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| 栉孔扇贝对麻痹性贝类毒素的生理响应及转录组分析 |
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李瑾祯1,2, 董晨帆2, 吴海燕3, 邴晓菲4, 冯志华1, 谭志军2,5
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1.江苏省海洋生物资源与环境重点实验室 江苏海洋大学 江苏 连云港 222000;2.中国水产科学研究院黄海水产研究所 农业农村部水产品质量安全检测与评价重点实验室 山东 青岛 266071;3.中国水产科学研究院黄海水产研究所 农业农村部水产品质量安全检测与评价重点实验室 山东 青岛 266072;4.中国水产科学研究院黄海水产研究所 农业农村部水产品质量安全检测与评价重点实验室 山东 青岛 266073;5.青岛海洋科学与技术试点国家实验室 山东 青岛 266071
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| 摘要: |
| 本研究将栉孔扇贝(Chlamys farreri)暴露于塔玛亚历山大藻(Alexandrium tamarense)，通过测定内脏团中毒素的蓄积含量、氧化应激酶活性及其基因转录调控变化，探究栉孔扇贝暴露于麻痹性贝类毒素(Paralytic shellfish toxin, PSTs)的初期应激响应机制。结果显示，PSTs在内脏团中迅速蓄积，实验第6天时毒素含量最高，实验第30天时毒素残留量高达62.4%；PSTs引发栉孔扇贝体内脂质过氧化，过氧化物酶(POD)、超氧化物歧化酶(SOD)及谷胱甘肽过氧化物酶(GSH-Px)显著应激(P<0.05)。透射电镜下观察组织病变，发现空泡化、染色质聚集和核质固缩等结构损伤。通过加权基因共表达网络分析鉴定细胞凋亡和谷胱甘肽代谢解毒通路显著应激上调，映射ALOX5、AfGST-σ11、caspase-8及Bax 4个关键转录因子。综上可知，除抗氧化应激外，栉孔扇贝可激活特征性细胞凋亡和以谷胱甘肽解毒代谢反应抵抗PSTs毒性作用。本研究可为深入探索栉孔扇贝应激与代谢PSTs的特征机制提供科学依据。 |
| 关键词: 麻痹性贝类毒素 栉孔扇贝 生理响应 细胞凋亡 转录组 |
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| Transcriptomic analysis and stress response of Chlamys farreri to paralytic shellfish toxins |
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LI Jinzhen1,2, DONG Chenfan2, WU Haiyan3, BING Xiaofei4, FENG Zhihua1, TAN Zhijun2,5
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1.Provincial Key Laboratory of Marine Biological Resources and Environment, Jiangsu Ocean University, Lianyungang 222000, China;2.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, National Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, Qingdao 266071, China;3.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, National Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, Qingdao 266072, China;4.Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, National Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality, Ministry of Agriculture and Rural Affairs, Qingdao 266073, China;5.Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071, China
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| Abstract: |
| Paralytic shellfish toxins (PSTs) are some of the most harmful algal neurotoxins in the world. They easily accumulate in bivalve shellfish and are transmitted through the food chain, causing symptoms such as nausea and vomiting, muscle paralysis, dyspnea and even asphyxiation in consumers, leading to food poisoning in humans. Therefore, a widely accepted limit standard of 800 μg STXeq/kg has been established as a safe limit for PSTs. PSTs are produced by some microalgae, among which Alexandrium tamarense is one of the predominant toxic algae found along the coast of China. It was found that the detection rate and over-standard rate of Chlamys farreri in bivalve shellfish sold in China are relatively high. PSTs are mainly stored in visceral masses and are characterized by their fast accumulation and slow metabolism.
PSTs are neurotoxins that exert their toxic effects by blocking sodium channels and inhibiting nerve conduction. Studies have shown that PSTs can cause stress responses in bivalves, including production of a large amount of reactive oxygen species (ROS), antioxidant stress (including enzymatic and non-enzymatic defense), imbalance of intracellular redox homeostasis, and cell damage (i.e., lipid peroxidation). As one of the main products of lipid peroxidation, the content of malondialdehyde (MDA) can directly reflect tissue and cell membrane damage caused by PSTs. In addition, superoxide dismutase (SOD) and peroxidase (POD) are often used as indicators to evaluate the level of antioxidation, and glutathione peroxidase (GSH-Px) plays a key role in antioxidant defense. The changes in lipid peroxidation and antioxidant enzymes are commonly used in existing studies to reflect the injury and degree of stress in organisms. Some studies have also shown that PSTs can cause tissue damage and induce abnormal gene expression in C. farreri, but research on the changes of gene expression and regulatory mechanism of PST-induced tissue damage in C. farreri is still lacking. This information is important for establishing and perfecting food safety risk assessment technology.
In this study, C. farreri was exposed to a strain of A. tamarense (AT5-3). We measured the toxin accumulation, oxidative stress kinase activity, and its transcriptional regulation in the visceral mass of C. farreri in control and experimental groups. Further, the ultrastructure of the visceral mass in the control group and the experimental group was observed to explore the initial stress response mechanism of C. farreri exposed to PSTs.
The 2-year-old scallop C. farreri was selected as the experimental animal. AT5-3 was cultured in L1 medium at temperatures of (20±1) ℃, light intensity of 54 μ/Em2·s, and a light-dark ratio of 12 h:12 h in the laboratory, and Chlorella vulgaris was cultured simultaneously. The experimental group was fed with algal solution in the exponential growth period and the cell density was 4×104–4.2×104 cells/mL. The control group was fed with the same amount of C. vulgaris. The experiment lasted 20 days, of which the first six (days 0–6) were the exposure stage and the remaining 14 days (days 7–20) were the metabolic stage. During the exposure stage, C. farreri were fed regularly with the AT5-3 strain in exponential growth period twice a day with a feeding dose of 8×106 cells/items/day. The same amount of C. vulgaris was fed in the metabolic stage.
The results of toxin accumulation showed that paralytic shellfish toxins could accumulate rapidly in the visceral mass of C. farreri, but the metabolic rate was slow. The toxin content was highest on day 6 of the experiment, and the maximum accumulated content was approximately 15 times higher than the limit standard. The toxin residue reached its highest level (62.4%) on 20th day of the experiment.
The results of enzyme activity tests showed that the stress due to MDA, GSH-Px, and POD in the visceral mass was significantly increase (P<0.05), and the SOD activity was significantly inhibited (P<0.05) after a brief increase. The results also showed that PSTs could induce lipid peroxidation in C. farreri, and POD, SOD, and GSH-Px were significantly stressed to eliminate the adverse effects of PSTs.
The pathological changes in the visceral mass were observed under a transmission electron microscope and included vacuolation, chromatin aggregation, and nucleoplasmic pyknosis. Tissue damage worsened as the exposure time increased and although the toxin content in C. farreri decreased after the exposure period, the tissue damage was further aggravated. The results of transcriptome analysis showed that 933 differentially expressed genes (DEGs) were screened from the visceral mass of C. farreri after PST exposure. The results of KEGG and GO annotations showed that DEGs were mainly annotated in amino acid metabolism, energy metabolism, and other metabolic processes. Weighted gene co-expression network analysis showed that apoptosis and the glutathione metabolic detoxification pathway were significantly up-regulated, mapping ALOX5, AfGST-σ11, caspase-8 and Bax4 key transcription factors. In the experimental group, the expression of ALOX5 and AfGST-σ11 increased significantly when the accumulation rate was highest (P<0.05). The expression of caspase-8 and Bax4 was highest when toxin accumulation was high, which was significantly higher than that in the control group (P<0.05).
In summary, PSTs can cause lipid peroxidation stress and cell damage to C. farreri. In addition to antioxidant stress, C. farreri can activate characteristic apoptosis and resist PSTs toxicity by glutathione detoxification metabolism. However, this effect is limited and the persistent damage caused by high residual PSTs cannot be eliminated. This study provides a basis for further study on the potential toxicity of PSTs to the scallop C. farreri and its immunomolecular mechanisms. |
| Key words: Paralytic shellfish toxin Chlamys farreri Physiological response Apoptosis Transcriptome |
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