【作者】 张皓；

【导师】 贡成良；

【作者基本信息】 苏州大学 ， 生态学， 2015， 硕士

【摘要】 南美白对虾是当今世界养殖产量最高的三大虾类之一,具有生长快,抗病能力强的特点,已逐渐成为我国南方的主要养殖虾种;鲤科鱼类是鱼类最大的一科,分布很广,我国为世界上鲤科鱼类种类最丰富的国家。养殖环境、肠道微生物群落的结构与水生动物疾病的产生有着十分重要的关联,水生动物的体表、肠道微生物群落的组成结构可以被养殖环境微生物群落所影响,从而影响水生动物的健康。为了探讨养殖环境微生物群落的变化规律,解析南美白对虾、鲤科鱼类疾病的发生与养殖环境、肠道微生物的关系,本研究通过高通量测序技术调查了南美白对虾、鲤科鱼(常规鱼)养殖池塘水体、底泥、肠道微生物群落结构和多样性,探讨了水体、底泥微生物群落与环境因子的互作。1、南美白对虾养殖环境微生物群落动态变化及与水环境的互作对南美白对虾养殖池塘的水体及底泥样品的微生物宏基因组分析显示,在6个不同时期采集的样品中,鉴定到31(8)门、66(19)纲、90(61)目、213(96)科、697(213)属的细菌(真菌),其中细菌(真菌)有6(15)个属在所采集的样品中均能检测到;同一时间点采集的样品中,底泥中微生物的丰富度和多样性指数值高于水体,各样品中细菌的丰富度和多样性指数值高于真菌。在同一时间采集的水样和泥样中,优势微生物种群的组成和丰度在二类样品中无明显对应关系。水体、底泥中的微生物群落与环境因子间的相关性分析显示,水体中的氨氮(AN)、化学需氧量(COD)、总氮(TN)对水体中细菌种群影响最大;水体中的致病菌假单胞菌属Pseudomonas的丰度与TN、总磷(TP)正相关,与p H负相关,而致病菌气单胞菌属Aeromonas则与p H正相关,与TN、亚硝酸盐(Nitrite)负相关;致病菌黄杆菌属Flavobacterium、益生菌蛭弧菌属Bdellovibrio与TP、COD、p H成正相关,与AN成负相关,益生菌芽孢杆菌属Bacillus则与TP、COD、AN成正相关,与Nitrite成负相关;水体中的水温(WT)、TN和COD对泥样细菌种群的影响最大,泥样中致病菌假单胞菌属Pseudomonas与TN、COD成正相关,和WT成负相关,致病菌气单胞菌属Aeromonas、埃希氏菌属Escherichia与WT成正相关,与TN、COD成负相关,益生菌芽孢杆菌属Bacillus、链球菌属Streptococcus、肠球菌属Enterococcus、拟杆菌属Bacteroides刚好与之相似;致病菌黄杆菌属Flavobacterium与TN、COD正相关,与WT负相关;益生真菌酵母菌属Saccharomyces、曲霉菌属Aspergillus和致病真菌镰刀菌属Fusarium的丰度与TN、Nitrite成显著正相关,与WT显著负相关。2、常规鱼养殖环境微生物群落动态变化及与水环境的互作对常规鱼养殖池塘的水样和泥样分析结果显示,在5个不同时期采集的水样中,鉴定到26门、57纲、73目、148科、380属的细菌;在3个不同时期采集的泥样中,鉴定到33门、68纲、90目、203科、648属的细菌,其中18个细菌属在3个同时采集的水样和泥样中均检测。同一时间点采集的泥样中,细菌群落的多样性指数值始终高于水样。在同一时间采集的水样和泥样中,优势细菌种群的组成和丰度在二类样品中没有明显的对应关系。水体中细菌群落与环境因子间的相关性分析显示,影响水体中细菌种群的水质因子主要是COD、p H和WT,水体中致病菌假单胞菌属Pseudomonas的丰度与TN、TP成正相关,与p H值成负相关,而致病菌气单胞菌属Aeromonas则与p H成正相关,与TN、Nitrite成负相关;益生菌芽孢杆菌属Bacillus、致病菌黄杆菌属Flavobacterium与TP、COD成正相关,与Nitrite成负相关。3、养殖水体微生物群落对鲤鱼、鲢鱼肠道菌群结构的影响对养殖池塘水体、鲤鱼、鲢鱼肠道微生物的宏基因组分析结果显示,在2个不同时间点采集的水样中,鉴定到25门、48纲、72目、153科、376属的细菌;在2014年4月28日、7月11日采集的鲤鱼肠道样品中,共鉴定到24门、47纲、68目、157科、415属的细菌,在2014年7月11日采集的鲢鱼肠道样品中,鉴定到15门、29纲、40目、80科、133属的细菌。在检测的水样中,变形菌门Proteobacteria、蓝细菌门Cyanobacteria/Chloroplast、放线菌门Actinobacteria、拟杆菌门Bacteroidetes和疣微菌门Verrucomicrobia是主要优势门(>10%);在鱼肠道中,变形菌门Proteobacteria、蓝细菌门Cyanobacteria/Chloroplast、厚壁菌门Firmicutes、疣微菌门Verrucomicrobia、浮霉状菌门Planctomycetes是主要优势门,水样和肠道的优势细菌群的差异较小。鲤鱼-7中,Spartobacteria_genera_incertae_sedis的丰度最高,其次则是未分类Unclassified属、Gp IIa、巴斯德氏菌属Pasteuria,在鲢鱼-7中,未分类Unclassified属的丰度最高,其次是Spartobacteria_genera_incertae_sedis。在2014年7月11日所采集的水样、鲤鱼和鲢鱼肠道样品中均可检测到的细菌有64个属,代表了所采集的样品总细菌群落丰富度的18.36%;在2014年4月28日、2014年7月11日所采集的水样和鲤鱼肠道样品中均可检测到的细菌71个属,代表了所采集的样品总细菌群落丰富度的12.31%;2014年4月28日采集的鲤鱼肠道中细菌群落的丰富度为所有样品中最高。在水样和肠道样品中可检测到的潜在致病性细菌有气单胞菌属Aeromonas、假单胞菌属Pseudomonas、黄杆菌属Flavobacterium、克雷伯氏菌属Klebsiella和弧菌属Vibrio。研究结果加深了对养殖水体、底泥、水生动物肠道间微生物种群相互影响的理解,为通过调控环境因子改善养殖环境中的微生物群落结构、抑制致病微生物,减轻南美白对虾、常规鱼传染病的发生提供了线索。更多还原

【Abstract】 Pacific white shrimp, Litopeneaus vannamei is one of the world’s famous three shrimp species which has the highest aquaculture production. Litopeneaus vannamei has the characteristics of fast growth and strong disease-resistant ability and gradually become the main farmed shrimp species in south of China.Fishes belongs to Cyprinidae is one of the biggest family and distributed widely, whereas, Cyprinidae fish species are the most abundant in our country.The occurrence of aquatic animal diseases has a close relationship with the aquaculture environment and occurrence of microbial community in the gut of animals.In order to investigate the presence of total microbial communities in the aquaculture environment, parsing the relationship between L.vannamei, Cyprinidae fish diseases and aquaculture environment and presence of microbes in gut. The study investigated the microbial communities structure and diversity in water, sediments, guts of L.vannamei and Cyprinidae fish aquaculture ponds by high-throughput sequencing technology.Results of the study discussed the interaction between environmental factors and microbial communities in water and sediments.1. The dynamic changes of microbial communities in L.vannamei aquaculture environment and interactions with water environment.The metagenomic analysis of water and sediment samples in L.vannamei aquaculture pond showed that the samples which were collected in six different periods,appraised to 31(8) phyla, 66(19) classes, 90(16) orders, 213(96) families, 697(213)genera of bacteria(fungi) including 6(15) genera of bacteria(fungi). The richness and diversity index values of microbes in sediments are higher than water, whereas,abundance and diversity index values of bacteria in each sample was higher than fungi.The collection of water and sediment samples at the same time,composition andabundance of dominant microorganisms has no significant association between two different types of samples.The correlation analysis between environmental factors and microbial communities in water and sediments reported that ammonia nitrogen(AN), chemical oxygen demand(COD), total nitrogen(TN) showed highest impact on bacterial populations in water.The abundance of pathogenic Pseudomonas was positive correlation with TN,total phosphorus(TP),negative correlation with p H,whereas,pathogenic Aeromonas was positive correlation with p H,negative correlation with TN,Nitrite. Pathogenic Flavobacterium and beneficial Bdellovibrio were showed positive correlation with TP,COD,p H,negative correlation with AN.Beneficial Bacillus was positive correlation with TP,COD,AN,and negative correlaiton with Nitrite.In case of sediment samples,water temperature(WT), TN and COD have been recorded significant impact on bacterial population. Pathogenic Pseudomonas were closely positive correlated with TN and COD, whereas, negative correlation was observed with WT. Pathogenic Aeromonas and Escherichia were positively correlated with WT and negative correlated with TN and COD. Additionally, beneficial Bacillus, Streptococcus, Enterococcus and Bacteroides were showed similar results. Pathogenic Flavobacterium was positively correlated with TN and COD, and negatively correlated with WT. The plenty of beneficial Saccharomyces, Aspergillus and pathogenic Fusarium were noted significant positive correlation with TN and Nitrite, whereas it was significantly negative correlated with WT.2. The dynamic changes of microbial communities in conventional fish aquaculture environment and interactions with water environment.According to the results of water and sediment samples from conventional fish aquaculture pond, 26 phyla, 57 classes, 73 orders, 148 families and 380 genera of bacteria were analyzed in water samples which were collected at five different time intervals. Additionally, 33 phyla, 68 classes, 90 orders, 203 families and 648 genera of bacteria were recorded from sediment samples which were collect ed at three different periods. Further, 18 bacterial genera were detected in water and sediment samples which were received at same time from three different places. Diversity index value ofsediment samples was always higher than water samples. Water samples and sediment samples which were collected at the same time, composition and abundance of dominant bacterial populations have not confirmed any obvious correspondence between two types of samples. Correlation analysis of bacterial communities in water and environmental factors showed that main water quality factors which interacts bacterial populations were COD, p H, WT. The richness of pathogenic Pseudomonas in water was positively correlated with TN,TP, and negative correlation with p H value,whereas, the pathogenic Aeromonas was positively correlated with p H, and negative correlation with TN,Nitrite.Beneficial Bacillus and pathogenic Flavobacterium were positively correlated with p H value,COD and negative correlation with Nitrite.3. The influence of aquaculture water microorganisms to microbial communities structure in Cyprinus carpio and Hypophthalmichthys molitrix guts.Metagenomic analysis of aquaculture water and intestinal microflora of Cyprinus carpio and Hypophthalmichthys molitrix recorded 25 phyla,48 classes,72 orders,153 families and 376 genera of bacteria in water samples which were collected in two different time intervals. Whereas, all Cyprinus carpio gut samples identified 24 phyla,47 classes, 68 orders,157 families and 415 genera which were on 28,201411,2014,Hypophthalmichthys molitrix-7 gut sample identified 15 phyla, 29 classes, 40 orders,80 families and 133 genera which was collected on July 11, 2014. In tested water samples, Proteobacteria, Cyanobacteria/Chloroplast, Actinobacteria, Bacteroidetes and Verrucomicrobia were major dominant Phyla(>10%) and in fish guts,Proteobacteria, Cyanobacteria/Chloroplast, Firmicutes, Verrucomicrobia and Planctomycetes were important dominant Phyla. There was a minor difference noted in water samples and intestinal dominant bacteria communities. In Cyprinus carpio-7,Spartobacteria genera incertae sedis has the highest abundance, followed by Unclassified, Gp IIa, and Pasteuria, whereas, in case of Hypophthalmichthys molitrix-7,Unclassified has the highest richness, followed by Spartobacteria genera incertae sedis.64 genera of bacteria was identified in the water,Cyprinus carpio and Hypophthalmichthys molitrix gut samples which was collected on July 11, 2014 and it represents 18.36 % richness of total bacterial communities in collectedsamples.Further, 71 genera of bacteria was noticed in water and Cyprinus carpio gut samples which was taken on April 28,2014 and July 11, 2014,represent 12.31 %richness of total bacterial communities. Maximum abundance was recorded in Cyprinus carpio gut samples which were collected on April 28, 2014. In water and gut samples,potential pathogenic bacteria were identified,namely, Aeromonas, Pseudomonas,Flavobacterium, Klebsiella and Vibrio. The outcome of the present investigation provide a authentic clue that through regulating environmental factors, it is feasible to improve the microbial community structure, inhibit pathogenic microorganisms in the aquaculture environment and reduce the occurrence of diseases in L.vannamei and conventional fishes.更多还原