State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, Fujian Province, China.
State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, Fujian Province, China; Marine Biodiversity and Global Change Research Center, College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, Fujian Province, China.
Fish Shellfish Immunol. 2019 Mar;86:653-661. doi: 10.1016/j.fsi.2018.11.066. Epub 2018 Nov 28.
Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg protein). However, cathepsin B was detected of 63.32 U mg protein and alkaline phosphatase was only 0.12 U mg protein. Moreover, protease showed a higher percentage of activity than antiprotease. A. clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria, particularly against Aeromonas hydrophila and Vibrio parahaemolyticus but showed no effect on Gram-positive bacteria at the tested concentrations. The bactericidal activity functioned within a short time in a distinct time- and dose-dependent manner. SEM showed that after treated with A. clarkii skin mucus, the V. parahaemolyticus cells distorted and piled together, and the filaments appeared and became into cotton-shaped or quasi-honeycomb texture to adhere cells. Meanwhile, A. clarkii skin mucus showed an apparent antiparasitic activity against C. irritans theronts with a distinct dose- and time-dependent relationship. LM and SEM observation showed that after treated with skin mucus, the theronts quickly stopped their swimming and cilia movement, cells became rounded, cilia shed, small bubbles formed on the surface, cell nucleolus enlarged, cytoskeleton deformed, cell membranes ruptured and cell content leaked out. Antibacterial activity was not affected by 30-90 °C heat treatment but was slightly suppressed by 100 °C. In the pH treatment groups, antibacterial activity was not affected by the moderate pH treatment of 5.0-8.0, but slightly suppressed by weak acid and weak base. Therefore, we speculated that the skin mucus of A. clarkii might be a potential source of novel antibacterial and antiparasitic components for fish or human health-related applications. This study broadened our understanding of the role of skin mucus in the innate immune system and provided a basis for the further isolation and purification of active substances.
最近，黏膜表面，尤其是鱼类的皮肤及其分泌的黏液，引起了免疫学家的极大兴趣。雀鲷科的双锯鱼（Amphiprion clarkii）与海葵共生，由于其丰富的皮肤黏液的保护，对常见的海水细菌疾病和寄生虫具有良好的抵抗力。在本研究中，分析了双锯鱼皮肤黏液的免疫相关酶（溶菌酶、蛋白酶、抗蛋白酶、组织蛋白酶 B、碱性磷酸酶和过氧化物酶）活性、对两种革兰氏阳性菌和五种革兰氏阴性菌的抗菌活性、对海洋白点病病原体（刺激隐核虫游仆虫）的抗寄生虫活性以及皮肤黏液的理化稳定性（对 pH 值和热）。结果表明，溶菌酶和过氧化物酶的水平非常相似（在 2 到 4 U/mg 蛋白之间）。然而，组织蛋白酶 B 的含量为 63.32 U/mg 蛋白，碱性磷酸酶仅为 0.12 U/mg 蛋白。此外，蛋白酶的活性百分比高于抗蛋白酶。双锯鱼皮肤黏液对革兰氏阴性菌具有很强的抗菌活性，特别是对嗜水气单胞菌和副溶血弧菌，但在测试浓度下对革兰氏阳性菌没有影响。杀菌活性在短时间内以明显的时间和剂量依赖性方式发挥作用。SEM 显示，经双锯鱼皮肤黏液处理后，副溶血弧菌细胞变形并堆积在一起，出现丝状并变成棉状或准蜂窝状结构以黏附细胞。同时，双锯鱼皮肤黏液对刺激隐核虫游仆虫表现出明显的抗寄生虫活性，具有明显的剂量和时间依赖性关系。LM 和 SEM 观察表明，经黏液处理后，游仆虫迅速停止游动和纤毛运动，细胞变圆，纤毛脱落，表面形成小气泡，核仁增大，细胞骨架变形，细胞膜破裂，细胞内容物泄漏。抗菌活性不受 30-90°C 热处理的影响，但受 100°C 热处理的轻微抑制。在 pH 值处理组中，皮肤黏液的抗菌活性不受 5.0-8.0 的温和 pH 值处理的影响，但受弱酸和弱碱的轻微抑制。因此，我们推测双锯鱼的皮肤黏液可能是鱼类或人类健康相关应用中新型抗菌和抗寄生虫成分的潜在来源。本研究拓宽了我们对皮肤黏液在先天免疫系统中作用的认识，为进一步分离和纯化活性物质提供了依据。
