免疫增强剂党参对仿刺参肠道菌群结构的影响

采用平板计数法和聚合酶链式反应( PCR)-变性梯度凝胶电泳( DGGE)技术分析免疫增强剂党参对仿刺参( Apostichopus japonicas)肠道菌群结构的影响。将初始体重为(18.00±2.00) g的仿刺参随机分为2组(对照组、试验组),每组6个重复,每个重复12只。对照组投喂海泥、鼠尾藻粉按照1∶1的质量比配制的饵料,试验组饵料中以鼠尾藻粉质量的2%添加党参,连续投喂28 d。结果表明：应用免疫增强剂党参不仅能够显著提高仿刺参的特定生长率( P<0.05),降低其饵料系数(P<0.05),而且能够显著增加仿刺参肠道内容物中异养菌的数量(P<0.05);序列统计分析显示投喂党参后仿刺参肠道细菌优质序列比例显著增加( P<0.05),试验组达97.57%,对照组仅为80.22%;Beta多样性分析反映投喂党参后仿刺参肠道微生态环境发生了变化,其多样性系数范围在14.91%~15.47%、15.47%~16.21%、14.91%~16.21%;丰度分析显示投喂党参后仿刺参肠道内容物中变形菌门( Proteobacteria)和拟杆菌门( Bacteroidetes)丰度提高,疣微菌门( Verrucomicrobia)、放线菌门( Actinobacteria)和厚壁菌门( Firmiaites)丰度降低;聚类分析显示试验组与对照组肠道菌群结构相似性系数为0.97。由此可见,免疫增强剂党参可提高仿刺参的特定生长率,降低饵料系数,增加肠道异养菌数量和优势菌群丰度,优化肠道微生态环境。     

Assessment of gut microbiota in different developmental stages of Malaysian Mahseer (Tor tambroides)

Malaysian Mahseer (Tor tambroides) has a good prospect for aquaculture because of its high market demand. However, there is a scarce information on gut microbiota associated with Malaysian Mahseer unlike other fish species. Therefore, we constructed and compared gut microbiota in different developmental stages (larval, juvenile, fingerling, yearling, and adult) using culture dependent and PCR‐DGGE fingerprinting technique for better understanding of gut microbiota composition associated with T. tambroides. Culturable gut microbiota composition in all developmental stages were composed of β‐ and γ‐Proteobacteria, and Bacilli. Biodiversity analysis of culturable gut microbiota showed that larval, juvenile, and adult stages have higher diversity than fingerling and yearling stages. Ward's linkage cluster analysis showed that culturable gut microbiota composition in larval and juvenile stages were close to adult stages, whereas fingerling and yearling stage composed same cluster. PCR‐DGGE fingerprinting technique showed that unculturable gut microbiota were constituted by α‐and γ‐Proteobacteria, and Actinobacteria. Ward's linkage cluster analysis showed that unculturable gut microbiota composition in both larval and juvenile stages were distinct from other developmental stages. Our results revealed that gut microbiota composition were varied in different developmental stages of Malaysian Mahseer and continuous shifts of gut microbiota from larval to adult stages.     

肠道菌群与草鱼个体大小相关性研究

[目的]探讨草鱼肠道菌群与草鱼个体大小之间的相关性。[方法]采用DGGE方法对同一批孵化并在同一池塘中养殖的个体大小显著差异的草鱼肠道样品进行分析。[结果]无论大个体草鱼还是小个体草鱼,其肠道优势菌群均为厚壁菌门和变形菌门,但2组草鱼在其肠道菌群的结构和多样性等方面存在差异。大个体草鱼肠道中存在着更多比例的厚壁菌门以及具有纤维素降解能力的细菌,而小个体草鱼肠道中存在较多的潜在致病菌。[结论]草鱼肠道微生物可以作为内在因素影响草鱼的生长性能,这一结果也为进一步开发可促进草鱼生长性能的益生菌奠定了研究基础。     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Intestinal Microbiota in Large Yellow Croaker,Larimichthys crocea,at Different Ages

The polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S ribosomal RNA gene was used to investigate bacterial communities in the intestines of large yellow croaker at six different ages (12 d, 18 d, 26 d, 40 d, 3 mo, and 1 yr old) as well as within the corresponding feed and culture water. In addition, Illumina Miseq sequencing was utilized to compare intestinal microbiota between 12‐d‐old and 1‐yr‐old individuals. PCR‐DGGE results revealed that the culture water had the highest bacterial diversity, followed by the feed, while the intestines had the lowest diversity. The intestinal microbiota at six ages changed severely; however, the change did not follow any trend. The large yellow croaker intestines harbored specific bacterial communities that differed from those in both feed and water. Illumina Miseq sequencing results revealed that the diversity of intestinal bacteria in 12‐d‐old fish was higher than that in 1‐yr‐old fish, and the bacterial composition differed significantly between them. γ‐Proteobacteria and Pseudoalteromonas supplied the most abundant phylum and genus in the 12‐d‐old fish intestine. However, in the 1‐yr‐old fish intestine, Firmicutes and Clostridium were the most dominant, respectively. The study may contribute to a better understanding of gut microbiota and dynamics of the large yellow croaker and the relationship with their surrounding environment.     

基于16SrDNA比较研究混养三角帆蚌和鲢鳙对池塘养殖水体微生物群落结构的影响

为了研究混养三角帆蚌(Hyriopsis cumingii)和鲢(Hypophthalmichthys molitrix)鳙(Aristichthys nobilis)对池塘养殖水体微生物群落结构的影响,分别在浙江海盐、江西九江、上海浦东开展了罗氏沼虾-三角帆蚌混养、草鱼—三角帆蚌混养、罗氏沼虾-三角帆蚌-鲢鳙鱼混养三组系列实验。利用DGGE技术对养殖水体的16S r DNA进行指纹图谱分析。三组实验共鉴定出57条不同条带,每组实验中混养三角帆蚌或鲢鳙实验组的平均条带数均高于单养水体水样平均条带数。Shannon多样性指数分析结果显示,各养殖水体混养三角帆蚌、鲢鳙后,多样性指数均有所增加。PCA分析和DGGE聚类结果显示,在无鲢鳙的情况下,蚌与主养物种(罗氏沼虾、草鱼)混养实验组的16S r DNA图谱聚为一类,显示出蚌对于微生物群落结构影响的贡献;在同时引入鲢鳙和蚌的情况下,16S r DNA指纹图谱则是按照有无鲢鳙聚为两类,提示在本实验中鲢鳙对于水体微生物的影响要大于蚌。对DGGE图谱其中20条显著条带进行回收、测序和系统发育分析,结果表明,所获序列主要分属于放线菌门(Actinobacteria,25%)、蓝藻门(Cyanobacteria,25%)、α-变形菌亚门(Alphaproteobacteria,15%)、β-变形菌亚门(Betaproteobacteria,15%)、γ-变形菌亚门(Gammaproteobacteria,10%)、ε-变形菌亚门(Epsilonproteobacteria,5%)、裸藻叶绿体(Euglenales,5%)。     

Characterization of Nitrate-Reducing and Amino Acid–Using Bacteria Prominent in Nitrotoxin-Enriched Equine Cecal Populations

Horses can be at risk for nitropoisoning by consuming plants containing 3-nitro-1-propionic acid or 3-nitro-1-propanol and to a lesser extent by plants containing nitrate. Populations of equine cecal microbes enriched for enhanced rates of 3-nitro-1-propionic acid (NPA) or nitrate metabolism were cultured for NPA- or nitrate-metabolizing bacteria on basal enrichment medium or tryptose soy agar supplemented with either 5-mM NPA or nitrate and under H₂:CO₂ (20:80) as the energy source. After 72 hours, separated colonies picked from plates, or roll tubes were cultured in fresh broth medium for 72 hours and then identified by 16S rRNA gene sequencing. Isolates from the NPA-enriched populations were identified as Streptococcus lutetiensis (five strains), Escherichia coli (two strains), and Sporanaerobacter acetigens (one strain). Strains isolated from nitrate-enriched populations were identified as Escherichia coli (one strain) and Wolinella succinogenes (three strains). None of these strains degraded NPA. Enriched populations of equine cecal microbes, the isolated pure strains and the type strain of Denitrobacterium detoxificans, a competent NPA-metabolizing microbe, were examined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis indicated that none of the strains in the enriched population of equine cecal microbes were similar to D. detoxificans. However, we report for the first time the isolation of the anaerobic amino acid–using Sporanaerobacter acetigenes from the equine cecum.     

Manipulation of rumen fermentation and ecology of swamp buffalo by coconut oil and garlic powder supplementation

This experiment was conducted to investigate the effect of coconut oil and garlic powder supplementation on digestibility of nutrients, rumen fermentation, rumen ecology, rumen microorganism and methanogen diversity. Four, 3-year old, rumen fistulated swamp buffalo bulls were randomly assigned in a 4 × 4 Latin square design to receive four dietary treatments; 7% coconut oil, 7% coconut oil with 50 g/day of garlic powder, 7% coconut oil with 100 g/day of garlic powder and non-supplemented (control). During the experiment, concentrate was offered at 0.5% of BW while rice straw was given on ad libitum basis. It was found that supplementation of 7% coconut oil had significantly influenced on total DM intake, OM, NDF and ADF digestibilites while supplementation of 7% coconut oil with garlic powder (50 and 100 g/day) were not significantly different when compared with the control. Dietary supplementations did not affect on rumen pH, NH₃–N concentrations. Blood urea nitrogen (BUN) was significantly higher in supplemented groups. Total VFA concentration, proportion of acetate and acetate to propionate ratio were reduced by supplementation. Proportion of propionate was increased (P < 0.05) when supplemented with 7% coconut oil and 7% coconut oil with 100 g/day of garlic powder. Methane production was dramatically reduced (P = 0.005) in supplemented treatments and was 10% reduced in 7% coconut oil supplementation. Amylolytic and proteolytic bacteria were increased (P = 0.007 and P = 0.024) while protozoal population decreased by 68–75% (P < 0.01) by supplementation. Total bacterial population was increased by supplementation while total fungi and total methanogens were not significantly different among treatments. Percentage of cellulolytic bacterial population was not different among treatments. However, dietary supplementation reduced (P < 0.001) percentage of F. succinogenes population. However, methanogen diversity was not changed using PCR–DGGE as technique. Thirteen separate bands were found in the DGGE gel. Based on this study, supplementation with 7% coconut oil plus 100 g/day of garlic powder could be efficiently utilized in the rumen and thus, could provide good fermentation end products and improve rumen ecology for the host swamp buffaloes particularly in reducing 9% methane gas production without changing nutrient digestibilities.     

土壤微生物多样性的分子生态学研究方法

传统的平板培养法分离培养和鉴定土壤微生物只能反映极少数微生物的信息,种类只占土壤微生物种类总数的0.1%～1%,分子生态学方法应用于土壤微生物的鉴定显示出极大的优越性.着重阐述了土壤微生物多样性的研究内容、意义及目前的采用分子生态学的方法研究土壤微生物多样性,尤其以DGGE（denaturing gradient gel electrophoresis）分子生物学技术以及RAPD（Random amplified polymorphic DNA ）随机扩增的多态性分析方法更为精确和快速,为土壤微生物多样性研究提供了一个更加广阔的前景.