牙鲆（Paralichthys olivaceus）仔稚幼鱼肠道菌群结构变化及定植规律研究
发布时间：2018-08-18 07:35
【摘要】：本文研究了工厂化人工育苗条件下牙鲆(Paralichthys olivaceus)仔稚幼鱼肠道菌群结构变化及定植规律,并分析了牙鲆仔稚幼鱼肠道菌群结构与饵料和水环境中菌群的关系,以及探讨了肠道菌群形成机制,旨在为牙鲆人工育苗和养殖过程中肠道生理健康标准提供理论依据。主要研究结果如下:1.采用MiSeq 16S rRNA高通量测序技术和生物信息学分析方法,构建牙鲆工厂化人工育苗模式下仔稚幼鱼阶段6个不同时期16S rRNA基因测序文库,共获得7462个OTU,42个菌门,972个菌属。初孵仔鱼G1样品中变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为主要优势菌,相对丰度分别为55.20%、13.37%和21.58%;9日龄G2和21日龄G3肠道的优势菌群结构较单一,变形菌门(90%)成为此时期肠道的绝对优势菌群;45日龄后,变形菌门的相对丰度显著降低,厚壁菌门和拟杆菌门的相对丰度明显增大,成为肠道菌群的优势菌群。属水平上,仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化,在9日龄和21日龄时期变形菌门中的弧菌属(Vibrio)相对丰度最高,到45日龄时期后其相对丰度锐减到最低水平;在80日龄时期,拟杆菌门中的拟杆菌属(Bacteroides)和普氏菌属(Prevotella)相对丰度达到较高水平,成为肠道优势菌属;在115日龄时期,厚壁菌门中有8个菌属均发展成为优势菌属。2.通过16S rRNA高通量测序分析,探明了牙鲆工厂化人工育苗饵料系列中各种饵料样品微生物菌群结构特征及其与仔稚幼鱼菌群结构的关系。轮虫饵料F2中变形菌门(78.21%)和拟杆菌门(13.33%)最多,而厚壁菌门(2.28%)和蓝藻菌门(Cyanobacterium)(1.47%)的相对丰度很低;共有14个优势菌属。卤虫饵料F3中的优势菌为变形菌门、拟杆菌门和厚壁菌门,三者OTU相对丰度之和大于98.26%;11个优势菌属中弧菌属(28.34%)和假交替单胞菌属(12.36%)的相对丰度最高;轮虫和卤虫两种活生物饵料的优势菌属差异明显,仅假交替单胞菌属和红杆菌属(Rhodobacter)为共有优势菌属。配合饲料F4中的优势菌属分布较为均匀,共有12个,大多属于厚壁菌门芽孢杆菌纲,其中乳球菌属(Lactococcus)和葡萄球菌属(Staphylococcus)、栖热菌属(Thermus)三者相对丰度最高,均大于6%。配合饲料F5中的优势菌属为腐败希瓦菌属(Shewanella)、嗜冷杆菌属(Psychrobacter)和不动杆菌属(Acinetobacter),与F4相比差别较大;配合饲料F6中蓝藻菌门的丰度达到最高为74%,成为绝对优势菌属。水体样品的菌群结构在门分类水平上变化不大,优势菌群都是变形菌门、拟杆菌门、厚壁菌门,但是各时期样品优势菌属变化明显。1日龄W1样品中菌群多样性丰富,有14个优势菌属;9日龄W2样品中,变形菌门是绝对优势菌群,拟杆菌门中的黄杆菌目(Flavobacteriales)的相对丰度增加到32.4%,厚壁菌门中的乳球杆菌的丰度增加到1.96%,成为优势菌属;21日龄W3样品中拟杆菌门中的优势菌相对丰度降低,而γ-变形菌门的总丰度高达73.45%,其中,弧菌属和假交替单胞菌属的相对丰度最高,分别为20.7%和13.7%;45日龄W4样品中黄杆菌目的相对丰度约为17.34%,但优势菌的种类减少到2个;80日龄水体W5中优势菌属的种类减少,而优势菌的相对丰度增加,其中γ-变形菌中的假交替单胞菌属的相对丰度达到38.94%;115日龄W6中除了乳球菌属的相对丰度增加成为优势菌属,菌群结构变化不大,与W5时期组成相似。3.对6个取样阶段饵料、水与肠道样品菌群的相似关系分析。牙鲆在摄食活饵料轮虫和卤虫阶段,肠道菌群与饵料的相似性较高,分别为17.61%、41.11%;在转换配合饲料后,肠道菌群与配合饲料的菌群相似性逐渐降低,115日龄时,与饵料的相似性仅为3.34%,而且共有优势菌属消失。初孵仔鱼样品与水体菌群的相似性最高为26.77%,共有优势菌属3个;在开口后9日龄至80日龄的4个时期,肠道菌群与水体菌群的相似性均在7%左右;115日龄时肠道菌群与水体菌群的相似性降到1.15%,而且两者没有共同的优势菌属存在。对肠道与饵料样品、肠道与水体样品进行RELATE分析,结果显示肠道菌群与饵料之间的相关性(Spearman correlation Rho 0.588,P0.05)远高于肠道菌群与水体之间的相关性(Spearman correlation Rho 0.15,P0.5);说明在牙鲆仔稚幼鱼肠道菌群的形成过程中,饵料中的微生物对肠道菌群的影响大于水中微生物的影响,其对肠道菌群的影响起主要作用。4.牙鲆仔稚幼鱼肠道菌群结构变化规律。牙鲆开口后,肠道菌群发生明显的演替,仔稚幼鱼肠道优势菌群的种类和数量都发生了较大变化。摄食活饵料阶段变形菌门主要定植在肠道内,弧菌属相对丰度最高;45日龄时期后变形菌门相对丰度逐渐减低,拟杆菌门和厚壁菌门丰度增加;在80日龄至115日龄时期,拟杆菌门的拟杆菌属、普氏菌属,成为肠道优势菌属,以及厚壁菌门的8个菌属成为优势菌属,此时的这些优势菌属是在稚幼鱼时期逐渐发育形成的。分析了仔稚幼鱼肠道菌群结构与工厂化养殖、池塘养殖、野生鱼肠道菌群结构,发现本试验条件下80-115日龄的牙鲆幼鱼肠道菌群的核心菌群与野生和养殖成鱼的肠道菌群组成在门水平上结构基本相同,而在属水平上由于可能摄食饵料的不同存在一定的差异。研究结果揭示了工厂化人工育苗模式下牙鲆仔稚幼鱼肠道菌群结构及演替规律。
[Abstract]:The changes of intestinal flora structure and colonization of juvenile Paralichthys olivaceus were studied under industrial artificial rearing conditions. The relationship between intestinal flora structure of juvenile Paralichthys olivaceus and the flora in diet and aquatic environment was analyzed, and the formation mechanism of intestinal flora was discussed, aiming at artificial rearing and culture of Paralichthys olivaceus. The main results are as follows: 1. Using MiSeq 16S rRNA high-throughput sequencing technology and bioinformatics analysis method, a 16S rRNA gene sequencing Library of juvenile and juvenile fish in six different stages was constructed under the industrial artificial seedling rearing mode of flounder. A total of 7462 OTUs, 42 phyla and 972 genera of bacteria were obtained. Proteobacteria, Firmicutes and Bacteroidetes were the predominant bacteria in G1 samples of larvae, with relative abundance of 55.20%, 13.37% and 21.58%, respectively. The predominant bacteria in the gut of 9-day-old G2 and 21-day-old G3 were relatively simple, and Proteobacteria (90%) became the predominant bacteria in the gut at that time. The relative abundance of P. Formosa decreased significantly, and the relative abundance of P. sclerenchyma and P. Pseudomonas increased significantly, which became the dominant flora of intestinal flora. The relative abundance of Bacteroides and Prevotella in Pseudomonas decreased sharply to the lowest level after the age of 115 days. At the age of 80 days, the relative abundance of Bacteroides and Prevotella in Pseudomonas reached a higher level and became the dominant genus of intestinal tract. At the age of 115 days, 8 genera of Pseudomonas developed into dominant genus. 2. Sequence analysis showed that the microbial community structure of all kinds of industrial artificial rearing diets for Paralichthys olivaceus and their relationship with the microbial community structure of larvae and juveniles were investigated.Proteus (78.21%) and Bacteroides (13.33%) were the most common in the rotifer diet F 2, but the relative abundance of Cyanobacterium (1.47%) and Pseudomonas (2.28%) was very low. There were 14 dominant bacteria genera in Artemia bait F3. The dominant bacteria in Artemia bait F3 were Proteus, Pseudomonas and Thickwall. The relative abundance of OTU of the three genera was more than 98.26%; the relative abundance of Vibrio (28.34%) and Pseudoalternating bacteria (12.36%) was the highest in 11 dominant genera; the dominant bacteria in Rotiform and Artemia baits were significantly different, except Pseudomonas. Alternating bacteria and Rhodobacter are the common dominant bacteria. The distribution of dominant bacteria in formula feed F4 is more uniform, and there are 12, mostly belonging to Bacillus sclerenchyma. The relative abundance of Lactococcus, Staphylococcus and Thermus is the highest, both of which are more than 6%. The dominant bacteria in compound feed F5 were Shewanella, Psychrobacter and Acinetobacter, which were different from F4. The highest abundance of cyanobacteria in compound feed F6 was 74% and became the absolute dominant bacteria. Potential flora were proteus, Bacteroides and sclerenchyma, but the dominant genera of samples changed obviously in different stages. The diversity of bacteria in W1 samples at 1 day old was abundant, and there were 14 dominant genera. The abundance of Lactococcus in the phylum increased to 1.96% and became the dominant bacteria; the relative abundance of dominant bacteria in the phylum decreased in 21-day-old W3 samples, while the total abundance of Proteus gamma reached 73.45%. The relative abundance of Vibrio and Pseudoalternating bacteria was the highest, 20.7% and 13.7% respectively; the objective phase of Flavobacterium in 45-day-old W4 samples. The relative abundance of the dominant bacteria was about 17.34%, but the species of the dominant bacteria was reduced to 2; the species of the dominant bacteria decreased in W5 at 80 days of age, and the relative abundance of the dominant bacteria increased, of which the relative abundance of Pseudoalternating bacteria in Gamma-Proteus reached 38.94%; the relative abundance of the dominant bacteria in W6 at 115 days of age became dominant except Lactococcus. The similarity between the intestinal flora and the diet was 17.61% and 41.11% respectively in the feeding stages of rotifers and artemia, and decreased gradually after changing the diet. At 115 days of age, the similarity between intestinal flora and water flora was only 3.34%, and the common dominant bacteria disappeared. The similarity decreased to 1.15%, and there was no common dominant bacteria. The RELATE analysis of intestinal and bait samples, intestinal and water samples showed that the correlation between intestinal flora and bait (Spearman correlation Rho 0.588, P 0.05) was much higher than that between intestinal flora and water (Spearman correlation Rho 0.05). 15, P 0.5), indicating that during the formation of intestinal flora of juvenile Paralichthys olivaceus, the influence of microorganisms in the diet on intestinal flora was greater than that in the water, and the effect on intestinal flora played a major role. The species and quantity of predominant flora in fish intestine changed greatly. Proteus was mainly colonized in intestine during the feeding stage, and the relative abundance of Vibrio was the highest; the relative abundance of Proteus decreased gradually after 45 days of age, and the abundance of Bacteroides and Sclerenchyma increased; Bacteroides of Bacteroides increased from 80 to 115 days of age. The dominant bacteria were composed of 8 genera of Pseudomonas and 8 genera of Sclerenchyma. The intestinal flora structure of juvenile and juvenile fish was analyzed in this paper. The core flora of intestinal flora of juvenile Paralichthys olivaceus was similar to that of wild and cultured adults at the gate level, but there were some differences at the generic level due to the different possible feeds.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
本文编号：2188777
[Abstract]:The changes of intestinal flora structure and colonization of juvenile Paralichthys olivaceus were studied under industrial artificial rearing conditions. The relationship between intestinal flora structure of juvenile Paralichthys olivaceus and the flora in diet and aquatic environment was analyzed, and the formation mechanism of intestinal flora was discussed, aiming at artificial rearing and culture of Paralichthys olivaceus. The main results are as follows: 1. Using MiSeq 16S rRNA high-throughput sequencing technology and bioinformatics analysis method, a 16S rRNA gene sequencing Library of juvenile and juvenile fish in six different stages was constructed under the industrial artificial seedling rearing mode of flounder. A total of 7462 OTUs, 42 phyla and 972 genera of bacteria were obtained. Proteobacteria, Firmicutes and Bacteroidetes were the predominant bacteria in G1 samples of larvae, with relative abundance of 55.20%, 13.37% and 21.58%, respectively. The predominant bacteria in the gut of 9-day-old G2 and 21-day-old G3 were relatively simple, and Proteobacteria (90%) became the predominant bacteria in the gut at that time. The relative abundance of P. Formosa decreased significantly, and the relative abundance of P. sclerenchyma and P. Pseudomonas increased significantly, which became the dominant flora of intestinal flora. The relative abundance of Bacteroides and Prevotella in Pseudomonas decreased sharply to the lowest level after the age of 115 days. At the age of 80 days, the relative abundance of Bacteroides and Prevotella in Pseudomonas reached a higher level and became the dominant genus of intestinal tract. At the age of 115 days, 8 genera of Pseudomonas developed into dominant genus. 2. Sequence analysis showed that the microbial community structure of all kinds of industrial artificial rearing diets for Paralichthys olivaceus and their relationship with the microbial community structure of larvae and juveniles were investigated.Proteus (78.21%) and Bacteroides (13.33%) were the most common in the rotifer diet F 2, but the relative abundance of Cyanobacterium (1.47%) and Pseudomonas (2.28%) was very low. There were 14 dominant bacteria genera in Artemia bait F3. The dominant bacteria in Artemia bait F3 were Proteus, Pseudomonas and Thickwall. The relative abundance of OTU of the three genera was more than 98.26%; the relative abundance of Vibrio (28.34%) and Pseudoalternating bacteria (12.36%) was the highest in 11 dominant genera; the dominant bacteria in Rotiform and Artemia baits were significantly different, except Pseudomonas. Alternating bacteria and Rhodobacter are the common dominant bacteria. The distribution of dominant bacteria in formula feed F4 is more uniform, and there are 12, mostly belonging to Bacillus sclerenchyma. The relative abundance of Lactococcus, Staphylococcus and Thermus is the highest, both of which are more than 6%. The dominant bacteria in compound feed F5 were Shewanella, Psychrobacter and Acinetobacter, which were different from F4. The highest abundance of cyanobacteria in compound feed F6 was 74% and became the absolute dominant bacteria. Potential flora were proteus, Bacteroides and sclerenchyma, but the dominant genera of samples changed obviously in different stages. The diversity of bacteria in W1 samples at 1 day old was abundant, and there were 14 dominant genera. The abundance of Lactococcus in the phylum increased to 1.96% and became the dominant bacteria; the relative abundance of dominant bacteria in the phylum decreased in 21-day-old W3 samples, while the total abundance of Proteus gamma reached 73.45%. The relative abundance of Vibrio and Pseudoalternating bacteria was the highest, 20.7% and 13.7% respectively; the objective phase of Flavobacterium in 45-day-old W4 samples. The relative abundance of the dominant bacteria was about 17.34%, but the species of the dominant bacteria was reduced to 2; the species of the dominant bacteria decreased in W5 at 80 days of age, and the relative abundance of the dominant bacteria increased, of which the relative abundance of Pseudoalternating bacteria in Gamma-Proteus reached 38.94%; the relative abundance of the dominant bacteria in W6 at 115 days of age became dominant except Lactococcus. The similarity between the intestinal flora and the diet was 17.61% and 41.11% respectively in the feeding stages of rotifers and artemia, and decreased gradually after changing the diet. At 115 days of age, the similarity between intestinal flora and water flora was only 3.34%, and the common dominant bacteria disappeared. The similarity decreased to 1.15%, and there was no common dominant bacteria. The RELATE analysis of intestinal and bait samples, intestinal and water samples showed that the correlation between intestinal flora and bait (Spearman correlation Rho 0.588, P 0.05) was much higher than that between intestinal flora and water (Spearman correlation Rho 0.05). 15, P 0.5), indicating that during the formation of intestinal flora of juvenile Paralichthys olivaceus, the influence of microorganisms in the diet on intestinal flora was greater than that in the water, and the effect on intestinal flora played a major role. The species and quantity of predominant flora in fish intestine changed greatly. Proteus was mainly colonized in intestine during the feeding stage, and the relative abundance of Vibrio was the highest; the relative abundance of Proteus decreased gradually after 45 days of age, and the abundance of Bacteroides and Sclerenchyma increased; Bacteroides of Bacteroides increased from 80 to 115 days of age. The dominant bacteria were composed of 8 genera of Pseudomonas and 8 genera of Sclerenchyma. The intestinal flora structure of juvenile and juvenile fish was analyzed in this paper. The core flora of intestinal flora of juvenile Paralichthys olivaceus was similar to that of wild and cultured adults at the gate level, but there were some differences at the generic level due to the different possible feeds.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4
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