Effect of low temperature stress on antioxidant stress, apoptosis and histological structure of gills in cobia (Rachycentron canadum)
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摘要: 军曹鱼 (Rachycentron canadum) 鳃组织对水温变化敏感。为探究低温胁迫对军曹鱼幼鱼鳃组织的影响，揭示该鱼应对低温胁迫的响应机制，实验设置2个低温胁迫组 (18 ℃、21 ℃) 和1个对照组 (28 ℃)，比较分析鳃组织在胁迫后第0、第4和第7天的氧化应激状态、细胞凋亡和组织结构情况。结果显示，低温胁迫下鳃组织超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT) 和谷胱甘肽过氧化物酶 (GSH-Px) 活性显著低于对照组 (P<0.05)，丙二醛 (MDA) 浓度显著高于对照组 (P<0.05)；低温胁迫组鳃组织凋亡相关基因bax、caspase-9、caspase-3、p53和mdm2表达量在第4和第7天时相较于对照组显著升高 (P<0.05)，bcl-2基因表达量显著降低 (P<0.05)；TUNEL检测显示低温组鳃组织细胞凋亡率升高；组织学分析表明低温胁迫下鳃组织出现了不同程度的鳃小片排列紊乱、基部增生、融合，上皮细胞和泌氯细胞空泡化等现象。研究表明，低温胁迫抑制了军曹鱼幼鱼的鳃组织抗氧化酶活性，造成氧化损伤，进一步诱导细胞凋亡，破坏鳃组织结构完整性。Abstract: The gill tissue of cobia (Rachycentron canadum) is sensitive to water temperature changes. In order to investigate the effect of low temperature stress on juvenile cobia, and reveal its response mechanism to low temperature stress, we designed two low temperature groups (18 ℃ and 21 ℃) and one control group (28 ℃), to analyze the antioxidant responses, expression of apoptosis-related genes and histological structure of the gills on 0, 4th and 7th day after the stress. The results show that the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the low-temperature groups were significantly lower than those in the control group (P<0.05), while the malondialdehyde (MDA) mass concentration in the low-temperature groups was significantly higher than that in the control group (P<0.05). The expression of apoptosis-related genes bax, caspase-9, caspase-3, p53 and mdm2 increased significantly in the low-temperature groups, while the expression of Bcl-2 decreased significantly on 4th and 7th day (P<0.05). The TUNEL results reveal that low-temperature treatment increased the cell apoptosis rate of the gills, and caused lesions including fusion of secondary lamellae, necrosis of epithelial cell and hyperplasia of chloride cells. The results indicate that low-temperature stress causes oxidative stress, induces apoptosis and damages the structural integrity of the gills, which suggests that normal physiological functions of juvenile cobia can be affected by low temperature significantly.
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Key words:
- Rachycentron canadum /
- Low temperature stress /
- Gill /
- Antioxidant enzyme /
- Apoptosis /
- Organization structure
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图 1 低温胁迫对军曹鱼幼鱼鳃组织超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶活性及丙二醛质量摩尔浓度的影响
Figure 1. Effects of low temperature stress on activities of SOD, CAT, GSH-Px and MDA molality in gill of R. canadum
图 2 低温胁迫对军曹鱼幼鱼鳃组织凋亡相关基因表达量的影响
Figure 2. Relative expression of genes in gill of R. canadum at low temperature stress
图 3 低温胁迫下军曹鱼幼鱼鳃组织细胞凋亡情况
注：a—c分别表示28 ℃(对照组)在第0、第4和第7 天时鳃组织细胞凋亡图；d—e分别表示21 ℃胁迫第0、第4和第7 天时鳃组织细胞凋亡图；g—i分别表示18 ℃胁迫第0、第4和第7 天时鳃组织细胞凋亡图；红色箭头表示凋亡，标尺=100 μm。
Figure 3. Apoptosis of gill of R. canadum at low temperature stress
Note: a−c. Apoptosis of the gill at 28 ℃ on 0, 4th and 7th day (Control group); d−e. Apoptosis of the gill at 21 ℃ on 0, 4th and 7th day; g−i. Apoptosis of the gill at 18 ℃ on 0, 4th and 7th day. The red arrows indicate apoptotic cells; bar=100 μm.
图 4 低温胁迫对军曹鱼幼鱼鳃组织结构的影响
注：a—c. 分别表示为28 ℃ (对照组) 在第0、第4和第7 天鳃组织显微结构图；d—e. 分别表示为21 ℃胁迫第0、第4和第7 天鳃组织显微结构图；g—i. 分别表示为18 ℃胁迫第0、第4和第7 天鳃组织显微结构图；标尺=50 μm。
Figure 4. Histopathological features in gill tissue of R. canadum after exposure to low temperature stress
Note: a–c. Microscopical gill structure at 28 ℃ on 0, 4th and 7th day (Control group); d–e. Microscopical gill structure at 21 ℃ on 0, 4th and 7th day; g–i. Microscopical gill structure of at 18 ℃ at 0, 4th and 7th day; bar=50 μm.
表 1 引物序列
Table 1. Primer sequence
引物Primer 引物序列 (5'—3')Primer sequence (5'−3') 基因序列Accession No. caspase-9-F GTGGAGCTCCTGCTGTTCAT OP546050 caspase-9-R ACGGGCTGGCATCCATTTTA caspase-3-F ACCAGACAGTGGACCAGATAA OP546051 caspase-3-R GTGGAGAAGGCATAAAGGAAG bcl-2-F CCACCACGGCGAAGAGAAGATT OP546048 bcl-2-R CTGCGGTGTCATCTCCTCCTTG p53-F GAGACCTTCAGGAAGTACCAGC OP546053 p53-R TCTCCGGTTTGTCCTTGTTGG bax-F GCAGAGTGGTCGCACTGTTCT OP546049 bax-R AATGCCCTCCCAGCCTCCTT mdm2-F ATCCTCGCAAGAGGTTGGTG OP546052 mdm2-R TCCACAGAGGAAAGCGTCAC β-actin-F AGGGAAATTGTGCGTGAC EU266 539.1 β-actin-R AGGCAGCTCGTAGCTCTT 
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