氨氮和微囊藻毒素-LR联合作用对斑马鱼肠道免疫和菌群的影响  

Effects of Combined Exposure to Ammonia and Microcystin-LR on I ntestinal Immunity and Microbial Community in Zebrafish(Danio rerio)

作　　者：况宇 何亚 欧阳康 杨慧 王良牟 李大鹏[1,2,3,4] 李莉[1,2,3,4] Kuang Yu;He Ya;Ouyang Kang;Yang Hui;Wang Liangmou;Li Dapeng;Li Li(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt,Ministry of Education,Wuhan 430070,China;Hubei Provincial Engineering Laboratory for Pond Aquaculture,Wuhan 430070,China;Freshwater Aquaculture Collaborative Innovation Center of Hubei Province,Wuhan 430070,China)

机构地区：[1]华中农业大学水产学院,武汉430070 [2]长江经济带大宗水生生物产业绿色发展教育部工程研究中心,武汉430070 [3]池塘健康养殖湖北省工程实验室,武汉430070 [4]淡水水产健康养殖湖北省协同创新中心,武汉430070

出　　处：《生态毒理学报》2024年第3期287-305,共19页Asian Journal of Ecotoxicology

基　　金：国家自然科学基金资助项目(32071621);国家重点研发计划课题(2023YFD2400505);国家现代农业产业技术体系(CARS-45-24)。

摘　　要：氨氮和微囊藻毒素-LR(MCLR)是水生环境中普遍存在的污染物。为探讨两者对斑马鱼肠道潜在的协同效应,实验将成年雌性斑马鱼分别暴露于氨氮(30 mg·L^(-1))、MCLR(10μg·L^(-1))以及两者混合(30 mg·L^(-1)+10μg·L^(-1))的环境中,持续30 d。组织病理学分析显示:氨氮暴露导致肠绒毛面积减少;MCLR暴露导致肠道绒毛破裂,空泡化面积增加;而联合暴露对肠组织损伤更为严重。这些变化伴随着肠道中溶菌酶和β-防御素的含量及相关基因表达的显著降低,表明斑马鱼肠道免疫功能受到抑制。此外,氨氮和MCLR的单独及联合处理还激活NOD1/2和TLR4a/4b信号通路,导致促炎因子IL-1β和TNF-α的表达水平和蛋白含量上升,进而可能诱发肠道炎症反应。肠道菌群分析结果进一步显示,氨氮和MCLR处理显著改变斑马鱼肠道内菌群的平衡,即氨氮增加厚壁菌门(Firmicutes)丰富度,MCLR增加放线菌门(Actinobacteria)丰富度但降低变形菌门(Proteobacteria)丰富度,而氨氮和MCLR联合作用增加肠道致病菌群假单胞菌属(Pseudomonas)和分枝杆菌属(Mycobacterium)丰富度。进一步,两者联合暴露还导致肠道中产生短链脂肪酸的菌群丰度和短链脂肪酸含量显著降低。综上所述,氨氮和MCLR联合处理对斑马鱼肠道免疫及菌群稳态产生了协同的负面影响,其对水生动物和水生态系统的健康构成了不容忽视的潜在风险。Ammonia and microcystin-LR(MCLR)are commonly found pollutants in aquatic environments around the world.To explore the potential synergistic effects of ammonia and MCLR on the zebrafish intestine,adult f emale zebrafish were exposed to total ammonia nitrogen(30 mg·L^(-1))、MCLR(10μg·L^(-1))and a mixture of both(30 mg·L^(-1)+10μg·L^(-1))for a period of 30 d.Histopathological analysis showed that ammonia exposure led to a decrease in intestinal villis area,while MCLR exposure caused villus rupture and increased the ratio of vacuolization in intestines.The combination of ammonia and MCLR resulted in more serious damage in the intestine.A mmonia alone and in combination with MCLR significantly reduced the contents and related gene expression l evels of intestinal lysozyme andβ-defensin compared with the control group,indicating suppressed intestinal i mmunity in zebrafish.Moreover,the single and combined exposure to ammonia and MCLR increased the mRNA expression levels and contents of intestinal inflammatory factors IL-1βand TNF-αby activating NOD1/2 and TLR4a/4b signal pathways,leading to intestinal inflammation.Results of intestinal microbiota showed that both ammonia and MCLR caused changes in diversity.Ammonia nitrogen increased the richness of Firmicutes,while MCLR increased the richness of Actinobacteria and decreased the richness of Proteobacteria.The combined e xposure to ammonia and MCLR significantly increased the abundance of Pseudomonas and Mycobacterium.F urthermore,their combined exposure reduced the number of OTUs of short-chain fatty acids producers and the contents of short-chain fatty acids in the intestine.In conclusion,the combination of ammonia and MCLR has a synergistic effect on the intestinal immunity and structure of intestinal flora in zebrafish,and their combined effects on the health of aquatic animals and the water ecosystem’s integrity should not be underestimated.

关 键 词：氨氮 微囊藻毒素-LR 斑马鱼 肠道菌群 肠道免疫 

分 类 号：X171.5[环境科学与工程—环境科学]