Subseafloor microbial communities associated with rapid turbidite deposition in the Gulf of Mexico continental slope (IODP Expedition 308)

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA , and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 × 10⁷ cells mL⁻¹ or copies g⁻¹ sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin.     

High‐throughput sequencing of fecal DNA to identify insects consumed by wild Weddell's saddleback tamarins (Saguinus weddelli,Cebidae, Primates) in Bolivia

The genus Saguinus represents a successful radiation of over 20 species of small‐bodied New World monkeys. Studies of the tamarin diet indicate that insects and small vertebrates account for ～16–45% of total feeding and foraging time, and represent an important source of lipids, protein, and metabolizable energy. Although tamarins are reported to commonly consume large‐bodied insects such as grasshoppers and walking sticks (Orthoptera), little is known concerning the degree to which smaller or less easily identifiable arthropod prey comprises an important component of their diet. To better understand tamarin arthropod feeding behavior, fecal samples from 20 wild Bolivian saddleback tamarins (members of five groups) were collected over a 3 week period in June 2012, and analyzed for the presence of arthropod DNA. DNA was extracted using a Qiagen stool extraction kit, and universal insect primers were created and used to amplify a ～280 bp section of the COI mitochondrial gene. Amplicons were sequenced on the Roche 454 sequencing platform using high‐throughput sequencing techniques. An analysis of these samples indicated the presence of 43 taxa of arthropods including 10 orders, 15 families, and 12 identified genera. Many of these taxa had not been previously identified in the tamarin diet. These results highlight molecular analysis of fecal DNA as an important research tool for identifying anthropod feeding patterns in primates, and reveal broad diversity in the taxa, foraging microhabitats, and size of arthropods consumed by tamarin monkeys. Am J Phys Anthropol 156:474–481, 2015. © 2014 Wiley Periodicals, Inc.     

基于宏基因组学解析不同质量等级中温大曲微生物组的异质性

【目的】中温大曲是浓香型白酒酿造中的糖化发酵剂和生香剂,目前其质量评价主要依赖于感官和理化特征。已有研究采用扩增子测序初步探索了大曲质量等级与微生物群落组成之间的关联性,但是微生物群落功能对大曲质量的影响尚缺乏深入研究。【方法】本研究采用宏基因组学解析和对比了不同质量等级大曲的微生物群落组成和潜在功能的差异,结合理化性质,聚焦分析了酶活、乳酸和乙酸代谢相关的关键酶基因丰度。【结果】两种等级大曲的真菌群落组成差异显著。一级大曲含有更高相对丰度的丝状真菌,主要分布在曲霉属(Aspergillus, 21.6%)、罗萨氏菌属(Rasamsonia, 6.8%)、拟青霉属(Paecilomyces, 5.0%)和篮状菌属(Talaromyces, 4.4%)等。一级大曲中与环境信息处理和细胞过程相关途径的基因相对丰度普遍显著高于二级大曲。一级大曲中薄层菌属(Hymenobacter)和曲霉属等提供了更高基因丰度的α-淀粉酶,使其具有显著较高的液化力。二级大曲则含有更高相对丰度的横梗霉属(Lichtheimia, 11.8%)、根霉属(Rhizopus, 13.4%)和毕赤酵母属(Pichia, 7.2%)以及乳酸菌类微生物,如伴生乳杆菌属(Companilactobacillus)、魏斯氏菌属(Weissella)和黏液乳杆菌属(Limosilactobacillus)等。二级大曲中与代谢相关途径的基因相对丰度显著较高,如碳水化合物代谢、能量代谢和核苷酸代谢等。二级大曲中较多的乳酸菌类群有利于提高乙醇脱氢酶基因丰度,同时,糖多孢菌属(Saccharopolyspora)、葡萄球菌属(Staphylococcus)、曲霉属和高温放线菌属(Thermoactinomyces)等提供了更多的羧酸酯酶基因丰度,分别使其具有较高的发酵力和酯化力。此外,二级大曲中较多的乳酸菌还可能产生更多的乳酸脱氢酶,降解乳酸,且更高基因丰度的乙酸代谢相关酶类可促进乙酸分解代谢,使得二级大曲酸度显著低于一级大曲。【结论】本研究在基因水平上研究了不同质量等级大曲酶活和酸度差异的微生物基础,可为建立完善的大曲质量评价体系以及理性地调控群落功能提供理论支撑。     

Salt marsh sediment diversity: a test of the variability of the rare biosphere among environmental replicates

Much of the phylogenetic diversity in microbial systems arises from rare taxa that comprise the long tail of taxon rank distribution curves. This vast diversity presents a challenge to testing hypotheses about the effects of perturbations on microbial community composition because variability of rare taxa among environmental replicates may be sufficiently large that it would require a prohibitive degree of sequencing to discern differences between samples. In this study we used pyrosequencing of 16S rRNA tags to examine the diversity and within-site variability of salt marsh sediment bacteria. Our goal was to determine whether pyrosequencing could produce similar patterns in community composition among replicate environmental samples from the same location. We hypothesized that repeated sampling from the same location would produce different snapshots of the rare community due to incomplete sequencing of the taxonomically rich rare biosphere. We demonstrate that the salt marsh sediments we sampled contain a remarkably diverse array of bacterial taxa and, in contrast to our hypothesis, repeated sampling from within the same site produces reliably similar patterns in bacterial community composition, even among rare organisms. These results demonstrate that deep sequencing of 16s tags is well suited to distinguish site-specific similarities and differences among rare taxa and is a valuable tool for hypothesis testing in microbial ecology.     

MAPLE 2.3.0: an improved system for evaluating the functionomes of genomes and metagenomes

MAPLE is an automated system for inferring the potential comprehensive functions harbored by genomes and metagenomes. To reduce runtime in MAPLE analyzing the massive amino acid datasets of over 1 million sequences, we improved it by adapting the KEGG automatic annotation server to use GHOSTX and verified no substantial difference in the MAPLE results between the original and new implementations.     

Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice

This study is the first to use a metagenomics approach to characterize the phylogeny and functional capacity of the canine gastrointestinal microbiome. Six healthy adult dogs were used in a crossover design and fed a low-fiber control diet (K9C) or one containing 7.5% beet pulp (K9BP). Pooled fecal DNA samples from each treatment were subjected to 454 pyrosequencing, generating 503 280 (K9C) and 505 061 (K9BP) sequences. Dominant bacterial phyla included the Bacteroidetes/Chlorobi group and Firmicutes, both of which comprised ∼35% of all sequences, followed by Proteobacteria (13–15%) and Fusobacteria (7–8%). K9C had a greater percentage of Bacteroidetes, Fusobacteria and Proteobacteria, whereas K9BP had greater proportions of the Bacteroidetes/Chlorobi group and Firmicutes. Archaea were not altered by diet and represented ∼1% of all sequences. All archaea were members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in K9C, but none in K9BP. Less than 0.4% of sequences were of viral origin, with >99% of them associated with bacteriophages. Primary functional categories were not significantly affected by diet and were associated with carbohydrates; protein metabolism; DNA metabolism; cofactors, vitamins, prosthetic groups and pigments; amino acids and derivatives; cell wall and capsule; and virulence. Hierarchical clustering of several gastrointestinal metagenomes demonstrated phylogenetic and metabolic similarity between dogs, humans and mice. More research is required to provide deeper coverage of the canine microbiome, evaluate effects of age, genetics or environment on its composition and activity, and identify its role in gastrointestinal disease.     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.     

Development and validation of an experimental life support system for assessing the effects of global climate change and environmental contamination on estuarine and coastal marine benthic communities

An experimental life support system (ELSS) was constructed to study the interactive effects of multiple stressors on coastal and estuarine benthic communities, specifically perturbations driven by global climate change and anthropogenic environmental contamination. The ELSS allows researchers to control salinity, pH, temperature, ultraviolet radiation (UVR), tidal rhythms and exposure to selected contaminants. Unlike most microcosms previously described, our system enables true independent replication (including randomization). In addition to this, it can be assembled using commercially available materials and equipment, thereby facilitating the replication of identical experimental setups in different geographical locations. Here, we validate the reproducibility and environmental quality of the system by comparing chemical and biological parameters recorded in our ELSS with those prevalent in the natural environment. Water, sediment microbial community and ragworm (the polychaete Hediste diversicolor) samples were obtained from four microcosms after 57 days of operation. In general, average concentrations of dissolved inorganic nutrients (NO₃^?; NH₄⁺ and PO₄^?3) in the water column of the ELSS experimental control units were within the range of concentrations recorded in the natural environment. While some shifts in bacterial community composition were observed between in situ and ELSS sediment samples, the relative abundance of most metabolically active bacterial taxa appeared to be stable. In addition, ELSS operation did not significantly affect survival, oxidative stress and neurological biomarkers of the model organism Hediste diversicolor. The validation data indicate that this system can be used to assess independent or interactive effects of climate change and environmental contamination on benthic communities. Researchers will be able to simulate the effects of these stressors on processes driven by microbial communities, sediment and seawater chemistry and to evaluate potential consequences to sediment toxicity using model organisms such as Hediste diversicolor.     

基因芯片数据预处理方法(LnMR和RAln)的评估和比较

【目的】评估并比较两种基因芯片数据预处理方法(LnMR和RAln)。【方法】以西藏高寒草甸草原夏季放牧实验和中国东部农田土壤移栽与玉米种植交互作用实验的两套基因芯片数据为例,利用等级-丰度曲线、均匀度指数、单因素方差分析、Q-Q图、α多样性和响应比等统计方法评估预处理效果。【结果】两种方法均能有效缩小极值和信号差异,改善信号分布,减小随机误差,提高数据正态性,增强实验结果的趋势,使芯片数据更适于进一步统计分析。两种预处理方法各有特点,LnMR法更适合检测不同处理间微生物结构差异,RAln法可以一定程度上消除基因芯片测定的系统误差。【结论】LnMR法和RAln法是两种行之有效的基因芯片预处理方 法,在实际分析中,研究者应根据研究需要合理选择。     

The density and diversity of gymnamoebae associated with terrestrial moss communities (Bryophyta: Bryopsida) in a northeastern U.S. forest

Moss communities are commonly found in temperate forests and form a nearly continuous understory in some high latitude forests. However, little is known about the microbial component of these communities, especially the non-testate amoeboid protists. Fifty morphospecies of naked amoebae were identified in samples collected at eight sites in a northeastern North American forest. The mean number (+/-SE) of morphospecies found per sample site based on laboratory cultures was 17+/-2.1. The density of amoebae expressed as number/g dry weight of moss ranged from 3.5+/-0.04 x 10(3) to 4.3+/-0.2 x 10(4) and was positively correlated with the moss moisture content (r=0.9, P<0.001, df=26). Densities of gymnamoebae in the moss are generally higher than found in the surrounding soil, but this may be due in part to the greater weight of soil per unit volume compared with moss. The percentage of encysted forms was inversely related to the moisture content of the moss sample.     

Fractionation of sediment phosphate with chelating compounds: a simplification, and comparison with other methods

The NTA/EDTA fractionation of sediment-bound, inorganic phosphate was improved and made more effective. The iron-bound phosphate is now extracted with Ca-EDTA plus dithionite and followed by an extraction of calcium-bound phosphate by Nat-EDTA at pH = 4.5. A comparison is made with two other sequential extraction procedures: the SEDEX and the Hieltjes & Lijklema extractions. The EDTA extractions have an advantage over the other two methods.     

A Window to the Subsurface: Microbial Diversity in Hot Springs of a Sulfidic Cave (Kaklik,Turkey)

Caves are windows to the extreme habitats of deep subsurface, and provide answers of unknowns about the underground life. Furthermore, sulfidic caves are important analogues for the early Earth environments, since some environmental conditions are common, such as high sulfur concentration, high temperature and oxygen-poor conditions. Kaklik Cave (Denizli, Turkey) with its travertine formation, carbonate- and sulfur-rich thermal springs, exhibits a unique ecosystem as a sulfidic cave. This study represents the first molecular survey of the microbial community in the Kaklik Cave, Turkey using high-throughput 16S rRNA gene amplicon sequencing analysis. An average of 859–2,416 operational taxonomic units per sample were observed including 25 bacterial phyla and 3 archaeal phyla. The bacterial diversity profiles were generally dominated by Epsilonproteobacteria and Gammaproteobacteria. At the carbonate-rich hot spring, that formed travertine structure, 9.7% of sequence reads affiliated with Thiofaba spp. In contrast, 38.74% of the total sequence reads at the sulfidic hot spring samples associated with the genus Sulfurimonas and Sulfurovum. In the archaeal community composition, Thermoplasmata was the most abundant group in all sampling areas. The 454-pyrotag results provide leads about ammonia-, nitrite- and sulfur-oxidation as well as sulfur-reduction, carbon dioxide fixation, and nitrogen fixation.     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

Metagenomics,paratransgenesis and the Anopheles microbiome: a portrait of the geographical distribution of the anopheline microbiota based on a meta-analysis of reported taxa

Anophelines harbour a diverse microbial consortium that may represent an extended gene pool for the host. The proposed effects of the insect microbiota span physiological, metabolic and immune processes. Here we synthesise how current metagenomic tools combined with classical culture-dependent techniques provide new insights in the elucidation of the role of the Anopheles-associated microbiota. Many proposed malaria control strategies have been based upon the immunomodulating effects that the bacterial components of the microbiota appear to exert and their ability to express anti-Plasmodium peptides. The number of identified bacterial taxa has increased in the current “omics” era and the available data are mostly scattered or in “tables” that are difficult to exploit. Published microbiota reports for multiple anopheline species were compiled in an Excel^® spreadsheet. We then filtered the microbiota data using a continent-oriented criterion and generated a visual correlation showing the exclusive and shared bacterial genera among four continents. The data suggested the existence of a core group of bacteria associated in a stable manner with their anopheline hosts. However, the lack of data from Neotropical vectors may reduce the possibility of defining the core microbiota and understanding the mosquito-bacteria interactive consortium.     

Comparison of responses to alarm calls by patas (Erythrocebus patas) and vervet (Cercopithecus aethiops) monkeys in relation to habitat structure

We studied responses to alarm calls of sympatric patas (Erythrocebus patas) and vervet (Cercopithecus aethiops) monkeys in relation to habitat structure, with the intention of understanding the relationship between the environment and predator avoidance. Patas and vervet monkeys are phylogenetically closely related and overlap in body size. However, while patas monkeys are restricted to nonriverine habitats at our study site, vervets use both nonriverine and riverine habitats, allowing us to "vary" habitat structure while controlling for effects of group size, composition, and phylogeny. Patas monkeys in the nonriverine habitat responded to mammalian predator alarm calls with a greater variety of responses than did vervets in the riverine habitat, but not when compared with vervets in the nonriverine habitat. Ecological measurements confirm subjective assessments that trees in the riverine habitat are significantly taller and occur at lower densities than trees in the nonriverine habitat. Despite the lower density of trees in the riverine habitat, locomotor behavior of focal animals indicates that canopy cover is significantly greater in the riverine than the nonriverine habitat. Differences in responses to alarm calls by the same groups of vervets in different habitat types, and convergence of vervets with patas in the same habitat type, suggest that habitat type can be a significant source of variation in antipredator behavior of primates.     

Bacteria,phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.     

Mitochondrial genome of the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) and comparison with other Lepidopterans

We present the complete sequence of the mitochondrial genome (mitogenome) of the cotton bollworm Helicoverpa armigera. The 15,347-bp mitogenome of H. armigera was arranged in the same order described for all other sequenced lepidopterans, which differs from the most common type found in insects, due to the movement of trnM to a position 5'-upstream of trnI. The gene overlap in the H. armigera mitogenome is totally 23 bp in six locations. The H. armigera mitogenome has a total of 175 bp of intergenic spacer sequences spread over 14 regions ranging in size from 1 to 45 bp. The nucleotide composition of the whole mitogenome of H. armigera is highly A+T biased, accounting for 80.97%, with a slightly positive AT skewness and negative GC skewness, indicating the occurrence of more A than T, C more than G. The protein-encoding genes have typical mitochondrial start codons, except for cox1, which contains the unusual CGA. The cox1, cox2, and nad4 genes have incomplete stop codons (T). The lrRNA and srRNA genes are 1395 and 794-bp long, respectively. All tRNAs have a typical cloverleaf structure of mitochondrial tRNAs, except for trnS1(AGN), the dihydrouridine arm of which could not form a stable stem-loop structure. The H. armigera A+T-rich region contains a conserved structure combining the motif ATAGA and a 19-bp poly-T stretch, but absence of the 9-bp poly-A element upstream of trnM.