应用微生物分子生态学方法研究对虾肠道细菌组成及其变化规律
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摘要
对虾肠道中存在大量的微生物群落,它们对宿主的营养、生理、免疫和疾病防御起着重要的作用。近来微生态制剂开始用于对虾养殖中以促进其生长、提高免疫力和减少病害发生。水产微生态制剂作为抗生素的替代品,代表水产养殖病害防治当前和未来的发展方向,但是其被水产动物口服后的作用机理并不是十分清楚。了解对虾肠道微生物区系中微生物的组成、结构及其生理功能如何,在此基础上可以进一步研究外源有益微生物制剂对其影响,将有助于微生态制剂在水产养殖中的有效应用与推广。
本论文克服传统培养法的缺陷,应用PCR-DGGE法和16S rDNA文库法研究分析中国明对虾和日本囊对虾肠道微生物组成,鉴定其优势种群；比较分析芽孢杆菌和柠檬酸等对日本囊对虾肠道微生物的影响；比较分析水产常用抗生素氟苯尼考对日本囊对虾肠道微生物的影响,为养殖对虾肠道微生物区系的调控技术提供理论依据。主要研究结果如下：
1.健康成年中国明对虾肠道微生物样品经DGGE分离得到22个不同位置的条带,鉴定后分别属于变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)两大类群,分别为冷杆菌属,不动杆菌属,假单胞菌属,希万氏菌属,海洋螺菌属,弧菌属,Thalassobius属,肠球菌属：构建16S rRNA克隆文库,克隆子经测序比对后分别属于变形菌门(Proteobacteria)和拟
杆菌门(Bacteroidetes)两大类群,分别为肠杆菌属,弧菌属,希万氏菌属,亮发菌属,红细菌属,弓形杆菌属,Loktanella属,玫瑰杆菌属,黄杆菌属,不可培养细菌。这2种方法都能有效的反映肠道微生物多样性状况,且克隆文库法比DGGE法更完整和全面。结合使用这2种方法,初步反映了中国明对虾肠道微生物多样性信息。结果表明,中国明对虾肠道微生物属于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)三大类群,其中变形细菌为优势菌群。
2.日本囊对虾肠道正态微生物区系组成种类主要有红细菌属,肠杆菌属,黄杆菌属,鞘脂单胞菌属,噬纤维素菌属,海神单胞菌属,十八杆菌属,青枯菌属,硫还原菌属,脱硫弧菌属,玫瑰杆菌属,弧菌属和不可培养细菌13个属,分别属于α-proteobacteria,β-proteobacteria,γ-proteobacteria,δ-proteobacteria, Bacteroidetes和不可培养细菌六大类群。
3.日本囊对虾在使用添加芽孢杆菌的饲料喂养后,肠道微生物种类主要有肠杆菌属,鞘脂单胞菌属,黄色单胞菌属,黄杆菌属,弧菌属,新月形单胞菌属,红细菌属,冷弯菌属,玫瑰杆菌属,芽孢杆菌属,丙酸杆菌属,噬纤维素菌属,海神单胞菌属和不可培养细
菌,属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Clostridia, Actinobacteria和Bacilli,其中肠杆菌数量明显增多；
添加芽孢杆菌的饲料喂养后改为普通饲料,肠道微生物种类主要有弧菌属,红细菌属,节杆菌属,Olleya, Lacinutrix, Oceanistipes,普雷沃氏菌属,泛菌属,梨形菌属和不可培养细菌,分别属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Bacteroidetes, Actinobacteria, Planctomycetacia;
添加芽孢杆菌的饲料喂养后改为饥饿喂养,肠道微生物种类主要有Lacinutrix, Oceanistipes, Aquimarina, Olleya, Thalassobius,玫瑰杆菌属,节杆菌属,弧菌属和不可培养细菌,分别属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Actinobacteria。
4.日本囊对虾在使用添加柠檬酸的饲料喂养后肠道微生物种类主要有肠杆菌属,黄杆菌属,红杆菌属,不动杆菌属,脱硫叶菌属,海神单胞菌属,芽孢杆菌属,玫瑰杆菌属,螺旋体属,弧菌属和不可培养细菌,属于α-proteobacteria,γ-proteobacteria,δ-proteobacteria, Flavobacteria, Bacillii和Spirochaetes。肠道微生物多样性增加。
5.日本囊对虾在使用添加氟苯尼考的饲料喂养后肠道微生物主要包括Aranicola, Granulosicoccus, Kocuria,弧菌属,节杆菌属,产黄杆菌属,微杆菌属和不可培养细菌,属于α-proteobacteria,γ-proteobacteria, Actinobacteria。肠道微生物种类减少。
综上所述,本研究从微生物分子生态学角度,分析中国明对虾和日本囊对虾肠道微生物组成,并探讨抗生素、益生菌和酸化剂等对日本囊对虾肠道微生物区系的影响,研究结果为微生态制剂在水产养殖中的应用和推广提供了理论依据。
The intestinal microflora of shrimp consists of numerous bacterial species that play an important role in the nutrition, physiology, immunity and disease resistance of the host. Recently, many probiotics have been introduced into shrimp culture as bio-friendly agents to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms and increase the production yield. Aquatic probiotics, considered as the substitute of antibiotics, represent the orientation of the preventing and curing of aquacultural animal diseases. However, to understand the relationship between the intestinal microbiota and health, and the metabolic potential of the microbes, and to evaluate the effect of different diets, treatments or medical conditions on the microbiota, fundamental knowledge of the intestinal microbial populations is needed. It will be helpful to the development, popularization and application of aquatic probiotics.
In the study reported here, PCR-DGGE and 16S rDNA clone library analysis approaches were applied to characterize the diversity of the microbial community in the gut samples of healthy adult Chinese shrimp and kuruma shrimp. The primary goal of the present study was to use molecular methods to characterize the intestinal microbial community in shrimp and evaluate the effect of probiotics, antibiotics and acidulant on the gut microbiota of kuruma shrimp. The present study will be helpful to further understand the significance of probiotics in auqaculture. The results are as follows:
1. The use of PCR-DGGE and clone library analyses has been shown as appropriate methods for describing the intestinal microbiota of Chinese shrimp (Fenneropenaeus chinensis). And the two different molecular biological methods gave similar results. According to two methods, the gut microbiota of was composed of Firmicutes, Proteobacteria and Bacteroidetes. The results of our study indicated that the predominant bacterial population in the intestine of Chinese shrimp was Proteobacteria and Vibrio sp. was the most abundant bacteria.
2. The normal gut microbiota of kuruma shrimp consisted of the genera Rhodobacter, Aranicola, Flavobacterium, Sphingomonas, Cellulophaga, Neptunomonas, Octadecabacter, Ralstonia, Desulfurella, Desulfovibrio, Roseobacter, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,β-proteobacteria,γ-proteobacteria,δ-proteobacteria, Bacteroidetes and uncultured bacterium groups.
3. The addition of Bacillus spp. to the feed of kuruma shrimp led to increased intersubject
variation and total diversity. The gut microbiota was composed of the genera Aranicola, Sphingomonas, Xanthomonas, Flavobacterium, Vibrio, Selenomonas, Rhodobacter, Psychroflexus, Roseobacter, Bacillus, Propionibacterium, Cellulophaga, Neptunomonas and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria, Clostridia, Actinobacteria and Bacilli groups.
After feeding without Bacillus spp., the gut flora consisted of the genera Vibrio, Rhodobacter, Arthrobacter, Olleya, Lacinutrix, Oceanistipes, Prevotella, Pantoea, Pirellula and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria, Bacteroidetes, Actinobacteria and Planctomycetacia groups.
Or after starvation, the gut flora was coposed of Lacinutrix, Oceanistipes, Aquimarina, Olleya, Thalassobius, Roseobacter, Arthrobacter, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria and Actinobacteria groups.
4. The addition of citric acid to the feed of kuruma shrimp led to increased gut microbial diversity. The gut microbiota was composed of the genera Aranicola, Flavobacterium, Rhodobacter, Acinetobacter, Desulfobulbus, Neptunomonas, Bacillus, Roseobacter, Spirochaeta, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria,δ-proteobacteria, Flavobacteria, Bacilli and Spirochaetes groups.
5. The addition of florfenicol to the feed of kuruma shrimp led to decreased gut microbial diversity. The gut microbiota was composed of the genera Aranicola, Granulosicoccus, Kocuria, Vibrio, Arthrobacter, Rhodanobacter, and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria and Actinobacteria groups.
Overall, the present study characterized the diversity of the microbial community in the gut samples of healthy adult Chinese shrimp and kuruma shrimp and evaluated evaluate the effect of probiotics, antibiotics and acidulant on the gut microbiota of kuruma shrimp. The results will be helpful to the development, popularization and application of aquatic probiotics.
本论文克服传统培养法的缺陷,应用PCR-DGGE法和16S rDNA文库法研究分析中国明对虾和日本囊对虾肠道微生物组成,鉴定其优势种群；比较分析芽孢杆菌和柠檬酸等对日本囊对虾肠道微生物的影响；比较分析水产常用抗生素氟苯尼考对日本囊对虾肠道微生物的影响,为养殖对虾肠道微生物区系的调控技术提供理论依据。主要研究结果如下：
1.健康成年中国明对虾肠道微生物样品经DGGE分离得到22个不同位置的条带,鉴定后分别属于变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)两大类群,分别为冷杆菌属,不动杆菌属,假单胞菌属,希万氏菌属,海洋螺菌属,弧菌属,Thalassobius属,肠球菌属：构建16S rRNA克隆文库,克隆子经测序比对后分别属于变形菌门(Proteobacteria)和拟
杆菌门(Bacteroidetes)两大类群,分别为肠杆菌属,弧菌属,希万氏菌属,亮发菌属,红细菌属,弓形杆菌属,Loktanella属,玫瑰杆菌属,黄杆菌属,不可培养细菌。这2种方法都能有效的反映肠道微生物多样性状况,且克隆文库法比DGGE法更完整和全面。结合使用这2种方法,初步反映了中国明对虾肠道微生物多样性信息。结果表明,中国明对虾肠道微生物属于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)三大类群,其中变形细菌为优势菌群。
2.日本囊对虾肠道正态微生物区系组成种类主要有红细菌属,肠杆菌属,黄杆菌属,鞘脂单胞菌属,噬纤维素菌属,海神单胞菌属,十八杆菌属,青枯菌属,硫还原菌属,脱硫弧菌属,玫瑰杆菌属,弧菌属和不可培养细菌13个属,分别属于α-proteobacteria,β-proteobacteria,γ-proteobacteria,δ-proteobacteria, Bacteroidetes和不可培养细菌六大类群。
3.日本囊对虾在使用添加芽孢杆菌的饲料喂养后,肠道微生物种类主要有肠杆菌属,鞘脂单胞菌属,黄色单胞菌属,黄杆菌属,弧菌属,新月形单胞菌属,红细菌属,冷弯菌属,玫瑰杆菌属,芽孢杆菌属,丙酸杆菌属,噬纤维素菌属,海神单胞菌属和不可培养细
菌,属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Clostridia, Actinobacteria和Bacilli,其中肠杆菌数量明显增多；
添加芽孢杆菌的饲料喂养后改为普通饲料,肠道微生物种类主要有弧菌属,红细菌属,节杆菌属,Olleya, Lacinutrix, Oceanistipes,普雷沃氏菌属,泛菌属,梨形菌属和不可培养细菌,分别属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Bacteroidetes, Actinobacteria, Planctomycetacia;
添加芽孢杆菌的饲料喂养后改为饥饿喂养,肠道微生物种类主要有Lacinutrix, Oceanistipes, Aquimarina, Olleya, Thalassobius,玫瑰杆菌属,节杆菌属,弧菌属和不可培养细菌,分别属于α-proteobacteria,γ-proteobacteria, Flavobacteria, Actinobacteria。
4.日本囊对虾在使用添加柠檬酸的饲料喂养后肠道微生物种类主要有肠杆菌属,黄杆菌属,红杆菌属,不动杆菌属,脱硫叶菌属,海神单胞菌属,芽孢杆菌属,玫瑰杆菌属,螺旋体属,弧菌属和不可培养细菌,属于α-proteobacteria,γ-proteobacteria,δ-proteobacteria, Flavobacteria, Bacillii和Spirochaetes。肠道微生物多样性增加。
5.日本囊对虾在使用添加氟苯尼考的饲料喂养后肠道微生物主要包括Aranicola, Granulosicoccus, Kocuria,弧菌属,节杆菌属,产黄杆菌属,微杆菌属和不可培养细菌,属于α-proteobacteria,γ-proteobacteria, Actinobacteria。肠道微生物种类减少。
综上所述,本研究从微生物分子生态学角度,分析中国明对虾和日本囊对虾肠道微生物组成,并探讨抗生素、益生菌和酸化剂等对日本囊对虾肠道微生物区系的影响,研究结果为微生态制剂在水产养殖中的应用和推广提供了理论依据。
The intestinal microflora of shrimp consists of numerous bacterial species that play an important role in the nutrition, physiology, immunity and disease resistance of the host. Recently, many probiotics have been introduced into shrimp culture as bio-friendly agents to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms and increase the production yield. Aquatic probiotics, considered as the substitute of antibiotics, represent the orientation of the preventing and curing of aquacultural animal diseases. However, to understand the relationship between the intestinal microbiota and health, and the metabolic potential of the microbes, and to evaluate the effect of different diets, treatments or medical conditions on the microbiota, fundamental knowledge of the intestinal microbial populations is needed. It will be helpful to the development, popularization and application of aquatic probiotics.
In the study reported here, PCR-DGGE and 16S rDNA clone library analysis approaches were applied to characterize the diversity of the microbial community in the gut samples of healthy adult Chinese shrimp and kuruma shrimp. The primary goal of the present study was to use molecular methods to characterize the intestinal microbial community in shrimp and evaluate the effect of probiotics, antibiotics and acidulant on the gut microbiota of kuruma shrimp. The present study will be helpful to further understand the significance of probiotics in auqaculture. The results are as follows:
1. The use of PCR-DGGE and clone library analyses has been shown as appropriate methods for describing the intestinal microbiota of Chinese shrimp (Fenneropenaeus chinensis). And the two different molecular biological methods gave similar results. According to two methods, the gut microbiota of was composed of Firmicutes, Proteobacteria and Bacteroidetes. The results of our study indicated that the predominant bacterial population in the intestine of Chinese shrimp was Proteobacteria and Vibrio sp. was the most abundant bacteria.
2. The normal gut microbiota of kuruma shrimp consisted of the genera Rhodobacter, Aranicola, Flavobacterium, Sphingomonas, Cellulophaga, Neptunomonas, Octadecabacter, Ralstonia, Desulfurella, Desulfovibrio, Roseobacter, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,β-proteobacteria,γ-proteobacteria,δ-proteobacteria, Bacteroidetes and uncultured bacterium groups.
3. The addition of Bacillus spp. to the feed of kuruma shrimp led to increased intersubject
variation and total diversity. The gut microbiota was composed of the genera Aranicola, Sphingomonas, Xanthomonas, Flavobacterium, Vibrio, Selenomonas, Rhodobacter, Psychroflexus, Roseobacter, Bacillus, Propionibacterium, Cellulophaga, Neptunomonas and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria, Clostridia, Actinobacteria and Bacilli groups.
After feeding without Bacillus spp., the gut flora consisted of the genera Vibrio, Rhodobacter, Arthrobacter, Olleya, Lacinutrix, Oceanistipes, Prevotella, Pantoea, Pirellula and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria, Bacteroidetes, Actinobacteria and Planctomycetacia groups.
Or after starvation, the gut flora was coposed of Lacinutrix, Oceanistipes, Aquimarina, Olleya, Thalassobius, Roseobacter, Arthrobacter, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria, Flavobacteria and Actinobacteria groups.
4. The addition of citric acid to the feed of kuruma shrimp led to increased gut microbial diversity. The gut microbiota was composed of the genera Aranicola, Flavobacterium, Rhodobacter, Acinetobacter, Desulfobulbus, Neptunomonas, Bacillus, Roseobacter, Spirochaeta, Vibrio and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria,δ-proteobacteria, Flavobacteria, Bacilli and Spirochaetes groups.
5. The addition of florfenicol to the feed of kuruma shrimp led to decreased gut microbial diversity. The gut microbiota was composed of the genera Aranicola, Granulosicoccus, Kocuria, Vibrio, Arthrobacter, Rhodanobacter, and uncultured bacterium, which belonged toα-proteobacteria,γ-proteobacteria and Actinobacteria groups.
Overall, the present study characterized the diversity of the microbial community in the gut samples of healthy adult Chinese shrimp and kuruma shrimp and evaluated evaluate the effect of probiotics, antibiotics and acidulant on the gut microbiota of kuruma shrimp. The results will be helpful to the development, popularization and application of aquatic probiotics.
引文
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