东山岛星座短腹海鞘共附生微生物多样性研究

Microbe diversity associated with the ascidian Aplidium constellatum from the Dongshan Island

在前期研究发现星座短腹海鞘(Aplidium constellatum)脂溶性提取物具有降血脂功效的基础上,为深入了解该海鞘的生物学背景,开展了其共附生微生物的多样性研究,为进一步从该海鞘及相关共附生微生物中发现可能存在的降血脂活性次生代谢产物奠定基础。通过Illumina Mi Seq高通量测序技术,分析了星座短腹海鞘样品中共附生微生物的物种多样性和物种丰度信息。测序共获得37 911条序列,310个OTU;稀释曲线表明测序深度足够;多样性指数Simpson值为0.373,Shannon值为2.494。样品共附生细菌分属于24个门,优势门有变形菌门(Proteobacteria,78.86%)、放线菌门(Actinobacteria,6.01%)、拟杆菌门(Bacteroidetes,4.67%)、蓝藻门(Cyanobacteria,2.62%)、厚壁菌门(Firmicutes,1.79%)、柔膜菌门(Tenericutes,1.76%)、螺旋菌门(Spirochaetae,1.33%);分属于197个属,优势属有Ruegeria(60.83%)、SAR116_clade科下未知属(3.27%)、Rhodococcus(2.40%)、Cyanobacteria纲下未知属(2.26%)、Candidatus_Hepatoplasma(1.76%)、Pe M15目下未知属(1.61%)、Spirochaetaceae科下未分类属(1.33%)等10个。通过纯培养技术共分离获得15株可培养共附生菌,分属于8个属,分别为弧菌属Vibrio(7株)、希瓦氏菌属Shewanella(2株),以及Pseudomonas、Pseudovibrio、Thalassobius、Exiguobacterium、Flavobacterium、Thalassospira属各一株。本研究首次为星座短腹海鞘提供了准确的共附生微生物背景数据,为进一步开展活性次生代谢产物研究提供了丰富的微生物菌株材料;同时研究还提示该海鞘有水产病原菌感染,在开发利用过程中应格外关注水产病原菌的安全风险防控。

In our previous studies,ester-soluble extracts from the ascidian Aplidium constellatum showed hypolipidemic effect. Herein we studied the biodiversity of microbes associated with the samples of this ascidian for a better understanding of its biological background,and to prepare for the further research on hypolipidemic secondary metabolites of A. constellatum as well as its associated microorganisms. By means of Illumina Mi Seq high-throughput sequencing,we analyzed biodiversity,species abundance and diversity of A.constellatum symbiotic microbe. From the results,a total of 37 911 sequences and 310 OTUs were obtained in97% level of similarity,the length distribution of the trimmed sequences in 293-480,the 310 OTUs were annotated to 24 doors,41 families,84 orders,124 families,197 genera,249 species. The dilution curve indicated that the sequencing depth was sufficient; Ace index（ 310. 327）,Chao index（ 310. 091）,Simpson（ 0. 373） and Shannon（ 2. 494） indicated that the diversity index of the community species was rather high.Coverage was 99. 99%,showing that the results can represent the true situation of microbes. The symbiotic microbe belonged to 24 phylums,and the dominant phylums were Proteobacteria（ 78. 86%）,Actinobacteria（ 6. 01%）,Bacteroidetes（ 4. 67%）,Cyanobacteria（ 2. 62%）,Firmicutes（ 1. 79%）,Tenericutes（ 1. 76%）,and Spirochaetae（ 1. 33%）. Furthermore,these 24 phylums were classified into 197 genuses while the dominant genera included Ruegeria（ 60. 83%）, norank f SAR116 clade（ 3. 27%）,Rhodococcus（ 2. 40%）,norankcCyanobacteria（ 2. 26%）,CandidatusHepatoplasma（ 1. 76%）,noranko Pe M15（ 1. 61%）, and unclassified f Spirochaetaceae（ 1. 33%）, Thalassobius（ 1. 26%）,unclassifiedfRhodobacteraceae（ 1. 23%）,and unclassifiedfRhodospirillaceae（ 1. 08%）. respectively.Pure culture was also carried out and 15 bacteria strains were obtained. These strains belonged to 8 genera including 7 Vibrio strains,2 Shewanella strains,and each one of Pseudomonas,Pseudovibrio,Thalassobius,Exiguobacterium,Flavobacterium and Thalassospira genus. This study provided accurate data of associated microorganisms of A. constellatum for the first time,and provided abundant microbial strains for future studies on their bioactive secondary metabolites. In addition,the study also indicated that the ascidians were infected by aquatic pathogens. We should pay special attention to the safety risk of aquatic pathogens,and try to prevent and control them in the process of development and utilization of A. constellatum.