Monitoring of phytoplankton community structure using terminal restriction fragment length polymorphism (T-RFLP)

To establish molecular monitoring for the phytoplankton community in aquatic ecosystems, we analysed the terminal restriction fragment length polymorphism (T-RFLP) of small subunit ribosomal RNA gene (18S rDNA) sequences of nuclear genomes from the algal strains of culture collections and environmental samples of two freshwater reservoirs (Sangcheon reservoir and Seoho reservoir, Korea). Terminal restriction fragment (T-RF) length database was also constructed from twelve strains of algal culture collections to annotate and identify the phytoplankton species from T-RFLP profiles. Algal species in reservoirs were identified and monitored through the colony sequencing and T-RF length patterns of 18S rRNA. In this study, 41 unique clones were identified from two reservoirs including Chlorophyta, Cryptophyta, and Alveolata. In the case of Cryptomonas sp., we found significant linear relationships between T-RF peak areas and biovolumes by cell counting. Our results suggest that T-RFLP analysis can be a fast and quantitative monitoring tool for species changes in phytoplankton communities.     

洋河水库浮游植物组成及优势种演替规律研究

在洋河水库设置6个采样点, 对浮游植物进行了周年研究, 并在夏季进行了每周一次的加密采样, 以揭示水华期间藻类优势种演替规律。结果表明洋河水库全年共检测到浮游植物8门41属49种, 群落季节变化与温度密切相关。春季隐藻门的啮齿隐藻(Cryptomonas erosa)为优势, 夏季初期表层水温在25℃以下时, 绿藻门的波吉卵囊藻(Oocystis borgei)占主要优势; 当表层水温升至25℃以上, 微囊藻(Microcystis spp.)迅速取代其成为绝对优势。秋季硅藻门的克洛脆杆藻(Fragiaria crotomensis)和隐藻门的啮齿隐藻(C. erosa)为优势。空间分布上水库北部浅水区域隐藻和硅藻生物量普遍高于南部; 受东南风影响, 蓝藻生物量在西洋河口S2点位最大。CCA分析表明夏季水华主体微囊藻的生物量与氮浓度正相关, 螺旋鱼腥藻在夏季仅作为第二优势种短暂出现于西洋河口处, 其出现与否受到磷营养盐的限制。       

Targeted deep sequencing reveals high diversity and variable dominance of bloom-forming cyanobacteria in eutrophic lakes

Cyanobacterial blooms in eutrophic lakes are severe environmental problems worldwide. To characterize the spatiotemporal heterogeneity of cyanobacterial blooms, a high-throughput method is necessary for the specific detection of cyanobacteria. In this study, the cyanobacterial composition of three eutrophic waters in China (Taihu Lake, Dongqian Lake, and Dongzhen Reservoir) was determined by pyrosequencing the cpcBA intergenic spacer (cpcBA-IGS) of cyanobacteria. A total of 2585 OTUs were obtained from the normalized cpcBA-IGS sequence dataset at a distance of 0.05. The 238 most abundant OTUs contained 92% of the total sequences and were classified into six cyanobacterial groups. The water samples of Taihu Lake were dominated by Microcystis, mixed Nostocales species, Synechococcus, and unclassified cyanobacteria. Besides, all the samples from Taihu Lake were clustered together in the dendrogram based on shared abundant OTUs. The cyanobacterial diversity in Dongqian Lake was dramatically decreased after sediment dredging and Synechococcus became exclusively dominant in this lake. The genus Synechococcus was also dominant in the surface water of Dongzhen Reservoir, while phylogenetically diverse cyanobacteria coexisted at a depth of 10 m in this reservoir. In summary, targeted deep sequencing based on cpcBA-IGS revealed a large diversity of bloom-forming cyanobacteria in eutrophic lakes and spatiotemporal changes in the composition of cyanobacterial communities. The genus Microcystis was the most abundant bloom-forming cyanobacteria in eutrophic lakes, while Synechococcus could be exclusively dominant under appropriate environmental conditions.     

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.     

Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.     

INTRASPECIFIC COMPARISONS OF NITRATE UPTAKE IN THREE MARINE DIATOMS1

Short-term (within 5 min) and long-term (≤2 h) rates of nitrate uptake were determined for the marine diatoms, Nitschiella longissima (Cleve), Skeletonema costatum (Greville) Cleve and Asterionella japonica (Cleve). Pigment levels, cell carbon, nitrogen and cell volume were also determined for cells in the logarithmic and stationary phases of growth. For each species, one clone isolated from oligotrophic coastal water and one clone isolated from eutrophic coastal water were compared. Long-term NO₃^? uptake typically followed saturation kinetics describable by the Michaelis-Menten expression. Under experimental conditions, half-saturation constants ranged from 0.6 to 2.2 μM NO₃^?. Generally, the oligotrophic clones had lower Ks and V_max (on a per cell basis) than their eutrophic counterparts, though this was only statistically significant in one pair of clones. Eutrophic and oligotrophic clones also differed in their short-term response to nutrient addition; oligotrophic clones showed greatest rate of uptake at the lowest nitrate addition while uptake by eutrophic clones increased with increasing nitrate concentration. However, all clones had very similar V_max values expressed on a dry weight basis. Under N-starvation, cellular C and pigment levels (and N to a lesser extent) generally declined more in eutrophic than in oligotrophic clones. While the differences between inshore and offshore clones were not great, the results are consistent with the hypothesis that eutrophic waters support algae which grow faster and are less conservative biochemically than cells in oligotrophic waters.     

东北旱田土壤中Anabaena伴生细菌的分离与鉴定

[背景]鱼腥藻(Anabaena)在农田土壤中广泛分布,具有固碳和固氮功能。明确伴生细菌与蓝细菌的关系,对提高农田土壤中Anabaena的功能具有重要意义。[目的]从东北不同旱田土壤中分离Anabaena sp.PCC7120的伴生细菌,初步鉴定伴生细菌的分类归属,推测伴生细菌的功能,为明确旱田土壤蓝细菌与伴生细菌的关系提供数据支撑。[方法]采用平板分离、PCR-DGGE、克隆测序技术测定并分析不同旱田土壤中伴生细菌的16S rRNA基因序列,确定伴生细菌的分类地位。[结果]PCR-DGGE图谱显示东北旱田14个土样中分离获得Anabaena sp.PCC7120伴生细菌数量和种类不同;PCR-克隆测序获得伴生细菌的16S rRNA基因序列37条,可鉴定到种水平的菌株36条,主要归为鞘氨醇盒菌属(Sphingopyxis)、贪噬菌属(Variovorax)、黄杆菌属(Flavobacterium)和红球菌属(Rhodococcus)等,推测这些伴生细菌具有适应寡营养、富集微量元素、清除毒素等功效。[结论]东北旱田不同土壤中Anabaena sp.PCC7120伴生细菌种类和数量各异,这...     

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Distribution of Macrophytes in Relation to Environmental Factors in the Ter River,N. E. Spain

Most of the tested water quality factors increase downstream coinciding with changes in the aquatic vegetation, from typical species of fast-flowing oligotrophic waters to those of slower-flowing eutrophic waters. Several statistic analyses were carried out to relate physical and chemical parameters with plant distribution. Five vegetation types are found in a longitudinal distribution along the river system. They are dominated by bryophytes in the initial parts before the reservoirs and by higher plants in the final parts after the reservoirs.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Identification and Phylogenetic Analysis of Toxigenic Cyanobacteria by Multiplex Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA PCR was used to generate unique and identifying DNA profiles for members of the cyanobacterial genera Anabaena and Microcystis, which are responsible for much of the production of nuisance blooms in various freshwater systems, including recreational and drinking water supplies. A method based on the combination of two 10-mer oligonucleotides in a single PCR was developed to provide specific and repeatable DNA fingerprints for cyanobacterial isolates. The strain-specific randomly amplified polymorphic DNA profiles made it possible to discriminate among all toxigenic cyanobacteria studied to the three taxonomic levels of genus, species, and strain. Analysis of DNA typing results obtained by the described method clearly distinguishes between the genera Anabaena and Microcystis. The markers produced for each strain were also applied to a phylogenetic analysis to infer genetic relatedness in this group of prokaryotes.     

北京市各种水体蓝细菌物种多样性研究

2016至2018年的5月和9月研究了北京市五大水系53个水体蓝细菌的物种多样性。结果表明,共发现蓝细菌4目4科22属63种,优势种(IRI)为小型色球藻(Chroococcus minor(Kütz.) N?g.)、湖泊鞘丝藻(Lyngbya limentica Lemm.)、水华束丝藻(Aphanizomenon flos-aquae(L.) Rafls.)、小席藻(Phormidium tende(Menegh.) Gom.)、钝顶节旋藻(Arthrospira platensis)、小颤藻(Oscillatoria tenuis Ag.)、铜绿微囊藻(Microcystis aeruginosa)、水华微囊藻(Microcystis flos-aquae)。通过对北京各种水体的蓝藻优势种进行CCA分析,得出大部分优势种与活性磷、叶绿素a、温度、溶解氧、总碱度、总硬度、pH呈正相关,与TN、TP呈负相关,与蓝藻的特性较符合,其中活性磷是限制蓝细菌生长的重要因素。各水体蓝藻平均密度为1.32×10~6 ind/L,平均生物量为0.003 2 mg/L,多样性指数平均为0.45,2018年较2017年多数水体的密度和生物量下降,多样性指数上升,说明北京市水环境治理有所改善,但仍需继续加强治理。     

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota

Various molecular-biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.     

广东省大中型供水水库营养现状及浮游生物的响应

于2000年调查了广东省18座大中型供水水库的水质现状并探讨了浮游生物对营养水平的响应。总氮、总磷、透明度和叶绿素a分别为0．15～7.15mg／L、0．003～0．387mg／L、0．4～6．3m和0．6～32．3ug／L。总氮、总磷、透明度均与叶绿素a呈较高的相关性。根据这4个因子的综合加权营养状态指数为23．7～55．1,季节差异不大,大多数水库处于中营养状态。金藻在中-富及富营养型水库中没有分布,而蓝藻、绿藻、硅藻和甲藻在调查水库中均有比较广的营养生态位,但它们的密度及相对优势度在各营养型水库中有一定的差异。高营养水平水库有较高的细胞密度和叶绿素a含量。营养水平较低的水库浮游植物以硅藻-甲藻、硅藻-绿藻或金藻-硅藻为主;营养水平较高的水库以蓝藻-硅藻或蓝藻-绿藻为主,并有较高的裸藻密度。浮游动物基本上以桡足类为优势种群,但在中-富营养和富营养型水库中,哲水蚤种类比低营养型水库中少。枝角类优势种类在各营养型水库差别不大。轮虫对水体营养水平的响应相对比较显著。低营养水平水库的轮虫以广营养型、中营养型或寡中营养型种类为主,种类数目比较少;富营养和中-富营养型水库的轮虫以喜在中营养到富营养条件下生长的种类为主,且轮虫种类数目比较多。     

Molecular identification of fecal pollution sources in water supplies by host-specific fecal DNA markers and Terminal Restriction Fragment Length Polymorphism profiles of 16S rRNA gene

Specific fecal DNA markers were investigated for major pollution sources, cow, human, and pig, and occurrence of the identified markers was analyzed in river waters using Terminal Restriction Fragment Length Polymorphism (T-RFLP) techniques and sequencing of 16S rDNA of Bacteroides-Prevotella. The unique and specific DNA markers for cow and human were identified as a 222 bp and 60 bp peak in HaeIII T-RFLP profiles, respectively, and the pig-specific marker was not identified but the unique T-RFLP profile of pig could be used as a substitution. Human-specific marker was detected in most of the river waters tested (92.1%) and T-RFLP profiles of river waters were shown to be similar to those of human feces. Cluster analysis of T-RFLP data showed that the fecal sources were multiple (human plus cow and human plus dairy cow) in most of the river waters. The phylogenetic analysis for the clones recovered from the fecal and water samples showed that the clones from cow formed a discreet cluster from those of other sources. The other clones from human, pig, and river water formed two groups all together. The results of this study could be used to identify and control the fecal pollution source in the bodies of water in Korea.     

Ecological development and genetic diversity of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> from artificial reservoir in Russia

Microcystis aeruginosa is a well-known Cyanobacterium responsible for the formation of toxic water blooms around the world. Shallow, warm, and eutrophic reservoirs provide the most favourable conditions for M. aeruginosa development. Numerous studies have been devoted to this species, but there still is a necessity to develop additional approaches for the monitoring of cyanobacteria in reservoirs. In this study, M. aeruginosa in the water column of a hypereutrophic Siberian reservoir was investigated by fluorescence, light, and electron microscopy as well as genetic analysis using a mcyE marker. Here, we demonstrate the genetic diversity and features of the fluorescence spectra for different ecotypes of this species. We suggest that a fluorescence approach can be used to identify M. aeruginosa in a natural environment in order to increase the effectiveness of ecological monitoring and water quality evaluation.     

Toxic cyanobacteria (blue-green algae) in Finnish fresh and coastal waters

A survey of the occurrence of toxic blooms of cyanobacteria in Finnish fresh and coastal waters was made during 1985 and 1986. Toxicity of the freeze-dried water bloom samples was tested by mouse-bioassay (i.p.). Forty-four per cent (83/188) of the bloom samples were found to be lethally toxic. Hepatotoxic blooms (54) were almost twice as common as neurotoxic ones (29). Anabaena was the most frequently found genus in toxic and non-toxic blooms and it was present in all neurotoxic samples. Statistical associations were found between hepatotoxicity and incidence of Microcystis aeruginosa, M. viridis, M. wesenbergii, Anabaena flos-aquae and Anabaena spiroides. Neurotoxicity was statistically associated with Anabaena lemmermannii, Anabaena flos-aquae and Gomphosphaeria naegeliana. Isolation of strains of cyanobacteria confirmed the occurrence of hepatotoxic and neurotoxic strains of Anabaena, as well as hepatotoxic strains of Microcystis and Oscillatoria species.Toxic blooms caused cattle poisonings at three different lakes during the study period. Toxic blooms also occurred in drinking water sources. Our study shows that toxic cyanobacteria are more common in Finnish lakes than would be expected on the basis of animal poisonings. The results of this study show the existence of toxic cyanobacteria in Finnish water supplies and the need for their continued study as agents of water based disease.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Genetic diversity of cyanobacteria in four eutrophic lakes

Recent studies indicate genetic diversity of cyanobacteria in eutrophic lakes is not represented well by culture collections or morphology. Yet, few studies have investigated genetic richness and evenness of cyanobacteria using culture-independent methods. We compared the genetic structure of cyanobacteria supported by four neighboring eutrophic lakes during the ice-free season. The partial phycobilincpcB/A genes plus intergenic spacer (PC-IGS) was used as a genetic marker.Sequences were phylogeneticallygrouped by maximum likelihood into genotypes representing sub-genera of the major taxa. Genotypes fell into genera commonly observed by microscopy in these lakes including Microcystis, Aphanizomenon, Chroococcus, Anabaena, and Cylindrospermopsis. Only three genotypes were shared among all four lakes, despite significant water flowage between lakes.A Parsimony P-test indicated lakes were significantly (p=0.01) clustered on the maximum likelihood tree. Pairwise differences using Unifrac distance were moderately or not significant. Analysis of molecular variance (AMOVA) indicated genetic variation among all genotypes (φ=0.06, p<0.001) and 94% of variability occurred within lakes rather than between lakes (6%), explaining the lack of pairwise differences between lakes. Lorenze curves of genotype abundance in each lake showed genetic structure was only moderately uneven (Gini coefficients of 0.37-0.5) indicating lakes did not support dominant genotypes. Overall, results from this study suggest diversity of cyanobacteria is shaped by heterogeneity within lakes (temporally or spatially) and relatively even population structures.     

Fecal microbial diversity in a strict vegetarian as determined by molecular analysis and cultivation

Fecal microbial diversity in a strictly vegetarian woman was determined by the 16S rDNA library method, terminal restriction fragment length polymorphism (T-RFLP) analysis and a culture-based method. The 16S rDNA library was generated from extracted fecal DNA, using bacteria-specific primers. Randomly selected clones were partially sequenced. T-RFLP analysis was performed using amplified 16S rDNA. The lengths of T-RF were analyzed after digestion by HhaI and MspI. The cultivated bacterial isolates were used for partial sequencing of 16S rDNA. Among 183 clones obtained, approximately 29% of the clones belonged to 13 known species. About 71% of the remaining clones were novel "phylotypes" (at least 98% similarity of clone sequence). A total of 55 species or phylotypes were identified among the 16S rDNA library, while the cultivated isolates included 22 species or phylotypes. In addition, many new phylotypes were detected from the 16S rDNA library. The 16S rDNA library and isolates commonly included the Bacteroides group, Bifidobacterium group, and Clostridium rRNA clusters IV, XIVa, XVI and XVIII. T-RFLP analysis revealed the major composition of the vegetarian gut microbiota were Clostridium rRNA subcluster XIVa and Clostridium rRNA cluster XVIII. The dominant feature of this strictly vegetarian gut microbiota was the detection of many Clostridium rRNA subcluster XIVa and C. ramosum (Clostridium rRNA cluster XVIII).