Molecular analysis of bacterial community structure and diversity in unimproved and improved upland grass pastures

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector's curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the alpha subdivision of the class Proteobacteria (alpha-proteobacteria) in improved soils.     

nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea,an oligonitrophilic perennial grass

Rhizosphere associative dinitrogen fixation could be a valuable source of nitrogen in many nitrogen limited natural ecosystems, such as the rhizosphere of Molinia coerulea, a hemicryptophytic perennial grass naturally occurring in contrasted oligonitrophilic soils. The diversity of the dinitrogen-fixing bacteria associated with this environment was assessed by a cloning-sequencing approach on the nifH gene directly amplified from environmental DNA extracts. Seventy-seven randomly picked clones were analysed. One type of NifH sequence was dominant in both roots and surrounding soil, and represented 56% of all retrieved sequences. This cluster included previously described environmental clones and did not contain any NifH sequences similar to cultivated diazotrophs. The predominance of few NifH sequence types in the roots and the rhizosphere of Molinia coerulea indicate that the plant environment mediates a favourable niche for such dinitrogen-fixing bacteria.     

Zooplankton abundance and diversity in Central Florida grass carp ponds

The effect of the Asian grass carp (Ctenopharyngodon idella Val.) upon the zooplankton in three adjacent experimental ponds (0.139 ha each) was studied for one year. The ponds contained nine species of aquatic macrophytes. Grass carp were stocked into Pond 1 (65 per ha) and Pond 2 (611 per ha) three months after the study was started. At the time of stocking, physichochemical and biological parameters were similar among the ponds.Grass carp did not affect water quality and had little direct or indirect effect upon the zooplankton in the ponds. The abundance and species diversity of zooplankton (number of species, Shannon Index, and Simpson Index) were not significantly different (P < 0.05) between ponds prior to grass carp stocking. After stocking with grass carp, the number of species and species diversity were found to be significantly different (P < 0.05) between Pond 1 (low grass carp stocking rate) and Pond 2 (high grass carp stocking rate). Additionally, significant differences (P < 0.05) in populations between ponds were found for Lecane luna, Monostyla bulla, Lepadella ehrenbergii, Polyarthra sp., and Synchaeta sp. Temporal variation rather than grass carp was probably responsible for those differences. The number of zooplankton per group (Rotifera, Cladocera, and Copepoda) and species diversity were not significantly different (P<0.05) between ponds containing different stocking rates of grass carp.During the study, zooplankton were collected with a shallow-water sampler. No significant differences (P < 0.05) between collections were found for the sampler and a No. 20 mesh nylon zooplankton net.     

稻纵卷叶螟肠道细菌群落结构与多样性分析

【目的】为明确水稻主要害虫稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)幼虫肠道细菌的群落结构和多样性。【方法】利用Illumina Mi Seq技术对稻纵卷叶螟4龄幼虫肠道细菌的16S r DNA V3-V4变异区序列进行测序,应用USEARCH和QIIME软件整理和统计样品序列数和操作分类单元(operational taxonomic unit,OTU)数量,分析4龄幼虫肠道细菌的物种组成、丰度和多样性。【结果】稻纵卷叶螟4个数量不同的4龄幼虫样本(1,2,3和5头)共得165 386条reads,在97%相似度下可将其聚类为604个OTUs。总共注释到22个门,43个纲,82个目,142个科,204个属,244个种。其中在门水平上,主要优势菌为变形菌门(Proteobacteria)(相对丰度26%~34%)和放线菌门(Actinobacteria)(23%~32%);在纲水平上,主要优势菌为放线菌纲(Actinobacteria)(相对丰度23%~32%)、酸杆菌纲(Acidobacteria)(9%~11%)、α-变形菌纲(Alphaproteobacteria)(10%~13%)、β-变形菌纲(Betaproteobacteria)(6%~8%)和γ-变形菌纲(Gammaproteobacteria)(6%~12%);在科水平上,共有优势菌为类诺卡氏菌科(Nocardioidaceae)、丛毛单胞菌科(Comamonadaceae)、黄单胞菌科(Xanthomonadaceae)、鞘脂单胞菌科(Sphingomonadaceae)和芽单胞菌科(Gemmatimonadaceae)等。在属水平上,4个样本前5位优势属中,类诺卡氏属Nocardioides和鞘脂单胞菌Sphingomonas为共有优势属。稻纵卷叶螟肠道细菌Simpson指数、Shannon指数、Ace指数和Chao指数分别为0.16~0.65,0.94~3.22,212~488和210~490。【结论】稻纵卷叶螟幼虫肠道细菌多样性比较丰富,个体间微生物群落结构和多样性有差异。不同数量样本数据与总体数据有助于综合反映稻纵卷叶螟种群肠道微生物状况。本研究结果为进一步研究稻纵卷叶螟肠道微生物的功能及其在防治中的应用奠定了基础。     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Intensive culture of grass carp and hybrid grass carp larvae

Intensive culture of grass carp and hybrid grass carp (Ctenopharyngodon idella x Aristichthys nobilis ) larvae was conducted under Florida (U.S.A.) conditions. The influence of different rearing facilities (indoor tanks, outdoor tanks and cages) and different methods of fish feeding using live zooplankton and artificial food was tested. High survival (86–100%) and satisfactory grass carp growth (47–56 mg) were obtained in the outdoor tanks and cages during the 10–day experiment. It is believed, that the technique described can be used after some improvements for commercial–scale grass carp larvae rearing. Low survival (1–3%) was obtained in the hybrid culture experiment even though satisfactory growth rates (84–212 mg) were obtained after 13 days. High mortality was attributed to genetic abnormalities caused by hybridization. The hybrid does not appear to be a promising fish for culture.     

Fermented liquid feed enhances bacterial diversity in piglet intestine

Because of limitations imposed on the antibiotic use in animal industry, there is a need for alternatives to maintain the efficiency of production. One of them may be the use of fermented liquid feed (FLF) but how it affects gut ecology is poorly understood. We investigated the effect of three diets, standard dry feed (control), dry feed supplemented with antibiotics, and fermented liquid feed (FLF, fermented with Lactobacillus plantarum), on gut bacterial diversity in piglets. The structure of the ileal and caecal communities was estimated by sequencing the SSU rRNA gene libraries. Antibiotic-supplemented feed slightly increased bacterial diversity in the ileum but reduced it in the caecum while in FLF-fed animals bacterial diversity was elevated. The majority of bacterial sequences in the ileum of all three groups belonged to lactobacilli (92–98%). In the caecum the lactobacilli were still dominant in control and antibiotic-fed animals (59% and 64% of total bacterial sequences, respectively) but in FLF-fed animals they fell to 31% with the concomitant increase in the Firmicutes diversity represented by the Dorea, Coprococcus, Roseburia and Faecalibacterium genera. Thus FLF affects the gut ecology in a different way than antibiotics and contributes to the enhanced bacterial diversity in the gastrointestinal tract.     

草鱼、鲢和瓦氏黄颡鱼幼鱼感应流速的比较

在21±1℃水温下,使用自行制作的鱼类游泳行为测试槽,采用流速递增的方法测定了草鱼(Ctenopharyngodon idellus)(体长9.64±3.12 cm,体重20.97±16.71 g)、鲢(Hypophthalmichthys molitrix)(体长10.60±3.45 cm,体重24.37±18.61 g)和瓦氏黄颡鱼(Pelteobagrus vachelli)(体长11.54±1.46 cm,体重20.98±6.94 g)幼鱼个体的感应流速.结果表明:草鱼、鲢和瓦氏黄颡鱼幼鱼的感应流速分别是(8.21±0.68)、(6.74±0.84)和(33.11±3.06) cm·s-1,相对感应流速分别是(0.91±0.22)、(0.67±0.15)和(2.9±0.29) BL·s-1;鲢和草鱼幼鱼的感应流速差异不显著(P＞0.05),但瓦氏黄颡鱼比草鱼和鲢幼鱼感应流速大近4倍,差异显著(P＜0.05);因此,草鱼和鲢幼鱼较瓦氏黄颡鱼幼鱼对流速的感应更敏感.本实验结果可为设计鱼道、确定拖网速度等提供一定的科学指导.     

Stability and change in estuarine biofilm bacterial community diversity

Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain Bacteria in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Fla. Community profiling was achieved by using 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the benthos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T-RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in dissolved oxygen.     

马尾松林节肢动物群落的组成及多样性

在不采取任何营林和治虫措施的皖南九连山林场马尾松纯林中设林分相似的2块样地,面积均为22m×22m。1997年1月～1998年12月,每月1次垂直分层对两样地节肢动物群落的调查表明每样地植食性、捕食性、寄生性昆虫和蜘蛛类群的物种数占该样地总物种数的百分比分别约52%、14%、8%和20%;每样地的益害物种数之比都约为11.2;益害个体数之比是15.7和16.6;相同林间层次的优势目昆虫或优势种蜘蛛相同;每样地的物种数量都是以灌木草本层最多,依次为乔木层、枯枝落叶层和土壤层。两样地乔木层、灌木草本层、土壤层和枯枝落叶层的节肢动物平均个体数百分率分别为39.6%、33.1%、22.8%和4.6%。3～8月物种数和多样性指数较大,5、8月为物种数高峰期。主成分分析显示群落的稳定性较差。     

Microsatellite genetic diversity and differentiation of native and introduced grass carp populations in three continents

Grass carp (Ctenopharyngodon idella), a freshwater species native to China, has been introduced to about 100 countries/regions and poses both biological and environmental challenges to the receiving ecosystems. In this study, we analyzed genetic variation in grass carp from three introduced river systems (Mississippi River Basin in US, Danube River in Hungary, and Tone River in Japan) as well as its native ranges (Yangtze, Pearl, and Amur Rivers) in China using 21 novel microsatellite loci. The allelic richness, observed heterozygosity, and within-population gene diversity were found to be lower in the introduced populations than in the native populations, presumably due to the small founder population size of the former. Significant genetic differentiation was found between all pairwise populations from different rivers. Both principal component analysis and Bayesian clustering analysis revealed obvious genetic distinction between the native and introduced populations. Interestingly, genetic bottlenecks were detected in the Hungarian and Japanese grass carp populations, but not in the North American population, suggesting that the Mississippi River Basin grass carp has experienced rapid population expansion with potential genetic diversification during the half-century since its introduction. Consequently, the combined forces of the founder effect, introduction history, and rapid population expansion help explaining the observed patterns of genetic diversity within and among both native and introduced populations of the grass carp.     

Bacillus subtilis SC02 supplementation causes alterations of the microbial diversity in grass carp water

This study was conducted to evaluate the effects of Bacillus subtilis SC02 supplement on water quality, microbial community diversity and structure in a grass carp (Ctenopharyngodon idellus) culture. Our selected strain, B. subtilis SC02, significantly reduced ammonia, nitrite and total nitrogen levels in water over an extended period compared with the control group. Pyrosequencing showed that the Shannon diversity index (Shannon) and species richness estimators (Chao) of the treatment group were higher, indicating that bacterial richness was significantly increased in the treatment group. The phyla Proteobacteria, Bacteroidetes and Actinobacteria were dominant in the treatment groups, accounting for 45, 21.9 and 21.9 % of the sequence reads, respectively. However, in sharp contrast, the control fishes were predominantly occupied by Proteobacteria (82.1 %) and Firmicutes (8.4 %). At the genus level, the microbial communities were different between the control and treatment groups, although the two groups shared similar genera. Additionally, some genera (such as Tepidimonas, Variovorax, Roseomonas, Rubritepida, Nitrobacter, etc.) only appeared in the treatment group, and many other genera only existed in the control group. Therefore, we conclude that the addition of the SC02 strain in water improves water quality, which may ultimately be a result of changes in microbial community diversity in grass carp cultures.     

Changes in the bacterial community structure and diversity during bamboo retting

Microbial retting is a critical step in obtaining fiber bundles from bamboo culm using indigenous microorganisms. A cultivation-independent technique for monitoring the changes in bacteria community during bamboo retting was applied in this work. This technique involves genetic profiling of PCR-amplified small-subunit rRNA and the single-strand conformation polymorphism (SSCP) gel analysis of the PCR-amplified 16S rDNA fragments. The study revealed that both the structure and the diversity of investigated communities varied with the incubation periods and sample locations. The bacteria bands from SCCP gel profiles related to Bacillus sp. decreased in intensity, and Phaeospirillum sp. and Azospirillum brasilense completely disappeared during the 4(th) and 5(th) month of incubation, while the bands related to the Sphingomonas japonica, Alphaproteobacterium Ellin335 and Microbacterium sp. increased. The bands closely related to Sphingomonads, Brevundimonas brasilense, Pseudoclavibacter sp., Agrococcus jenensis and Oxalophagus oxalicus remained dominant during the whole incubation period. This study showed that the use of PCR assay targeting 16S rRNA and SCCP profiling provided valuable information on monitoring the bacteria dynamic changes occurring in the bacteria community during bamboo retting, which is crucial for controlling the quality of the retting process and improving the retting efficiency, and thus benefits for fiber recovery.     

The relationship between feeding preferences and consumption rates in grass carp and grass carp × bighead carp hybrids

The relationship between food preference hierarchy and consumption rate was examined for the grass carp, Ctenopharygodon idella , and its Fl hybrid (grass carp × bighead carp). Preferences of both types of fish were similar, although hybrids consumed vegetation at a much lower rate. For both, relative preference in feeding trials with mixed plant species was significantly correlated with consumption rate in monospecific feeding experiments. No correlations were found between preference and caloric content, protein content or relative water content. The hypothesis is proposed that preference hierarchies reflect relative handling times of different food types and that these fish maximize 'through-put' by preferentially consuming plant species which can be most rapidly ingested.     

武夷山茶园动物群落的组成结构和多样性

2004～2005年对武夷山茶园动物群落进行系统调查.结果表明,茶园动物隶属于3门、8纲、31目、252科、1188种.其中,植食者450种(占总物种数的37.88%)、捕食者323(占27.19%)、寄生者133种(占11.20%)、腐食者230种(占19.36%)、杂食者52种(占4.38%).在茶园动物群落中,同翅目、疥螨目、柄眼目、鞘翅目为生态优势类群,而柄眼目、直翅目和鳞翅目则为资源优势类群.98.99%的动物物种为稀有种或偶见种,而90.9l%的物种为资源次要种.动物群落及植食者、捕食者、寄生者和腐食者的种一多度关系同时符合对数正态分布和对数序列分布,而杂食者的种一多度关系则符合对数序列分布.动物群落及各营养类群的多样性、丰盛度及均匀性都呈现明显的季节消长规律,且其多样性指数(Shannon-Wiener指数)都与物种丰富度和均匀度一致.多样性指数的大小依次为:捕食性类群>寄牛性类群>总群落>杂食性类群>植食性类群>腐食性类群;均匀度大小则依次为:寄生性类群>杂食性类群>捕食性类群>总群落>植食性类群>腐食性类群.植食者和腐食者作为空间食物种类资源均能够显著促进群落容纳较多的天敌物种,天敌对这两类群的空间数量跟随效应强,而且捕食者、植食者和腐食者的多样性相互促进.     

Composition diversity and nutrition conditions for accumulation of polyhydroxyalkanoate (PHA) in a bacterial community from activated sludge

We investigated the effect of chemical oxygen demand (COD)/N ratio on polyhydroxybutyrate (PHB) accumulating ability in an anaerobic/aerobic cycle sequential batch reactor (SBR). Compared the COD/N ratio of 10, 20, 50, and 125, the COD/N of 125 was the most suitable nutritional proportion. When COD was 1,200 mg/L and COD/N/P was 1,200/9.6/30, activated sludge PHB synthesis reached a maximum of 64.2 % of the dry weight of the cells. The population of the activated sludge was detected periodically by denaturing gradient gel electrophoresis (DGGE). The predominant strains belonged to five genera: Bacteroidetes sp., Acinetobacter sp., Betaproteobacteria sp., Gammaproteobacteria sp., Arcobacter sp., and Bacillus sp. Pyrosequencing analysis of the 16S rRNA gene indicated that the PHB synthesis community was more diverse than that was detected by DGGE, specifically Acidobacteria (12.25 %), Alphaproteobacteria (10.78 %), Actinomycetales (9.68 %), Actinobacteria (5.15 %), Proteobacteria (4.04 %), and unclassified bacteria (24.14 %).     

Molecular characterization of glutathione peroxidase gene from the liver of silver carp, bighead carp and grass carp

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phytoplanktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.