高通量测序分析小头裸裂尻鱼皮肤和肠道的微生物多样性

Skin and intestinal microbial diversity of Herzensteinia microcephalus determined by high-throughput sequencing

【目的】解析小头裸裂尻鱼不同部位的微生物群落结构、物种组成、多样性特征以及菌群功能差异。【方法】通过Illumina MiSeq扩增子高通量测序,分析小头裸裂尻鱼皮肤黏膜、肠道黏膜和肠道内容物3个部位微生物菌群组成差异,并通过Tax4Fun预测菌群潜在功能。【结果】皮肤黏膜微生物α多样性最高,其Shannon指数显著高于肠道黏膜(P<0.05)和肠道内容物(P<0.001)。主坐标分析表明,皮肤黏膜微生物显著区别于其他2个部位。在门水平小头裸裂尻鱼3个部位相对丰度前五的微生物类群均为放线菌门(Actinobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)和蓝藻门(Cyanobacteria),其中肠道内容物中放线菌门相对丰度(46.53%)显著高于肠道黏膜(29.23%,P<0.05)和皮肤黏膜(25.83%,P<0.01);肠道黏膜中变形菌门的相对丰度(40.33%)显著高于肠道内容物(26.10%,P<0.05)。对各部位相对丰度前10的菌群进行分析发现,小头裸裂尻鱼皮肤黏膜和肠道内容物菌群差异菌群更多,包括微杆菌科(Microbacteriaceae)、伯克氏菌科(Burkholderiaceae)和JG30-KF-CM45等6个差异菌科和冷杆菌属(Cryobacterium)、肉食杆菌属(Carnobacterium)、节杆菌属(Arthrobacter)等9个差异菌属,其中皮肤黏膜中相对丰度较高的菌属主要与有机化合物降解和抑菌作用相关。Tax4Fun功能预测发现,3个部位在第三级通路上表现出显著的差异特征,皮肤黏膜中的菌群在ABC转运蛋白代谢和组氨酸代谢通路富集,肠道黏膜在信号转导和甘油磷脂代谢通路显著富集,而肠道内容物则富集于脂肪酸合成、萜类化合物生物合成途径。【结论】小头裸裂尻鱼不同部位的菌群主要与环境相关,其中皮肤黏膜的微生物多样性最高,其微生物群主要与环境相关,而肠道黏膜和肠道内容物之间微生物多样性差异较小,除与环境相关菌群以外,还发现与自身生理特性、免疫以及营养物质的摄入与消化相关的菌群。阐明不同部位微生物分布特征,将为高原鱼类保护及生存环境改善提供基础数据和科学依据。

[Objective]To reveal the microbiota structure and diversity in different parts of Herzensteinia microcephalus and predict the functions of the microbiota.[Methods]High-throughput sequencing of 16S rRNA gene was performed to reveal the microbiota structure.Tax4Fun was employed to predict the functions of the microbiota.[Results]The microbiota in skin mucosa had the highest alpha diversity,with the Shannon index higher than that of the microbiota in intestinal mucosa(P<0.05)and intestinal content(P<0.001).Moreover,the microbiota in skin mucosa can be significantly distinguished from that in intestinal mucosa and intestinal content by principal co-ordinates analysis.Actinobacteria,Proteobacteria,Firmicutes,Chloroflexi,and Cyanobacteria were the top 5 phyla accounting for more than 75%of the total microbiota.Among them,Actinobacteria in intestinal content had higher relative abundance(46.53%)than that in intestinal mucosa(29.23%,P<0.05)and skin mucosa(25.83%,P<0.01).The relative abundance of Proteobacteria in intestinal mucosa(40.33%)was higher than in intestinal content(26.10%,P<0.05).The top 10 families and genera in each part were further analyzed,which showed that skin mucosa and intestinal content had more different microorganisms,including 6 families(e.g.,Microbacteriaceae,Burkholderiaceae,and JG30-KF-CM45)and 9 different genera(e.g.,Cryobacterium,Carnobacterium,and Arthrobacter).The genera with higher abundance in skin mucosa were associated with organic material degradation and bacterial inhibition.The microbiota in the three parts showed different functional characteristics on pathway level 3 as predicted by Tax4Fun.Specifically,the microbiota in skin mucosa were mainly involved in ABC transporters and histidine metabolism pathway,that in intestinal mucosa in a two-component system and glycerophospholipid metabolism pathway,and that in intestinal content in fatty acid biosynthesis and terpenoid backbone biosynthesis pathway.[Conclusion]The microbiota structures in different parts of H.microcephalus were related to the environment.The skin mucosa had the highest microbial diversity among the three parts,and the intestinal mucosa and intestinal content had similar microbial diversity.In addition,the microorganisms related to the environment,those associated with the host’s physiological characteristics,immunity,and digestion were identified.Revealing the characteristics of microbial distribution in different parts will provide basic data and a scientific basis for the protection and the living environment improvement of fish in the plateau regions.