Phylogenetic analysis of microbial communities in different regions of the gastrointestinal tract in Panaque nigrolineatus, a wood-eating fish.
ABSTRACT: The Neotropical detritivorous catfish Panaque nigrolineatus imbibes large quantities of wood as part of its diet. Due to the interest in cellulose, hemi-cellulose and lignin degradation pathways, this organism provides an interesting model system for the detection of novel microbial catabolism. In this study, we characterize the microbial community present in different regions of the alimentary tract of P. nigrolineatus fed a mixed diet of date palm and palm wood in laboratory aquaria. Analysis was performed on 16S rRNA gene clone libraries derived from anterior and posterior regions of the alimentary tract and the auxiliary lobe (AL), an uncharacterized organ that is vascularly attached to the midgut. Sequence analysis and phylogenetic reconstruction revealed distinct microbial communities in each tissue region. The foregut community shared many phylotypes in common with aquarium tank water and included Legionella and Hyphomicrobium spp. As the analysis moved further into the gastrointestinal tract, phylotypes with high levels of 16S rRNA sequence similarity to nitrogen-fixing Rhizobium and Agrobacterium spp. and Clostridium xylanovorans and Clostridium saccharolyticum, dominated midgut and AL communities. However, the hindgut was dominated almost exclusively by phylotypes with the highest 16S rRNA sequence similarity to the Cytophaga-Flavobacterium-Bacteroides phylum. Species richness was highest in the foregut (Chao(1) = 26.72), decreased distally through the midgut (Chao(1) = 25.38) and hindgut (Chao(1) = 20.60), with the lowest diversity detected in the AL (Chao(1) = 18.04), indicating the presence of a specialized microbial community. Using 16S rRNA gene phylogeny, we report that the P. nigrolineatus gastrointestinal tract possesses a microbial community closely related to microorganisms capable of cellulose degradation and nitrogen fixation. Further studies are underway to determine the role of this resident microbial community in Panaque nigrolineatus.
Project description:The Amazonian catfish, Panaque nigrolineatus, consume large amounts of wood in their diets. The nitrogen-fixing community within the gastrointestinal (GI) tract of these catfish was found to include nifH phylotypes that are closely related to Clostridium sp., Alpha and Gammaproteobacteria, and sequences associated with GI tracts of lower termites. Fish fed a diet of sterilized palm wood were found to contain nifH messenger RNA within their GI tracts, displaying high sequence similarity to the nitrogen-fixing Bradyrhizobium group. Nitrogenase activity, measured by acetylene reduction assays, could be detected in freshly dissected GI tract material and also from anaerobic enrichment cultures propagated in nitrogen-free enrichment media; nifH sequences retrieved from these cultures were dominated by Klebsiella- and Clostridium-like sequences. Microscopic examination using catalyzed reporter deposition-enhanced immunofluorescence revealed high densities of nitrogenase-containing cells colonizing the woody digesta within the GI tract, as well as cells residing within the intestinal mucous layer. Our findings suggest that the P. nigrolineatus GI tract provides a suitable environment for nitrogen fixation that may facilitate production of reduced nitrogen by the resident microbial population under nitrogen limiting conditions. Whether this community is providing reduced nitrogen to the host in an active or passive manner and whether it is present in a permanent or transient relationship remains to be determined. The intake of a cellulose rich diet and the presence of a suitable environment for nitrogen fixation suggest that the GI tract microbial community may allow a unique trophic niche for P. nigrolineatus among fish.
Project description:The Amazonian catfish, Panaque nigrolineatus have several physiological adaptions enabling the scraping and consumption of wood (xylivory), facilitating a detritivorous dietary strategy. Composed of lignocellulose, wood is a difficult substrate to degrade and as yet, it is unclear whether the fish obtains any direct nutritional benefits from wood ingestion and degradation. However, there are numerous systems that rely on microbial symbioses to provide energy and other nutritional benefits for host organisms via lignocellulose decomposition. While previous studies on the microbial community of P. nigrolineatus have focused upon the bacterial population, the role of fungi in lignocellulose degradation in the fish has not yet been examined. This study describes the detection of fungi within the fish gastrointestinal tract. Using next generation sequencing, the effects of diet on enteric fungal populations were examined in each gastrointestinal tract region. Fungal species were found to vary in different regions of the gastrointestinal tract as a function of diet. This study is the first to examine the fungal community in a xylivorous fish and results support the hypothesis that diet influences fungal distribution and diversity within the gastrointestinal tract of P. nigrolineatus.
Project description:To examine the effect of pathogens on the diversity and structure of plant-associated bacterial communities, we carried out a molecular analysis using citrus and huanglongbing as a host-disease model. 16S rRNA gene clone library analysis of citrus roots revealed shifts in microbial diversity in response to pathogen infection. The clone library of the uninfected root samples has a majority of phylotypes showing similarity to well-known plant growth-promoting bacteria, including Caulobacter, Burkholderia, Lysobacter, Pantoea, Pseudomonas, Stenotrophomonas, Bacillus, and Paenibacillus. Infection by "Candidatus Liberibacter asiaticus" restructured the native microbial community associated with citrus roots and led to the loss of detection of most phylotypes while promoting the growth of bacteria such as Methylobacterium and Sphingobacterium. In pairwise comparisons, the clone library from uninfected roots contained significantly higher 16S rRNA gene diversity, as reflected in the higher Chao 1 richness estimation (P </= 0.01) of 237.13 versus 42.14 for the uninfected and infected clone libraries, respectively. Similarly, the Shannon index of the uninfected clone library (4.46) was significantly higher than that of the infected clone library (2.61). Comparison of the uninfected clone library with the infected clone library using LIBSHUFF statistics showed a significant difference (P </= 0.05). Quantitative PCR analysis revealed that the bacterial community changes not only qualitatively but also quantitatively. The relative proportions of different groups of bacteria changed significantly after infection with the pathogen. These data indicate that infection of citrus by "Ca. Liberibacter asiaticus" has a profound effect on the structure and composition of the bacterial community associated with citrus roots.
Project description:Wood is consistently found in high levels in the gastrointestinal tract of the Amazonian catfish Panaque nigrolineatus, which, depending on environmental conditions, can switch between xylivorous and detritivorous dietary strategies. This is highly unusual among primary wood consumers and provides a unique system to examine the effect of dietary change in a xylivorous system. In this study, microbiome and predictive metagenomic analyses were performed for P. nigrolineatus fed either wood alone or a less refractory mixed diet containing wood and plant nutrition. While diet had an impact on enteric bacterial community composition, there was a high degree of interindividual variability. Members of the Proteobacteria and Planctomycetes were ubiquitous and dominated most communities; Bacteroidetes, Fusobacteria, Actinobacteria, and Verrucomicrobia also contributed in a tissue and diet-specific manner. Although predictive metagenomics revealed functional differences between communities, the relative abundance of predicted lignocellulose-active enzymes remained similar across diets. The microbiomes from both diets appeared highly adapted for hemicellulose hydrolysis as the predicted metagenomes contained several classes of hemicellulases and lignin-modifying enzymes. Enteric communities from both diets appeared to lack the necessary cellobiohydrolases for efficient cellulose hydrolysis, suggesting that cellobiose is not the primary source of dietary carbon for the fish. Our findings suggest that the P. nigrolineatus gut environment selects for an enteric community based on function, rather than a vertically transferred symbiotic relationship. This functional selection strategy may provide an advantage to an organism that switches between dietary strategies to survive a highly variable environment.
Project description:Direct sequencing of environmental DNA (metagenomics) has a great potential for describing the 16S rRNA gene diversity of microbial communities. However current approaches using this 16S rRNA gene information to describe community diversity suffer from low taxonomic resolution or chimera problems. Here we describe a new strategy that involves stringent assembly and data filtering to reconstruct full-length 16S rRNA genes from metagenomicpyrosequencing data. Simulations showed that reconstructed 16S rRNA genes provided a true picture of the community diversity, had minimal rates of chimera formation and gave taxonomic resolution down to genus level. The strategy was furthermore compared to PCR-based methods to determine the microbial diversity in two marine sponges. This showed that about 30% of the abundant phylotypes reconstructed from metagenomic data failed to be amplified by PCR. Our approach is readily applicable to existing metagenomic datasets and is expected to lead to the discovery of new microbial phylotypes.
Project description:The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. In a companion paper (T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune, Appl. Environ. Microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community. Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P. ephippiata larvae, determined with cultivation-independent techniques. Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P. ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil. The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper. Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum. The archaeal community, however, is less diverse. 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut. These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.
Project description:To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.
Project description:Taxonomic marker gene studies, such as the 16S rRNA gene, have been used to successfully explore microbial diversity in a variety of marine, terrestrial, and host environments. For some of these environments long term sampling programs are beginning to build a historical record of microbial community structure. Although these 16S rRNA gene datasets do not intrinsically provide information on microbial metabolism or ecosystem function, this information can be developed by identifying metabolisms associated with related, phenotyped strains. Here we introduce the concept of metabolic inference; the systematic prediction of metabolism from phylogeny, and describe a complete pipeline for predicting the metabolic pathways likely to be found in a collection of 16S rRNA gene phylotypes. This framework includes a mechanism for assigning confidence to each metabolic inference that is based on a novel method for evaluating genomic plasticity. We applied this framework to 16S rRNA gene libraries from the West Antarctic Peninsula marine environment, including surface and deep summer samples and surface winter samples. Using statistical methods commonly applied to community ecology data we found that metabolic structure differed between summer surface and winter and deep samples, comparable to an analysis of community structure by 16S rRNA gene phylotypes. While taxonomic variance between samples was primarily driven by low abundance taxa, metabolic variance was attributable to both high and low abundance pathways. This suggests that clades with a high degree of functional redundancy can occupy distinct adjacent niches. Overall our findings demonstrate that inferred metabolism can be used in place of taxonomy to describe the structure of microbial communities. Coupling metabolic inference with targeted metagenomics and an improved collection of completed genomes could be a powerful way to analyze microbial communities in a high-throughput manner that provides direct access to metabolic and ecosystem function.
Project description:Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur.
Project description:Investigations of gut microbiomes have shed light on the diversity and genetic content of these communities, and helped shape our understanding of how host-associated microorganisms influence host physiology, behavior, and health. Despite the importance of gut microbes to metazoans, our understanding of the changes in diversity and composition across the alimentary tract, and the source of the resident community are limited. Here, using community metagenomics and 16S rRNA gene sequencing, we assess microbial community diversity and coding potential in the foregut, midgut, and hindgut of a juvenile Panchlora cockroach, which resides in the refuse piles of the leaf-cutter ant species Atta colombica. We found a significant shift in the microbial community structure and coding potential throughout the three gut sections of Panchlora sp., and through comparison with previously generated metagenomes of the cockroach's food source and niche, we reveal that this shift in microbial community composition is influenced by the ecosystems in which Panchlora sp. occurs. While the foregut is composed of microbes that likely originate from the symbiotic fungus gardens of the ants, the midgut and hindgut are composed of a microbial community that is likely cockroach-specific. Analogous to mammalian systems, the midgut and hindgut appear to be dominated by Firmicutes and Bacteroidetes with the capacity for polysaccharide degradation, suggesting they may assist in the degradation of dietary plant material. Our work underscores the prominence of community changes throughout gut microbiomes and highlights ecological factors that underpin the structure and function of the symbiotic microbial communities of metazoans.