more quickly than that of ark shells exposed to 32[degrees]C (Fig. 1 A).
Figure 1B shows that SOD activity in the HL decreased significantly (P < 0.05) after 72 h of stress in ark shells exposed to 32[degrees]C compared with the initial value and returned to normal within the recovery period. No differences in HL SOD activity were observed in the 17[degrees]C or 27[degrees]C groups during the entire experiment (P > 0.05).
Glutathione peroxidase activity in CFH of ark shells exposed to 17[degrees]C and 27[degrees]C increased significantly (P < 0.01) 24 h after the acute temperature change as compared with the initial value and returned to baseline within the recovery period. A significant increase in GPX activity was observed in the CFH of ark shells after 12-h exposure to 32[degrees]C (P < 0.01), followed by a significant decrease (P <0.01) by the end of the recovery period (Fig. 2A).
Glutathione peroxidase activity in HL of ark shells exposed to 32[degrees]C showed a biphasic change with an increase at 12 h followed by a significant decrease (P < 0.01) during the recovery period. No significant effects of the temperature variation or stress duration were, however, observed on GPX activity in the other groups (Fig. 2B, P > 0.05).
Malondialdehyde content in CFH was significantly higher (P < 0.01) 48 h after the ark shells were exposed to 32[degrees]C compared with that of the initial value, followed by a significant decrease (P < 0.01) after 1 h of recovery. These same trends were detected between the 17[degrees]C and 27[degrees]C groups throughout the experiment but the differences were not significant (P > 0.05) (Fig. 3A).
As shown in Figure 3B, MDA contents in HL of stressed ark shells decreased significantly, regardless of temperature (P < 0.01), compared with the initial values after 1 h (both stress and recovery periods), followed by complete recovery by the end of the experiment.
This study was carried out to assess the activities of antioxidant enzymes (SOD and GPX) and MDA content in hemolymph of the ark shell Scapharca subcrenata during and after exposure to acute temperature changes. These biochemical parameters were chosen because temperature stress indirectly affects their levels, and they are commonly used biomarkers of oxidative stress (Liu et al. 2004, Chen et al. 2007, Li et al. 2010, Banni et al. 2009).
Reactive oxygen species are responsible for the oxidative stress that leads to cellular and metabolic alterations, including protein degradation (Viarengo et al. 1990) and membrane lipid peroxidation (Viarengo et al. 2000). The antioxidant defense system, including SOD, CAT, and GPX, inhibits the formation of oxyradicals (DiGiulio et al. 1989, Regoli 1998). The activities of these enzymes are, however, closely related to environmental factors (Di Giulio et al. 1989). For example, high or low temperatures are stressful depending on the species and can result in immunosuppression (Cheng et al. 2004, Monari et al. 2007, Gagnaire et al. 2006). Excess ROS are produced at higher temperatures, which accelerates the metabolic rate (Finkel & Holbrook 2000), leading to the accumulation of ROS at a rate faster than they can be removed. Accumulating ROS can decrease antioxidant enzyme activities (SOD, CAT, and GPX) (Fridovich 1978). Because SOD activity depends on temperature (Monari et al. 2007), a significant decrease in SOD activity was detected at 72 h in CFH and HL of ark shells exposed to 32[degrees]C, but no changes occurred at in 17[degrees]C or 27[degrees]C. Superoxide dismutase activities in HL and CFH of Scapharca subcrenata, however, did not decrease the oxidative damage of ark shell hemocytes over the entire stress application, even though SOD is the first and most important antioxidant defense (Downs et al. 2001). This contrasts with the transitory increase in SOD activity in the HL of Chlamys farreri to scavenge excess ROS after 1 h of thermal stress (Chen et al. 2007). Interestingly, no significant effects of the acute temperature change were observed in total SOD activity (Cu-Zn and Mn) in CFH or HL before 48 h. This result is consistent with Sun and Li (2000) who reported that Cu-Zn-SOD activity is very stable even at an extremely high temperature (80[degrees]C). It is also consistent with previous studies on scallops by Sun and Li (2000) and Chen et al. (2007). Therefore, the sustained low SOD activity during the recovery period, compared with that in the control ark shells, suggests that complete recovery from oxidative damage did not occur within 6 h and may require a much longer time. Glutathione peroxidase activities in CFH and HL of ark shells exposed to 32[degrees]C showed a biphasic change, with an increase during the initial 12 h followed by a decrease, and then a significant decrease by the end of recovery period. High GPX activity suggests that acute oxidant stress occurred with the acute temperature change and the decrease in GPX activity explains the simultaneous increase in SOD activity as a result of use of antioxidant enzymes. This may also be owing to the role of the antioxidant system in eliminating excess ROS.
Lipid peroxidation is a complex chain reaction of free radicals, leading to degradation of polyunsaturated fatty acids in the cell membrane (Halliwell & Gutteridge 1984). Malondialdehyde is the final product of lipid peroxidation and is an oxidative stress biomarker (Doyotte et al. 1997). In the present study, MDA content in the CFH increased when ark shells were transferred to 32[degrees]C, and a significantly higher value was detected after 48 h, which may have resulted from a diminished ability to remove ROS, as SOD and GPX activities decreased with time. As a result, ROS accumulated, leading to the increase in MDA content at 32[degrees]C. In addition, MDA contents in the CFH and HL of stressed ark shells, regardless of temperature, were at their lowest at 1 h of the recovery period, which may have been due to the enhanced CFH GPX activity to remove ROS when the ark shells were transferred back to 22[degrees]C. Kong et al. (2008), however, reported an increase in MDA content in gills of Scylla serrata during exposure to decreasing temperature. It is presumed that differences in species and sampled tissues, experimental settings, and duration and severity of the stress affected the outcomes of these experiments differently.
In conclusion, the homeostatic capabilities of Scapharca subcrenata transferred from 17[degrees]C to 27[degrees]C appeared to be compromised. The acute modulating ability of the antioxidant defense system was negatively affected during the 32[degrees]C thermal stress by suppressing SOD and GPX activities and increasing MDA content. Superoxide dismutase activity was restored to the initial level during the recovery period. Glutathione peroxidase activity decreased consistently after the acute temperature change and was accompanied by an increased SOD activity, which may have been due to the inducing effect of thyroid hormones on SOD activity and inhibition of GPX activity (Pereira et al. 1995). The roles of thyroid hormones in metabolic pathways and antioxidant enzyme activities are well known in many vertebrates (Asayama et al. 1987, Das & Chainy 2004, Zia-ur-Rahman et al. 2007, Nazifi et al. 2009). Nevertheless, no study has described the relationship between thyroid hormones or thyroid hormone intermediates and the antioxidant system in any invertebrate. Therefore, a useful experimental method to evaluate thyroid hormones or thyroid hormone intermediates in bivalve species is important to further elucidate their roles in the antioxidant defense system of thermally stressed bivalves and provide new insight into summer mortality of bivalves.
This study was supported by Program for Innovative Research Team (in Science and Technology) in University of Liaoning Province (No. 2007T016) and National Key Technology R & D Program in the 11th Five-Year Period (no. 2006BAD09A01).
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WEN ZHAO, * TINGTING HAN, JIE WEI, HONGYU PU, SHAN WANG AND XIA YUAN
Key Laboratory of Hydrobiology in Liaoning Province, College of Fisheries and Life Science, Dalian Ocean University, Heishijiao Street, Dalian 116023, Liaoning Province, China
* Corresponding author. E-mail: firstname.lastname@example.org
Please note: Some tables or figures were omitted from this article.
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|Author:||Zhao, Wen; Han, Tingting; Wei, Jie; Pu, Hongyu; Wang, Shan; Yuan, Xia|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2016|
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