Disentangling the assembly mechanisms of ant cuticular bacterial communities of two Amazonian ant species sharing a common arboreal nest

Bacteria living on the cuticle of ants are generally studied for their protective role against pathogens, especially in the clade of fungus‐growing ants. However, little is known regarding the diversity of cuticular bacteria in other ant host species, as well as the mechanisms leading to the composition of these communities. Here, we used 16S rRNA gene amplicon sequencing to study the influence of host species, species interactions and the pool of bacteria from the environment on the assembly of cuticular bacterial communities on two phylogenetically distant Amazonian ant species that frequently nest together inside the roots system of epiphytic plants, Camponotus femoratus and Crematogaster levior. Our results show that (a) the vast majority of the bacterial community on the cuticle is shared with the nest, suggesting that most bacteria on the cuticle are acquired through environmental acquisition, (b) 5.2% and 2.0% of operational taxonomic units (OTUs) are respectively specific to Ca. femoratus and Cr. levior, probably representing their respective core cuticular bacterial community, and (c) 3.6% of OTUs are shared between the two ant species. Additionally, mass spectrometry metabolomics analysis of metabolites on the cuticle of ants, which excludes the detection of cuticular hydrocarbons produced by the host, were conducted to evaluate correlations among bacterial OTUs and m/z ion mass. Although some positive and negative correlations are found, the cuticular chemical composition was weakly species‐specific, suggesting that cuticular bacterial communities are prominently environmentally acquired. Overall, our results suggest the environment is the dominant source of bacteria found on the cuticle of ants.     

Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations > 0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n = 24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.     

Gut microbiota instead of host genotype drive the specificity in the interaction of a natural host-parasite system

Specific interactions between parasite genotypes and host genotypes (G_p × G_h) are commonly found in invertebrate systems, but are largely lacking a mechanistic explanation. The genotype of invertebrate hosts can be complemented by the genomes of microorganisms living on or within the host (‘microbiota’). We investigated whether the bacterial gut microbiota of bumble bees (Bombus terrestris) can account for the specificity of interactions between individuals from different colonies (previously taken as host genotype proxy) and genotypes of the parasite Crithidia bombi. For this, we transplanted the microbiota between individuals of six colonies. Both the general infection load and the specific success of different C. bombi genotypes were mostly driven by the microbiota, rather than by worker genotype. Variation in gut microbiota can therefore be responsible for specific immune phenotypes and the evolution of gut parasites may be driven by interactions with ‘microbiota types’ as well as with host genotypes.     

Molecular characterization of the bacteria adherent to human colorectal mucosa

AIMS: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. METHODS AND RESULTS: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. CONCLUSIONS: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. SIGNIFICANCE AND IMPACT OF THE STUDY: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.     

Plant-microbe interactions: identification of epiphytic bacteria and their ability to alter leaf surface permeability

Bacteria were either isolated from leaf surfaces of Hedera helix or obtained from a culture collection in order to analyse their effect on barrier properties of isolated Hedera and Prunus laurocerasus cuticles. On the basis of the 16S rDNA sequences the genera of the six bacterial isolates from Hedera were identified as Pseudomonas sp., Stenotrophomonas sp. and Achromobacter. Water permeability of cuticles isolated from H. helix was measured before and after inoculation with the six bacterial strains. In addition water permeability of cuticles isolated from P. laurocerasus was measured before and after inoculation with the three bacterial strains Pseudomonas aeruginosa, Xanthomonas campestris and Corynebacterium fascians. Rates of water diffusing across isolated cuticles of both species significantly increased by up to 50% after inoculation with all bacterial strains. Obtained results show that epiphytic bacteria have the ability of increasing water permeability of Hedera and Prunus cuticles, which in turn should increase the availability of water and dissolved compounds in the phyllopshere. Consequently, living conditions in the habitat phyllosphere are improved. It can be concluded that the ability to change leaf surface properties will improve epiphytic fitness of leaf surface bacteria.     

空气环境中几种细菌检测与16S rRNA测序鉴定

采集空气环境微生物,根据菌落和革兰氏染色特征,分离5株细菌。提取分离株DNA,采用设计的16SrRNA引物扩增DNA片段;纯化PCR产物,进行测序反应并再纯化,上机读序。拼接与校对DNA序列,在NCBI网站进行Blast,鉴定细菌型别。5株菌株分别是表皮葡萄球菌、头状葡萄球菌、施氏假单胞菌、溶血葡萄球菌和大肠杆菌;其中溶血葡萄球菌是致病菌,其他4株是条件致病菌。这提示,应警惕空气环境中致病菌的感染。     

Combined use of 16S ribosomal DNA and automated ribosomal intergenic spacer analysis to study the bacterial community in catfish ponds

AIMS: To apply culture-independent techniques to explore the bacterial community composition in catfish pond water. METHODS AND RESULTS: 16S rDNA libraries were constructed and sequenced from 15 pond water samples. Automated ribosomal intergenic spacer analysis (ARISA) was used to fingerprint each bacterial community. A broad diversity in bacterial species composition was found by 16S rDNA analysis. Alphaproteobacteria was the most represented class in all ponds, followed by Gammaproteobacteria and Gram-positive high G + C content bacteria. Uniqueness of bacterial communities from each individual pond was confirmed by ARISA. Catfish pathogens were detected sporadically. CONCLUSIONS: Bacterial communities in a catfish aquaculture setting can vary from pond to pond at one given point. No correlation could be made between bacteria composition and fish strain or between bacterial profile and the presence of catfish pathogens in a particular pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the composition of bacterial communities in catfish ponds. Fish health specialists and catfish aquaculture managers should be aware of the wide differences in bacterial communities between ponds and include this variable in fish husbandry practices.     

A diet containing native or fermented wheat bran does not interfere with natural microbiota of laying hens

Wheat bran (WB) is an important side product of the milling industry and can serve as dietary fiber compound for monogastric animals. The aim of this study was to evaluate the influence of native or fermented WB on the gut physiology and microbiology of laying hens. To accomplish this, 24 laying hens were fed the following diets: conventional diet without WB; 15% native WB in the diet; 15% WB fermented with Pleurotus eryngii; and 15% WB fermented with P. eryngii and a lactic acid bacterial culture. Immediately after slaughtering, digesta samples were taken from the jejunum, ileum and cecum, respectively. Total DNA was extracted and subsequently investigated with 16S DNA amplicon sequencing. Neither native nor fermented WB supplementations negatively affected the feed conversion ratio, laying performance or the relative abundances and alpha-diversity of microbiota in the intestine. Effects of WB-based diets on gut morphology were only recognized in the jejunum (reduced villum height and mucosa thickness). Likewise, WB supplementation decreased the digestibility of DM and starch. Based on these findings, it was demonstrated that different WB variants are applicable without exerting practically negative consequences on performance or on gut microbiota. Fermentation improved the digestibility/retention of dietary fat and phosphorus. However, no further beneficial effects were observed. This study also allowed a more in-depth view on the laying hens’ gut microbiome and its variation within the gut segments.     

Geographical and ecological stability of the symbiotic mid‐gut microbiota in European firebugs,Pyrrhocoris apterus (Hemiptera,Pyrrhocoridae)

Symbiotic bacteria often play an essential nutritional role for insects, thereby allowing them to exploit novel food sources and expand into otherwise inaccessible ecological niches. Although many insects are inhabited by complex microbial communities, most studies on insect mutualists so far have focused on single endosymbionts and their interactions with the host. Here, we provide a comprehensive characterization of the gut microbiota of the red firebug (Pyrrhocoris apterus, Hemiptera, Pyrrhocoridae), a model organism for physiological and endocrinological research. A combination of several culture‐independent techniques (454 pyrosequencing, quantitative PCR and cloning/sequencing) revealed a diverse community of likely transient bacterial taxa in the mid‐gut regions M1, M2 and M4. However, the completely anoxic M3 region harboured a distinct microbiota consisting of facultative and obligate anaerobes including Actinobacteria (Coriobacterium glomerans and Gordonibacter sp.), Firmicutes (Clostri‐dium sp. and Lactococcus lactis) and Proteobacteria (Klebsiella sp. and a previously undescribed Rickettsiales bacterium). Characterization of the M3 microbiota in different life stages of P. apterus indicated that the symbiotic bacterial community is vertically transmitted and becomes well defined between the second and third nymphal instar, which coincides with the initiation of feeding. Comparing the mid‐gut M3 microbial communities of P. apterus individuals from five different populations and after feeding on three different diets revealed that the community composition is qualitatively and quantitatively very stable, with the six predominant taxa being consistently abundant. Our findings suggest that the firebug mid‐gut microbiota constitutes a functionally important and possibly coevolved symbiotic community.     

Comparative sequence analysis of bacterial symbionts from the marine sponges Geodia cydonium and Ircinia muscarum

Marine sponges (Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Recently, several studiesindicated that sponges are the most prolific source of biologically-active compounds produced by symbiotic microorganisms ratherthan by the sponges themselves. In the present study we characterized the bacterial symbionts from two Demospongiae, Irciniamuscarum and Geodia cydonium. We amplified 16S rRNA by PCR, using specific bacterial-primers. The phylogenetic analysisrevealed the presence of nine bacterial clones from I. muscarum and ten from G. cydonium. In particular, I. muscarum resultedenriched in Bacillus species and G. cydonium in Proteobacterium species. Since these bacteria were able to produce secondarymetabolites with potential biotechnological and biopharmaceutical applications, we hypothesized that I. muscarum and G. cydoniumcould be a considered as a “gold mine” of natural products.     

Phylogenetic analysis of bacteria associated with Laminaria saccharina

Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.     

植物内生细菌测定方法的研究进展

伴随全功能体(holobiont)和全基因组(hologenome)概念的出现,植物微生物群落被看作植物全功能体的重要组成部分,其结构和功能逐渐得到研究和解析.内生细菌是植物微生物群落的成员之一,由于其定殖在组织内部而与宿主的接触更为紧密,因而其与植物的相互作用也更加直接、高效且不容易受到环境条件变化的影响.本文介绍了...     

Identification of key factors in Accelerated Low Water Corrosion through experimental simulation of tidal conditions: influence of stimulated indigenous microbiota

Biotic and abiotic factors favoring Accelerated Low Water Corrosion (ALWC) on harbor steel structures remain unclear warranting their study under controlled experimental tidal conditions. Initial stimulation of marine microbial consortia by a pulse of organic matter resulted in localized corrosion and the highest corrosion rates (up to 12-times higher than non-stimulated conditions) in the low water zone, persisting after nine months exposure to natural seawater. Correlations between corrosion severity and the abundance and composition of metabolically active sulfate-reducing bacteria (SRB) indicated the importance and persistence of specific bacterial populations in accelerated corrosion. One phylotype related to the electrogenic SRB Desulfopila corrodens appeared as the major causative agent of the accelerated corrosion. The similarity of bacterial populations related to sulfur and iron cycles, mineral and tuberculation with those identified in ALWC support the relevance of experimental simulation of tidal conditions in the management of steel corrosion exposed to harbor environments.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

Comparative phylogeny of the ammonia monooxygenase subunit A and 16S rRNA genes of ammonia-oxidizing bacteria

A fragment of the ammonia monooxygenase gene (amoA) from 31 strains of ammonia-oxidizing bacteria (AOB) was sequenced and analysed phylogenetically. The results were compared with the phylogeny of 16S rDNA from AOB. For most groups of AOB we found a high consistency between the phylogenetic trees based on the 16S rDNA and amoA sequences. Although it is not a phylogenetic marker, using the amoA as a probe when studying microbial diversity will probably reduce the amount of non-AOB detected, compared to using rDNA based probes. The data presented in this paper extend and improve the basis for application of amoA in studies of AOB in the environment.     

Changes in composition of cuticular biochemicals of the facultatively polygynous ant Petalomyrmex phylax during range expansion in Cameroon with respect to social, spatial and genetic variation

In social insects, biochemicals found at the surface of the cuticle are involved in the recognition process and in protection against desiccation and pathogens. However, the relative contribution of evolutionary forces in shaping diversity of these biochemicals remains largely unresolved in ants. We determined the composition of epicuticular biochemicals for workers sampled in 12 populations of the ant Petalomyrmex phylax from Cameroon. Genetic variation at 12 microsatellite markers was used to infer population history and to provide null expectations under the neutrality hypothesis. Genetic data suggest a recent southward range expansion of this ant species. Furthermore, there is a decline southward in the numbers of queens present in mature colonies. Here, we contrast the pattern of biochemical variation against genetic, social and spatial parameters. We thus provide the first estimates of the relative contribution of neutral and selective processes on variation of ant cuticular profile. Populations in migration-drift disequilibrium showed reduction of within-population variation for genetic markers as well as for cuticular profiles. In these populations, the cuticular profile became biased towards a limited number of high molecular weight molecules. Within- and among-population biochemical variation was explained by both genetic and social variation and by the spatial distribution of populations. We therefore propose that during range expansion of P. phylax, the composition of epicuticular compounds has been affected by a combination of neutral processes - genetic drift and spatially limited dispersal - and spatially varying selection, social organization and environmental effects.     

Invasion of Spartina alterniflora on Zostera japonica enhances the abundances of bacteria by absolute quantification sequencing analysis

Plant invasion can alter soil organic matter composition and indirectly impact estuary ecology; therefore, it is paramount to understand how plant invasion influences the bacterial community. Here, we present an absolute quantification 16S rRNA gene sequencing to investigate the bacterial communities that were collected from Zostera japonica and Spartina alterniflora covered areas and Z. japonica degradation areas in the Yellow River Estuary. Our data revealed that the absolute quantity of bacteria in the surface layer was significantly (p < .05) higher than that in the bottom and degradation areas. Following the invasion of S. alterniflora, the abundances of Bacteroidia, Acidimicrobiaceae, and Dehalococcoidaceaewere enriched in the S. alterniflora sediment. In addition, variations in the composition of sediment bacterial communities at the phylum level were the most intimately related to total organic carbon (TOC), and the content of heavy metals could reduce the abundance of bacteria. This study provided some information to understand the effects of S. alterniflora invasion on Z. japonica from the perspective of microbiome level.     

Molecular methods of analysing bacterial diversity: the case of Rhizobia

The distribution of rhizobia, bacteria which nodulate the roots of leguminous plants, was surveyed for 91 species of the familyLeguminosae. A total of 117 strains of rhizobia were isolated, and 28 strains were obtained from culture collections. The sample total of 145 strains was discriminated by rapid ribosomal RNA (rRNA) sequencing. The partial sequences (157 bases from position (inEscherichia coli) 1220 to 1377 from 5 end) of 16S rRNA revealed the presence of 16 groups in these rhizobia. Further, DNA-DNA homology studies suggested that the differences of the 16 groups were enough to justify establishing at least 16 species.