牙鲆（Paralichthys olivaceus）仔稚幼魚腸道菌群結(jié)構(gòu)變化及定植規(guī)律研究
發(fā)布時間：2018-08-18 07:35
【摘要】：本文研究了工廠化人工育苗條件下牙鲆(Paralichthys olivaceus)仔稚幼魚腸道菌群結(jié)構(gòu)變化及定植規(guī)律,并分析了牙鲆仔稚幼魚腸道菌群結(jié)構(gòu)與餌料和水環(huán)境中菌群的關(guān)系,以及探討了腸道菌群形成機(jī)制,旨在為牙鲆人工育苗和養(yǎng)殖過程中腸道生理健康標(biāo)準(zhǔn)提供理論依據(jù)。主要研究結(jié)果如下:1.采用MiSeq 16S rRNA高通量測序技術(shù)和生物信息學(xué)分析方法,構(gòu)建牙鲆工廠化人工育苗模式下仔稚幼魚階段6個不同時期16S rRNA基因測序文庫,共獲得7462個OTU,42個菌門,972個菌屬。初孵仔魚G1樣品中變形菌門(Proteobacteria)、厚壁菌門(Firmicutes)和擬桿菌門(Bacteroidetes)為主要優(yōu)勢菌,相對豐度分別為55.20%、13.37%和21.58%;9日齡G2和21日齡G3腸道的優(yōu)勢菌群結(jié)構(gòu)較單一,變形菌門(90%)成為此時期腸道的絕對優(yōu)勢菌群;45日齡后,變形菌門的相對豐度顯著降低,厚壁菌門和擬桿菌門的相對豐度明顯增大,成為腸道菌群的優(yōu)勢菌群。屬水平上,仔稚幼魚腸道優(yōu)勢菌群的種類和數(shù)量都發(fā)生了較大變化,在9日齡和21日齡時期變形菌門中的弧菌屬(Vibrio)相對豐度最高,到45日齡時期后其相對豐度銳減到最低水平;在80日齡時期,擬桿菌門中的擬桿菌屬(Bacteroides)和普氏菌屬(Prevotella)相對豐度達(dá)到較高水平,成為腸道優(yōu)勢菌屬;在115日齡時期,厚壁菌門中有8個菌屬均發(fā)展成為優(yōu)勢菌屬。2.通過16S rRNA高通量測序分析,探明了牙鲆工廠化人工育苗餌料系列中各種餌料樣品微生物菌群結(jié)構(gòu)特征及其與仔稚幼魚菌群結(jié)構(gòu)的關(guān)系。輪蟲餌料F2中變形菌門(78.21%)和擬桿菌門(13.33%)最多,而厚壁菌門(2.28%)和藍(lán)藻菌門(Cyanobacterium)(1.47%)的相對豐度很低;共有14個優(yōu)勢菌屬。鹵蟲餌料F3中的優(yōu)勢菌為變形菌門、擬桿菌門和厚壁菌門,三者OTU相對豐度之和大于98.26%;11個優(yōu)勢菌屬中弧菌屬(28.34%)和假交替單胞菌屬(12.36%)的相對豐度最高;輪蟲和鹵蟲兩種活生物餌料的優(yōu)勢菌屬差異明顯,僅假交替單胞菌屬和紅桿菌屬(Rhodobacter)為共有優(yōu)勢菌屬。配合飼料F4中的優(yōu)勢菌屬分布較為均勻,共有12個,大多屬于厚壁菌門芽孢桿菌綱,其中乳球菌屬(Lactococcus)和葡萄球菌屬(Staphylococcus)、棲熱菌屬(Thermus)三者相對豐度最高,均大于6%。配合飼料F5中的優(yōu)勢菌屬為腐敗希瓦菌屬(Shewanella)、嗜冷桿菌屬(Psychrobacter)和不動桿菌屬(Acinetobacter),與F4相比差別較大;配合飼料F6中藍(lán)藻菌門的豐度達(dá)到最高為74%,成為絕對優(yōu)勢菌屬。水體樣品的菌群結(jié)構(gòu)在門分類水平上變化不大,優(yōu)勢菌群都是變形菌門、擬桿菌門、厚壁菌門,但是各時期樣品優(yōu)勢菌屬變化明顯。1日齡W1樣品中菌群多樣性豐富,有14個優(yōu)勢菌屬;9日齡W2樣品中,變形菌門是絕對優(yōu)勢菌群,擬桿菌門中的黃桿菌目(Flavobacteriales)的相對豐度增加到32.4%,厚壁菌門中的乳球桿菌的豐度增加到1.96%,成為優(yōu)勢菌屬;21日齡W3樣品中擬桿菌門中的優(yōu)勢菌相對豐度降低,而γ-變形菌門的總豐度高達(dá)73.45%,其中,弧菌屬和假交替單胞菌屬的相對豐度最高,分別為20.7%和13.7%;45日齡W4樣品中黃桿菌目的相對豐度約為17.34%,但優(yōu)勢菌的種類減少到2個;80日齡水體W5中優(yōu)勢菌屬的種類減少,而優(yōu)勢菌的相對豐度增加,其中γ-變形菌中的假交替單胞菌屬的相對豐度達(dá)到38.94%;115日齡W6中除了乳球菌屬的相對豐度增加成為優(yōu)勢菌屬,菌群結(jié)構(gòu)變化不大,與W5時期組成相似。3.對6個取樣階段餌料、水與腸道樣品菌群的相似關(guān)系分析。牙鲆在攝食活餌料輪蟲和鹵蟲階段,腸道菌群與餌料的相似性較高,分別為17.61%、41.11%;在轉(zhuǎn)換配合飼料后,腸道菌群與配合飼料的菌群相似性逐漸降低,115日齡時,與餌料的相似性僅為3.34%,而且共有優(yōu)勢菌屬消失。初孵仔魚樣品與水體菌群的相似性最高為26.77%,共有優(yōu)勢菌屬3個;在開口后9日齡至80日齡的4個時期,腸道菌群與水體菌群的相似性均在7%左右;115日齡時腸道菌群與水體菌群的相似性降到1.15%,而且兩者沒有共同的優(yōu)勢菌屬存在。對腸道與餌料樣品、腸道與水體樣品進(jìn)行RELATE分析,結(jié)果顯示腸道菌群與餌料之間的相關(guān)性(Spearman correlation Rho 0.588,P0.05)遠(yuǎn)高于腸道菌群與水體之間的相關(guān)性(Spearman correlation Rho 0.15,P0.5);說明在牙鲆仔稚幼魚腸道菌群的形成過程中,餌料中的微生物對腸道菌群的影響大于水中微生物的影響,其對腸道菌群的影響起主要作用。4.牙鲆仔稚幼魚腸道菌群結(jié)構(gòu)變化規(guī)律。牙鲆開口后,腸道菌群發(fā)生明顯的演替,仔稚幼魚腸道優(yōu)勢菌群的種類和數(shù)量都發(fā)生了較大變化。攝食活餌料階段變形菌門主要定植在腸道內(nèi),弧菌屬相對豐度最高;45日齡時期后變形菌門相對豐度逐漸減低,擬桿菌門和厚壁菌門豐度增加;在80日齡至115日齡時期,擬桿菌門的擬桿菌屬、普氏菌屬,成為腸道優(yōu)勢菌屬,以及厚壁菌門的8個菌屬成為優(yōu)勢菌屬,此時的這些優(yōu)勢菌屬是在稚幼魚時期逐漸發(fā)育形成的。分析了仔稚幼魚腸道菌群結(jié)構(gòu)與工廠化養(yǎng)殖、池塘養(yǎng)殖、野生魚腸道菌群結(jié)構(gòu),發(fā)現(xiàn)本試驗條件下80-115日齡的牙鲆幼魚腸道菌群的核心菌群與野生和養(yǎng)殖成魚的腸道菌群組成在門水平上結(jié)構(gòu)基本相同,而在屬水平上由于可能攝食餌料的不同存在一定的差異。研究結(jié)果揭示了工廠化人工育苗模式下牙鲆仔稚幼魚腸道菌群結(jié)構(gòu)及演替規(guī)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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4
本文編號：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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4
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