以宏觀基因組分析日本鰻、鱸鰻及太平洋雙色鰻腸道微生物相

Metagenomic analysis of the gut microflora in Anguilla japonica, Anguilla marmorata and Anguilla bicolor pacifica

張芳齊(Fang-Chi Chang)

指導教授 ： 韓玉山

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.02854

摘要

日本鰻為東亞地區重要的水產養殖物種。近三十年來，日本鰻鰻苗之產量長期趨勢有明顯下降的現象，因此，近年來各國遂興起異種鰻的養殖風潮。全世界共有19種及亞種的淡水鰻，其中有6種分布在台灣，包括有日本鰻、鱸鰻、太平洋雙色鰻、呂宋鰻、內唇鰻及西里伯斯鰻。目前以日本鰻、鱸鰻及太平洋雙色鰻為台灣地區重要的經濟養殖鰻種。然而，在大規模高密度的養殖情況下，魚隻緊迫，容易有疾病爆發和水質環境惡化，造成經濟損失。預防及控制疾病的方法，傳統使用化學性合成藥劑及抗生素，然而藥劑容易殘留環境中及進入食物鏈，有害動物體健康、留下具有抗藥性菌株、對宿主可能造成免疫抑制的影響等缺點。近幾十年逐漸傾向使用益生菌的方式，常用於水產業的益生菌有Clostridium(梭孢桿菌屬), Bacillus (桿菌屬), Enteroccus (腸球菌屬), Lactobacillus (乳酸菌屬), Shewanella (希瓦氏菌屬), Leuconostoc (明串珠菌屬), Lactococcus (乳酸球菌屬), Carnobacterium (肉食桿菌屬), Aeromonas (產氣單胞菌屬), Pseudomonas (假單孢菌屬)等。然而，這些篩選出來的益生菌能否確實在鰻魚腸道繁殖而達到其益生菌的效用不得而知。在腸道中有一複雜、穩定、動態的微生物族群，能夠製造消化酵素和營養素，還能夠抵禦病原菌，對於維持腸道正常功能扮演很重要的角色。因此，許多研究利用魚隻腸道分離的菌株做為益生菌。
本研究利用近十年蓬勃發展的次世代定序技術 (NGS, Illumina Mi-Seq) 分析野生日本鰻、鱸鰻、太平洋雙色鰻及養殖日本鰻病鰻的腸道微生物相，並且利用餵食益生菌實驗驗證腸道常見及優勢的菌屬能夠有效地在鰻魚腸道中附著。本研究發現鰻魚腸道中菌相會依不同環境及鰻魚種類有所變動，有趣的是，可以發現Clostridium (梭孢桿菌屬), Cetobacterium (鯨桿菌屬), Plesiomonas (鄰單胞菌屬), Serratia (沙雷菌屬), Shewanella (希瓦氏菌屬), Bradyrhizobium (慢生根瘤菌屬), Acinetobacter (不動菌屬) and Bacteroides (擬桿菌屬)是常見及優勢的菌屬。研究成果能夠提供一個基礎的資訊供選擇益生菌，以及往後的益生菌研究。

關鍵字

鰻魚 ； 次世代定序 ； 16S rRNA宏觀基因組 ； 腸道微生物相 ； 益生菌

並列摘要

The Japanese eel is an important aquaculture species in east Asia. In the past 3 decades, glass eel abundances have been declining significantly, so the culture of other eel species becomes more and more popular during recent several years. There are 19 species and subspecies freshwater eels known worldwide and are widely distributed in the world. Among them, 6 species, Anguilla japonica, A. marmorata, A. bicolor pacifica, A. luzonensis (A. huangi), A. interioris and A. celebesensis are reported from Taiwan. Currently, A. japonica, A. marmorata and A. bicolor pacifica are commercially important cultured eel species in Taiwan. In large-scale production facilities, where fishes are exposed to stressful conditions, problems related to diseases and deterioration of environmental conditions can result in serious economic losses. Prevention and control of diseases in the aquaculture traditionally use synthetic chemicals and antibiotics, while the use of antimicrobial agents produces potential hazards to public health, including accumulation of residues in the environment, generation of drug-resistance bacteria and immunosuppression of host etc. In the past decades, new alternative approaches gradually use probiotics to substitute the chemotherapy. The commonly used probiotics in fish culture practices belong to genus Clostridium, Bacillus, Enteroccus, Lactobacillus, Shewanella, Leuconostoc, Lactococcus, Carnobacterium, Aeromonas, Pseudomonas and several other species. However, the ability of these probiotics to colonize in gut mucosa of freshwater eels has been little studied. The intestinal tract harbors a complex, stable and dynamic collection of microorganisms that produces digestive enzymes and nutrients as well as prevents pathogens and plays an important role in the maintenance of normal gut function. Therefore, several studies evaluate the microflora isolated from many aquaculture species to select bacteria with potential to become probiotics.
In this study, we used remarkable advance of Next-generation sequencing technology (Illumina Mi-Seq) to analysis the intestinal microbiota of wild A. japonica, A. marmorata, A. bicolor pacifica and cultured sick A. japonica. The feeding experiment proved that the common and dominant bacterial genera found in intestine of wild eels have high efficiency to colonize in gut of cultured eels. This study found that microbial community of intestinal mucus varied with the environments and the eel species. Interestingly, there were some dominant bacterial genera shared in the gut of freshwater eels from different rivers and eel farm such as Clostridium, Cetobacterium, Plesiomonas, Serratia, Shewanella, Bradyrhizobium, Acinetobacter and Bacteroides. This research provides a baseline for use of probiotics and future probiotic studies.

並列關鍵字

Anguillid eel ； NGS ； 16S rRNA metagenomics ； intestinal microflora ； probiotics

參考文獻

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