【作者】 闫法军；

【导师】 董双林； 田相利；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2013， 博士

【摘要】 刺参（Apostichopus japonicus Selenka）养殖业是我国特色海水养殖产业之一，主要分布在辽宁和山东沿海。刺参养殖业发展迅速，2010年全国刺参养殖面积已达到16万公顷，产量达13万吨，产值接近200亿元。然而，相对于其迅速发展的规模，与刺参养殖技术相关的基础研究还比较滞后。目前，在刺参的养殖生产实践中，微生物修复技术已越来越受到人们的重视。微生物修复作用的强弱与养殖环境中微生物群落的生态结构与功能特征密切相关，反之，微生物群落的生态结构与功能特征能够反映环境的健康状况。因此，了解刺参养殖池塘环境微生物群落的结构和功能特征具有十分重要的意义。本研究采用PCR-DGGE指纹图谱技术和BIOLOG-ECO微平板培养法，研究了刺参养殖池塘水体、底泥和附着基沉积物中的微生物群落结构和功能以及它们与环境因子间的相互关系；另外，对几株前期分离筛选自刺参池塘环境中的潜在益生菌进行了饲喂刺参实验以探讨它们对刺参生长和免疫提高方面的功能作用。本研究旨在为刺参池塘环境微生态调控模式的建立提供基础资料。主要研究结果如下：1刺参池塘环境微生物群落结构的季节变化利用PCR-DGGE技术和冗余分析（RDA）方法研究了刺参（Apostichopusjaponicus）养殖池塘环境（水体、底泥、附着基）细菌微生物群落结构的季节变化及其与环境因子间的关系。结果表明：（1）刺参养殖水体中的优势菌群归属于α-Proteobacteria、 γ-Proteobacteria、Bacteroidetes、Cyanobacteria及一部分未知不可培养菌群（uncultured bacteria）。变形菌纲细菌（Proteobacteria）为各季节水体中的绝对优势菌群，其相对丰度在49.3-72.1%之间，其中以α-Proteobacteria含量最高；Cyanobacteria仅出现在秋季水体中，其丰度颇低。显著性方差分析结果发现， γ-Proteobacteria菌群丰度具有显著的季节变化。比较而言，水体细菌群落结构以秋季最复杂，冬季最简单。（2）底泥中的优势菌群归属于α-Proteobacteria、 γ-Proteobacteria、 δ-Proteobacteria、Bacteroidetes、Chloroflex、Actinobacteria和Sphingobacteria。变形菌纲细菌为各季节底泥中的绝对优势菌群，其相对丰度在49.6-55.1%之间，其中又以γ-Proteobacteria含量占绝对优势；Chloroflex菌群仅出现在春、秋季节样品中，且春季丰度较高；Actinobacteria菌群在春季样品中未检测到。显著性方差分析发现，Actinobacteria菌群丰度在夏季显著高于冬季。比较而言，底泥细菌群落结构以秋季最复杂。（3）附着基中的优势菌群归属于α-Proteobacteria、 γ-Proteobacteria、 δ-Proteobacteria、ε-Proteobacteria、Flavobacteria、Bacteroidetes、Cyanobacteria、Actinobacteria及uncultured bacteria。变形菌纲为各季节附着基中的绝对优势菌群，其相对丰度在54.6-60.2%之间，其中以α-Proteobacteria、-Proteobacteria含量最高；Flavobacteria相对丰度亦颇高，其中在春、夏季节含量仅次于α-Proteobacteria；Cyanobacteria在春季样品中未检测到，Actinobacteria在夏季样品中未检测到。显著性分析结果表明， γ-Proteobacteria、 ε-Proteobacteria和Flavobacteria3类菌群含量具有显著季节变化。附着基细菌群落结构在秋、冬季较春、夏季复杂。（4）RDA分析发现，T、TN、NO₃^-N、PO₄^-P和NO₂^-N是影响水体细菌群落结构季节变化的主要环境因子，PO₄^-P、NH4^-N、NO₃^-N和TP是影响底泥细菌群落结构季节变化的主要环境因子，TP、TN、TOC、SOM和NO₃^-N是影响附着基细菌群落结构季节变化的主要环境因子。本研究结论认为，刺参池塘水体、底泥和附着基细菌群落结构具有明显的不同的季节变化，相对而言，底泥菌群结构较水体和附着基稳定；水体、底泥和附着基菌群结构组成存在明显差异，其中以附着基菌群结构最复杂，生物多样性最丰富，水体中最简单；刺参池塘水体、底泥和附着基细菌群落结构季节变化与环境因子具有很好的相关性。2刺参池塘环境微生物群落功能多样性的季节变化利用BIOLOG技术和冗余分析（RDA）方法对刺参（Apostichopus japonicus）养殖池塘环境（水体、底泥、附着基）微生物群落功能多样性的季节变化及其与环境因子间的关系进行了研究。结果表明：（1）刺参池塘水体、底泥和附着基微生物对碳源总量和单类碳源的利用均具有显著的季节变化，总体表现为春、夏、秋季节高于冬季，其中水体微生物利用比例较高的碳源类型为聚合物，底泥微生物利用比例较高的碳源类型为聚合物、糖类、羧酸和氨基酸，附着基微生物利用比例较高的碳源类型为聚合物、糖类、氨基酸和胺。（2）主成分分析表明，刺参池塘水体、底泥和附着基微生物碳代谢方式均具有显著的季节变化。水体中，与主成分显著相关的碳源有15种，其中与主成分1显著相关的碳源主要有10种，分别属于聚合物、糖类、羧酸、氨基酸和胺；底泥中，与主成分显著相关的碳源有18种，其中与主成分1显著相关的主要有13种，分别属于糖类、羧酸和氨基酸；附着基中，与主成分显著相关的碳源有22种，其中与主成分1显著相关的主要有20种，分别属于聚合物、糖类、羧酸和氨基酸。（3）刺参池塘水体、底泥和附着基微生物多样性指数Shannon、McIntosh、Simpson和S-E均匀度均存在显著的季节变化，但不同指数之间的变化有较大差异。（4）RDA分析表明，TP、NO₃^-N、TN和PO₄^-P是影响刺参池塘水体微生物群落功能多样性季节变化的主要因素；TP、NO₃^-N和PO₄^-P是影响底泥微生物功能多样性季节变化的主要因素；SOM、NO₃^-N和TN是影响附着基微生物功能多样性季节变化的主要因素。结论认为，刺参池塘水体、底泥和附着基微生物功能多样性具有显著的不同的季节变化，这些变化与环境因子具有很好的相关性。3刺参池塘不同类型附着基微生物群落结构研究利用PCR-DGGE技术和冗余分析（RDA）方法研究了刺参（Apostichopusjaponicus）养殖池塘不同类型附着基（瓦片、网笼、塑料管）细菌微生物群落结构特征及其与环境因子间的关系。结果表明：（1）瓦片与网笼附着基中的优势菌群群落组成相同，均归属于α-Proteobacteria、 γ-Proteobacteria、Sphingobacteria、Chloroflex、Bacteroidetes、Cyanobacteria、Flavobacteria；而塑料管附着基中的优势菌群归属于α-Proteobacteria、 γ-Proteobacteria、Sphingobacteria、Chloroflex、Bacteroidetes、Actinobacteria、Flavobacteria。（2）黄杆菌纲细菌（Flavobacteria）为各附着基样品中的绝对优势菌群，其相对丰度均在45%以上，但在不同类型附着基之间无显著差异。变形菌纲（Proteobacteria）为各附着基中的第二大优势菌群，其相对丰度在瓦片中最低（20.7%），网笼中最高（34.7%），其中瓦片和塑料管中以γ-Proteobacteria占优势，网笼中以α-Proteobacteria占优势；显著性方差分析表明，变形菌纲相对丰度在不同类型附着基中具有显著差异，其中α-Proteobacteria在网笼中丰度（16.2%）显著高于瓦片（9.5%）和塑料管（6.4%），γ-Proteobacteria在塑料管中丰度（28.3%）显著高于瓦片（11.2%）和网笼（13.2%）。拟杆菌纲（Bacteroidetes）相对丰度在瓦片、网笼和塑料管中依次降低，其在瓦片中的含量（6.0%）显著高于塑料管（0.4%）；蓝藻纲（Cyanobacteria）细菌丰度（在塑料管中未检测到）在各附着基中的变化趋势与拟杆菌纲相同。Actinobacteria仅出现在塑料管附着基样品中。Sphingobacteria和Chloroflex相对丰度在不同附着基之间无显著差异。（3）RDA分析发现，TN、PO₄^-P、NO₃^-N和NH₄^-N是不同类型附着基细菌群落结构差异的主要影响环境因子。本研究结论认为，刺参池塘瓦片、网笼和塑料管附着基细菌群落结构不同，比较而言，瓦片和网笼附着基细菌群落结构特征最相似，而塑料管附着基中的菌群结构与瓦片和网笼中差异较大；刺参池塘瓦片、网笼和塑料管附着基细菌群落结构特征与环境因子具有很好的相关性。4刺参池塘不同类型附着基微生物群落功能多样性研究利用BIOLOG技术和冗余分析（RDA）方法对刺参（Apostichopus japonicus）养殖池塘不同类型附着基（瓦片、网笼、塑料管）微生物群落功能多样性及其与环境因子间的关系进行了研究。结果表明：（1）瓦片、网笼和塑料管附着基微生物对碳源总量和单类碳源的利用均具有明显差异，总体表现为塑料管>网笼>瓦片，其中塑料管和网笼附着基微生物对单类碳源的代谢强度均显著高于瓦片；塑料管附着基微生物利用比例较高的碳源类型为聚合物、糖类和氨基酸，网笼附着基微生物利用比例较高的碳源类型为聚合物、糖类和羧酸，瓦片附着基微生物利用比例较高的碳源类型为聚合物和羧酸。（2）主成分分析表明，瓦片、网笼和塑料管附着基微生物碳代谢方式均具有显著差异，这种差异主要体现在主成分1上；与主成分1显著相关的碳源有13种，分别属于聚合物（3种）、糖类（4种）、氨基酸（4种）、羧酸（1种）和胺（1种）。（3）瓦片、网笼和塑料管附着基微生物碳代谢多样性指数McIntosh和Simpson均存在显著差异，其中McIntosh指数在网笼和塑料管中显著高于瓦片，Simpson指数在塑料管中显著高于瓦片；而Shannon和S-E均匀度均无显著差异。（4）RDA分析表明，TN、PO₄^-P和NO₃^-N是不同类型附着基微生物功能多样性差异的主要影响环境因子。结论认为，刺参池塘瓦片、网笼和塑料管不同类型附着基微生物功能多样性具有显著的不同，这些差异与环境因子具有很好的相关性。5饲料中添加潜在益生菌对刺参生长和非特异性免疫酶活性的影响研究了饲喂1株马氏副球菌（Paracoccus marccui DB11）和1株芽孢杆菌（Bacillus baekryungensis YD13）对刺参（Apostichopus japonicus）生长和非特异性免疫酶活性的影响。将DB11或YD13分别以3个添加浓度水平（110⁴、110⁶和110⁸cfu/g）添加于基础饲料中饲喂刺参（初始体质量（5.340.11）g）60d（以基础饲料饲喂组为对照），试验它们对刺参生长和刺参不同体组织中非特异性免疫酶活性的影响。生长实验结果表明：与对照相比，饲料中添加110⁸cfu/g DB11、110⁴或110⁶cfu/g YD13均能显著提高刺参的增重率和特定生长率（P<0.05）。免疫实验结果表明：DB11在110⁸cfu/g高浓度下对刺参各体组织免疫酶活有最大提高作用，而YD13在110⁶cfu/g较低浓度下具有更好的酶活提高作用；刺参不同体组织的免疫酶活反应强度不同，表现为体腔液>呼吸树>肠道>体壁，因此体腔液对刺参的免疫功能起着非常重要的作用。结论认为，本研究的2株细菌具有提高刺参生长和免疫功能的潜力，因此在刺参养殖方面具有较好的应用前景。更多还原

【Abstract】 Sea cucumber （Apostichopus japonicus Selenka） aquaculture has been one ofChina’s special seawater aquaculture industry, which mainly located along the coastof Shandong and Liaoning island. This industry developed rapidly, of which thenational aquaculture area has reached160thousand hectares, the output reached130thousand tons and the economic value been nearly20billion yuan in the year of2010.However, compared to the rapid development of this industry, basic research relatedto it is still relatively lagging behind. In this thesis, microbial community structure andfunctional diversity in sea cucumber culture pond environment, including the water,bottom mud and the shelter, were studied by using the16S rDNA PCR denaturinggradient gel electrophoresis （PCR-DGGE） and the BIOLOG method. In addition, theeffect of three bacterial strains （previously isolated from sea cucumber culture pond）supplemented in diets on growth performance and immune response of sea cucumberwas also investigated. The main results are detailed as follows:1Seasonal changes of microbial community structure in sea cucumber culturepondSeasonal changes of microbial community structure in water, bottom mud andshelter of sea cucumber （Apostichopus japonicus） cultural ponds were studied byPCR-DGGE technique, and meanwhile the relationships between them andenvironmental factors were detected by using the redundancy analysis （RDA） method.The results were concluded as follows:（1） In the water samples, phylogenetic analysisof the cloned bands showed that bacterial phylotypes were closely related toα-Proteobacteria, γ-Proteobacteria, Bacteroidetes, Cyanobacteria and some uncultured bacteria. Proteobacteria were the dominant bacteria with high relativeamount of49.3-72.1%in different seasons, among which α-Proteobacteria were thehighest. Cyanobacteria were only detected in summer season with a very low amount.Significant variance analysis revealed that γ-Proteobacteria exhibited significantseasonal changes in amount. Comparatively, the water microbial community structurewas the most complex in autumn, while the simplest in winter.（2） In the bottom mudsamples, phylogenetic analysis of the cloned bands showed that bacterial phylotypeswere closely related to α-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria,Bacteroidetes, Chloroflex, Actinobacteria and Sphingobacteria. Proteobacteria werethe dominant bacteria with high relative amount of49.6-55.1%in different seasons,among which γ-Proteobacteria were the highest. Chloroflex bacteria were onlyfound in spring and autumn seasons, of which the amount in spring was higher.Actinobacteria existed in all seasons except spring, among which the relative amountof Actinobacteria in summer was significantly higher than in winter. Comparatively,in the bottom mud, the microbial community structure in autumn was the mostcomplex.（3） In the shelter samples, phylogenetic analysis of the cloned bands showedthat bacterial phylotypes were closely related to α-Proteobacteria, γ-Proteobacteria,δ-Proteobacteria, ε-Proteobacteria, Flavobacteria, Bacteroidetes, Cyanobacteria,Actinobacteria and uncultured bacteria. Proteobacteria were the dominant bacteriawith high relative amount of54.6-60.2%in different seasons, among which α-Proteobacteria and γ-Proteobacteria were the highest. The amount ofFlavobacteria was also high, and that in spring and summer was just slightly lowerthan that of α-Proteobacteria. Cyanobacteria were not examined in spring, andActinobacteria were not detected in summer. There existed significant seasonalchanges in amount of γ-Proteobacteria, ε-Proteobacteria and Flavobacteria. In theshelter, the microbial community structure in autumn and winter was more complexthan that in spring and summer.（4） RDA analysis revealed that the criticalenvironmental factors influencing the seasonal changes of water, bottom mud andshelter microbial community structure were T, TN, NO₃^-N, PO₄^-P, NO₂^-N; PO₄^-P,NH₄^-N, NO₃^-N, TP; and TP, TN, TOC, SOM, NO₃^-N, respectively. In conclusion, seasonal changes of microbial community structure in water, bottom mud and shelterof sea cucumber culture pond were different, which were relatively more stable inbottom mud than those in water and shelter. The microbial community compositionwas very different in water, bottom mud and shelter, and that in the shelter was themost complex, while that in the water was the simplest. The seasonal changes ofmicrobial community structure in water, bottom mud and shelter of sea cucumberculture pond were all closely relevant to their environmental factors.2Seasonal changes of microbial community functional diversity in sea cucumberculture pondSeasonal variations of microbial community functional diversity in water, bottommud and shelter of sea cucumber （Apostichopus japonicus） cultural ponds wereexamined using the BIOLOG technique, and the relationships between them andenvironmental factors were also detected by using the redundancy analysis （RDA）method. The results showed that the amount of total and different types of carbonsources utilized by water, bottom mud and shelter microbes in sea cucumber culturalponds varied seasonally, which was higher in spring, summer and autumn whencompared to the winter, respectively. The main types of carbon sources utilized werepolymers by water microbes, polymers, carbohydrates, carboxylic acids and aminoacids by sediment microbes, and polymers, carbohydrates, amino acids and amides byshelter microbes, respectively. Principal Component Analysis indicated that carbonmetabolism functional diversity of water, bottom mud and shelter microbes in seacucumber cultural ponds varied significantly over the seasonal courses. For watermicrobes,15categories of carbon sources were significantly related to the principalcomponents, among which10were significantly related to the principal component1and belonged to the polymers, carbohydrates, carboxylic acids, amino acids andamines. For bottom mud microbes,18categories of carbon sources were significantlyrelated to the principal components, among which13were significantly related to theprincipal component1and belonged to carbohydrates, carboxylic acids and aminoacids. For shelter microbes,22categories of carbon sources were significantly related to the principal components, among which22were related to the principal component1and belonged to the polymers, carbohydrates, carboxylic acids and amino acids.Significant seasonal changes were detected for all microbial diversity indexesincluding Shannon, McIntosh, Simpson and S-E in water, bottom mud and shelter ofsea cucumber cultural ponds, but differences were observed in seasonal variationsbetween all microbial diversity indexes. RDA analysis revealed that the criticalenvironmental factors influencing the seasonal changes of water, bottom mud andshelter microbial community functional diversity were TP, NO₃^-N, TN, PO₄^-P; TP,NO₃^-N, PO₄^-P; and SOM, NO₃^-N, TN, respectively. In conclusion, seasonal changesof microbial community functional diversity were marked and different in water,bottom mud and shelter of sea cucumber cultural ponds, which all correlated wellwith environmental factors.3Studies on microbial community structure in different types of shelters of seacucumber culture pondMicrobial community structures in different types of shelters includingearthenware, polyethylene mesh and plastic pipe in sea cucumber （Apostichopusjaponicus） cultural ponds were studied by PCR-DGGE technique, and therelationships between them and environmental factors were also investigated by usingthe redundancy analysis （RDA） method. The results were concluded as follows:（1） Inboth the earthenware and polyethylene mesh shelters, bacterial phylotypes wereclosely related to α-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Chloroflex,Bacteroidetes, Cyanobacteria and Flavobacteria. While in the plastic pipe shelter,bacterial phylotypes were closely related to α-Proteobacteria, γ-Proteobacteria,Sphingobacteria, Chloroflex, Bacteroidetes, Actinobacteria and Flavobacteria.（2）Flavobacteria were the first dominant bacteria with relative amount of more than45%in each of three types of shelter, for which, however, no significant differences wereobserved between different types of shelter. Proteobacteria were the second dominantbacteria in all shelters, and the relative amount of Proteobacteria was the lowest inearthenware （20.7%） and the highest in polyethylene mesh （34.7%）, respectively. In addition, γ-Proteobacteria were the dominant bacteria in both the earthenware andplastic pipe shelters, while α-Proteobacteria were dominant in polyethylene meshshelter. There were significant differences for amount of Proteobacteria in differenttypes of shelter, in which the amount of α-Proteobacteria in polyethylene mesh（16.2%） was higher than that in earthenware （9.5%） and plastic pipe （6.4%）, while theamount of γ-Proteobacteria in plastic pipe （28.3%） was higher than in earthenware（11.2%） and polyethylene mesh （13.2%）. The relative amount of Bacteroidetespresented a trend of decrease in shelters of earthenware, polyethylene mesh andplastic pipe, and that in earthenware （6.0%） was significantly higher than that inplastic pipe （0.4%）. Cyanobacteria had a similar changing trend with Bacteroidetes,which however were not detected in samples of plastic pipe. Conversely,Actinobacteria were only observed in plastic pipe shelter samples. Sphingobacteriaand Chloroflex both showed no significant changing in amount between differenttypes of shelters.（3） RDA analysis found that the critical environmental factorsresulting in the difference of microbial community structures in shelters ofearthenware, polyethylene mesh and plastic pipe were TN, PO₄^-P, NO₃^-N and NH₄^-N.In conclusion, microbial community structure was different in different types ofshelters of earthenware, polyethylene mesh and plastic pipe in sea cucumber culturepond, and comparatively that in earthenware and polyethylene mesh was much similarto each other, but both were very different from that in plastic pipe. The microbialcommunity structures in shelters of earthenware, polyethylene mesh and plastic pipeof sea cucumber culture pond were all closely relevant to their environmental factors.4Studies on microbial community functional diversity in different types ofshelters of sea cucumber culture pondMicrobial community functional diversity in different types of shelters includingearthenware, polyethylene mesh and plastic pipe in sea cucumber （Apostichopusjaponicus） cultural ponds were examined using the BIOLOG method, and therelationships between them and environmental factors were also detected by using theredundancy analysis （RDA） method. The results showed that the amount of total and different types of carbon sources utilized by earthenware, polyethylene mesh andplastic pipe shelter microbes in sea cucumber cultural ponds varied, which was thehighest in plastic pipe and the lowest in earthenware. And the amount of differenttypes of carbon sources utilized by polyethylene mesh or plastic pipe shelter microbeswas significantly higher than that utilized by earthenware shelter microbes. The maintypes of carbon sources utilized were polymers, carbohydrates and amino acids byplastic pipe shelter microbes; polymers, carbohydrates and carboxylic acids bypolyethylene mesh shelter microbes; and polymers and carboxylic acids byearthenware shelter microbes, respectively. Principal Component Analysis indicatedthat carbon metabolism functional diversity of shelter microbes in sea cucumbercultural ponds varied significantly among earthenware, polyethylene mesh and plasticpipe shelters. And this difference was presented by Principal Component1, to which13categories of carbon sources were significantly related which belonged to thepolymers （3categories）, carbohydrates （4categories）, amino acids （4categories）,carboxylic acids （1category） and amines （1category）. Significant differences weredetected for microbial diversity indexes including McIntosh and Simpson, except forShannon and S-E, among earthenware, polyethylene mesh and plastic pipe shelters ofsea cucumber cultural ponds. And McIntosh index was significantly higher inpolyethylene mesh and plastic pipe than that in earthenware. Simpson index wassignificantly higher in plastic pipe than in earthenware. RDA analysis revealed thatthe critical environmental factors resulting in the difference of shelter microbialcommunity functional diversity in earthenware, polyethylene mesh and plastic pipewere TN, PO₄^-P and NO₃^-N. In conclusion, microbial community functionaldiversities were different in different types of shelters of earthenware, polyethylenemesh and plastic pipe in sea cucumber cultural ponds, which was closely relevant tothe environmental factors.5Effects of probiotics supplemented in diets on growth performance andimmune response in sea cucumberThe present study assessed the growth performance and immune response in the juvenile sea cucumber Apostichopus japonicus by two potential probiotic strains,Paracoccus marcusii DB11and Bacillus baekryungensis YD13. The fresh cells ofDB11and YD13were respectively added to sea cucumber basal feed at three doses of1×10⁴,1×10⁶and1×10⁸CFU/g in diet, and administered orally to A. japonicus （initialmean wet weight5.34±0.11g）. After a60-day feeding trial with probioticsupplemented and unsupplemented control diets, A. japonicus fed with DB11at110⁸cfu/g, with YD13at110⁴and110⁶cfu/g, all exhibited significantly bettergrowth performance in weight gain rate and specific growth rate as compared to thecontrol （P<0.05）. Five non-specific immune parameters including superoxidedismutase （SOD）, catalase （CAT）, lysozyme （LSZ）, acid phosphatase （ACP） andalkaline phosphatase （ALP） activities were measured to evaluate the immune responsein different tissues （coelomic fluid, respiratory tree, intestine and body wall） of A.japonicus to supplemented DB11or YD13. Results showed that the enhancement ofimmune enzyme activities in different tissues of A. japonicus were the greatest whenfed with DB11at110⁸cfu/g and with YD13at110⁶cfu/g, respectively. Theimmune response in A. japonicus to supplement DB11and YD13was highlytissue-specific, and the immune stimulation intensity was coelomic fluid>respiratorytree>intestine>body wall, indicating that the coelomic fluid plays a key role in thenon-specific immunity of A. japonicas. To conclude, the probiotics, Paracoccusmarcusii DB11and Bacillus baekryungensis YD13, could improve the growth andimmune enzyme activities of A. japonicas so that they both have potential applicationprospect in sea cucumber aquaculture.更多还原