Host densities as determinants of abundance in parasite communities

Several epidemiological models predict a positive relationship between host population density and abundance of directly transmitted macroparasites. Here, we generalize these, and test the prediction by a comparative study. We used data on communities of gastrointestinal strongylid nematodes from 19 mammalian species, representing examination of 6670 individual hosts. We studied both the average abundance of all strongylid nematodes within a host species, and the two components of abundance, prevalence and intensity. The effects of host body weight, diet, fecundity and age at maturity and parasite body size were controlled for directly, and the phylogenetically independent contrast method was used to control for confounding factors more generally. Host population density and average parasite abundance were strongly positively correlated within mammalian taxa, and across all species when the effects of host body weight were controlled for. Controlling for other variables did not change this. Even when looking at single parasite species occurring in several host species, abundance was highest in the host species with the highest population density. Prevalence and intensity showed similar patterns. These patterns provide the first macroecological evidence consistent with the prediction that transmission rates depend on host population density in natural parasite communities.     

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.     

Host community similarity and geography shape the diversity and distribution of haemosporidian parasites in Amazonian birds

Identifying the mechanisms driving the distribution and diversity of parasitic organisms and characterizing the structure of parasite assemblages are critical to understanding host–parasite evolution, community dynamics, and disease transmission risk. Haemosporidian parasites of the genera Plasmodium and Haemoproteus are a diverse and cosmopolitan group of bird pathogens. Despite their global distribution, the ecological and historical factors shaping the diversity and distribution of these protozoan parasites across avian communities and geographic regions remain unclear. Here we used a region of the mitochondrial cytochrome b gene to characterize the diversity, biogeographical patterns, and phylogenetic relationships of Plasmodium and Haemoproteus infecting Amazonian birds. Specifically, we asked whether, and how, host community similarity and geography (latitude and area of endemism) structure parasite assemblages across 15 avian communities in the Amazon Basin. We identified 265 lineages of haemosporidians recovered from 2661 sampled birds from 330 species. Infection prevalence varied widely among host species, avian communities, areas of endemism, and latitude. Composition analysis demonstrated that both malarial parasites and host communities differed across areas of endemism and as a function of latitude. Thus, areas with similar avian community composition were similar in their parasite communities. Our analyses, within a regional biogeographic context, imply that host switching is the main event promoting diversification in malarial parasites. Although dispersal of haemosporidian parasites was constrained across six areas of endemism, these pathogens are not dispersal‐limited among communities within the same area of endemism. Our findings indicate that the distribution of malarial parasites in Amazonian birds is largely dependent on local ecological conditions and host evolutionary relationships.     

Host population density and body mass as determinants of species richness in parasite communities: comparative analyses of directly transmitted nematodes of mammals

Epidemiological theory predicts positive correlations between host population density or body mass and species richness among parasite communities. Here I test these predictions by a comparative study of communities of directly transmitted mammalian parasites, gastrointestinal strongylid nematodes. I use data from 45 species of mammals, representing examination of 17 200 individual hosts. The variable studied was the average number of gastrointestinal strongylid nematode species per host population, and three different methods were used to obtain estimates of parasite species richness that are unbiased by number of host individuals examined. Analyses were done using the phylogenetically independent contrast method. Host population density and parasite species richness were strongly positively correlated when the effects of host body weight had been controlled for. Controlling for other variables did not change this, and the relationship was found regardless of method used to correct for uneven sampling effort among host species. A positive relationship between parasite species richness and host body weight was also found, but the effect of host densities had to be controlled for to see this. These relationships between host traits and species richness of directly transmitted parasites are stronger than patterns found using data on indirectly transmitted mammalian parasites, and suggests that links between host traits and parasite species richness are stronger than previously suggested. The results are consistent with parasite species richness being positively linked to pathogen transmission rates and reductions in transmission rates possibly increasing extinction probabilities in parasite populations. The results also suggest that parasites may exert a cost of increases in rate of population energy usage, and thus show that pathogens may be important in generating independence between body mass and rate of population energy usage among host species.     

Loss of forest cover and host functional diversity increases prevalence of avian malaria parasites in the Atlantic Forest

Host phylogenetic relatedness and ecological similarity are thought to contribute to parasite community assembly and infection rates. However, recent landscape level anthropogenic changes may disrupt host-parasite systems by impacting functional and phylogenetic diversity of host communities. We examined whether changes in host functional and phylogenetic diversity, forest cover, and minimum temperature influence the prevalence, diversity, and distributions of avian haemosporidian parasites (genera Haemoproteus and Plasmodium) across 18 avian communities in the Atlantic Forest. To explore spatial patterns in avian haemosporidian prevalence and taxonomic and phylogenetic diversity, we surveyed 2241 individuals belonging to 233 avian species across a deforestation gradient. Mean prevalence and parasite diversity varied considerably across avian communities and parasites responded differently to host attributes and anthropogenic changes. Avian malaria prevalence (termed herein as an infection caused by Plasmodium parasites) was higher in deforested sites, and both Plasmodium prevalence and taxonomic diversity were negatively related to host functional diversity. Increased diversity of avian hosts increased local taxonomic diversity of Plasmodium lineages but decreased phylogenetic diversity of this parasite genus. Temperature and host phylogenetic diversity did not influence prevalence and diversity of haemosporidian parasites. Variation in the diversity of avian host traits that promote parasite encounter and vector exposure (host functional diversity) partially explained the variation in avian malaria prevalence and diversity. Recent anthropogenic landscape transformation (reduced proportion of native forest cover) had a major influence on avian malaria occurrence across the Atlantic Forest. This suggests that, for Plasmodium, host phylogenetic diversity was not a biotic filter to parasite transmission as prevalence was largely explained by host ecological attributes and recent anthropogenic factors. Our results demonstrate that, similar to human malaria and other vector-transmitted pathogens, prevalence of avian malaria parasites will likely increase with deforestation.     

Bacterial diversity across individual lichens

Symbioses are unique habitats for bacteria. We surveyed the spatial diversity of bacterial communities across multiple individuals of closely related lichens using terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing. Centers of lichens house richer, more consistent assemblages than species-poor and compositionally disparate lichen edges, suggesting that ecological succession plays a role in structuring these communities.     

Nested assemblages resulting from host size variation: the case of endoparasite communities in fish hosts

Nested species subsets are a common pattern in many types of communities found in insular or fragmented habitats. Nestedness occurs in some communities of ectoparasites of fish, as does the exact opposite departure from random assembly, anti-nestedness. Here, we looked for nested and anti-nested patterns in the species composition of communities of internal parasites of 23 fish populations from two localities in Finland. We also compared various community parameters of nested and anti-nested assemblages of parasites, and determined whether nestedness may result simply from a size-related accumulation of parasite species by feeding fish hosts. Nested parasite communities were characterised by higher prevalence (proportion of infected fish) and intensities of infection (number of parasites per fish) than anti-nested communities; the two types of non-random communities did not differ with respect to parasite species richness, however. In addition, the correlation between fish size and the number of parasite species harboured by individual fish was much stronger in nested assemblages than in anti-nested ones, where it was often nil. These results were shown not to be artefacts of sampling effort or host phylogeny. They apply to both assemblages of adult and larval parasites, which were treated separately. Since species of larval parasites are extremely unlikely to interact with one another in fish hosts, the establishment of nestedness appears independent of the potential action of interspecific interactions. The species composition of these parasite communities is not determined from within the community, but rather by the extrinsic influence of host feeding rates and how they amplify differences among parasite species in probabilities of colonisation or extinction. Nested patterns occur in parasite communities whose fish hosts accumulate parasites in a predictable fashion proportional to their size, whereas anti-nested communities occur in parasite communities whose fish hosts do not, possibly because of dietary specialisation preventing them from sampling the entire pool of parasite species available locally. Thus, nestedness in parasite communities may result from processes somewhat different from those generating nested patterns in free-living communities.     

Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism

An evaluation of 18 DNA restriction endonucleases for use in terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed by using richness and density indices in conjunction with computer simulations for 4,603 bacterial small-subunit rRNA gene sequences. T-RFLP analysis has become a commonly used method for screening environmental samples for precursory identification and community comparison studies due to its precision and high-throughput capability. The accuracy of T-RFLP analysis for describing a community has not yet been thoroughly evaluated. In this study, we attempted to classify restriction endonucleases based upon the ability to resolve unique terminal-restriction fragments (T-RFs) or operational taxonomic units (OTUs) from a database of gene sequences. Furthermore, we assessed the predictive accuracy of T-RFLP at fixed values of community richness (n = 1, 5, 10, 50, and 100). Classification of restriction endonuclease fidelity was performed by measuring richness and density for the entire database of T-RFs. Further analysis of T-RFLP accuracy for determining richness was performed by iterative, random sampling from the derived database of T-RFs. It became apparent that two constraints were influential for measuring the fidelity of a given restriction endonuclease: (i) the ability to resolve unique sequence variants and (ii) the number of unique T-RFs that fell within a measurable size range. The latter constraint was found to be more significant for estimating restriction endonuclease fidelity. Of the 18 restriction endonucleases examined, BstUI, DdeI, Sau96I, and MspI had the highest frequency of resolving single populations in model communities. All restriction endonucleases used in this study detected < or =70% of the OTUs at richness values greater than 50 OTUs per modeled community. Based on the results of our in silico experiments, the most efficacious uses of T-RFLP for microbial diversity studies are those that address situations where there is low to intermediate species richness (e.g., colonization, early successional stages, biofilm formation).     

North‐African house martins endure greater haemosporidian infection than their European counterparts

Afro‐Palearctic migrant species are exposed to parasites at both breeding and over‐wintering grounds. The house martin Delichon urbicum is one such migratory species facing high instances of blood parasite infection. In an attempt to determine whether breeding European house martins harbour similar blood parasite communities to populations breeding in North Africa, birds were sampled at their breeding grounds in Switzerland and Algeria. Moreover, haemosporidian prevalence and parasite communities were compared to published data sets on Spanish and Dutch breeding populations. This study furthermore wanted to establish whether co‐infection with multiple genera or lineages of parasites had negative e?ects on host body condition. Breeding house martins caught in Algeria showed a higher prevalence of avian haemosporidian parasites than did European populations. Swiss house martins showed a prevalence comparable to that of Spanish and Dutch populations. There were slight differences in the haemosporidian community between European and North‐African populations in terms of composition and abundance of each lineage. Similar to the Dutch house martins, but in contrast to the Spanish population, infection status and number of genera of parasites infecting single hosts did not in?uence Swiss house martin body condition.     

Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

Biogeography and Host Fidelity of Bacterial Communities in Ircinia spp. from the Bahamas

Research on sponge microbial assemblages has revealed different trends in the geographic variability and specificity of bacterial symbionts. Here, we combined replicated terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of 16S rRNA gene sequences to investigate the biogeographic and host-specific structure of bacterial communities in two congeneric and sympatric sponges: Ircinia strobilina, two color morphs of Ircinia felix and ambient seawater. Samples were collected from five islands of the Bahamas separated by 80 to 400 km. T-RFLP profiles revealed significant differences in bacterial community structure among sponge hosts and ambient bacterioplankton. Pairwise statistical comparisons of clone libraries confirmed the specificity of the bacterial assemblages to each host species and differentiated symbiont communities between color morphs of I. felix. Overall, differences in bacterial communities within each host species and morph were unrelated to location. Our results show a high degree of symbiont fidelity to host sponge across a spatial scale of up to 400 km, suggesting that host-specific rather than biogeographic factors play a primary role in structuring and maintaining sponge–bacteria relationships in Ircinia species from the Bahamas.     

Parasite assemblages of Double-crested Cormorants as indicators of host populations and migration behavior

The Double-crested Cormorant (Phalacrocorax auritus) is culled in many states because of the real and presumed damages it inflicts on farmed and recreational fisheries and other ecosystem services. Resident cormorant colonies breeding in the southeastern United States are protected in some areas, so it is important to distinguish these from co-occurring but unprotected migratory cormorants. Migratory P. auritus are likely to contain helminthic parasite communities that differ from those of non-migratory, resident birds, because they will encounter a wider variety of habitats and intermediate host communities during migrations. Here, we document five distinct assemblages of helminth parasites collected from 218 P. auritus culled from 11 sites in Alabama, Minnesota, Mississippi, and Vermont. The assemblages of P. auritus parasites are distinct among many sampling locations and can be used to correctly predict where a host cormorant has been feeding. We provide evidence for mixing of cormorants at a regional scale using discriminant analysis, which suggests there is a single population of migratory cormorants. Furthermore, our models strongly differentiate between migratory and resident P. auritus in the southeastern United States. In conjunction with species-by species latitudinal and longitudinal trends, our models could serve as effective tools for managers interested in both the population control of migratory cormorants and the conservation of non-migratory, resident birds. Finally, parasite counts per host are notoriously variable with many zeros and a few large numbers, leading many researchers to use simple prevalence in their analyses. We show that an intermediate level of data resolution, using species occurrence ranks within individual hosts, behaves well statistically and provides the greatest discrimination among distinct host groupings.     

Impact of Virioplankton on Archaeal and Bacterial Community Richness as Assessed in Seawater Batch Cultures

During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and Bacteria. One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton.     

Patterns of diversity and abundance of fleas and mites in the Neotropics: host‐related,parasite‐related and environment‐related factors

The effects of host‐related, parasite‐related and environmental factors on the diversity and abundance of two ectoparasite taxa, fleas (Insecta: Siphonaptera) and mites (Acari: Mesostigmata), parasitic on small mammals (rodents and marsupials), were studied in different localities across Brazil. A stronger effect of host‐related factors on flea than on mite assemblages, and a stronger effect of environmental factors on mite than on flea assemblages were predicted. In addition, the effects of parasite‐related factors on flea and mite diversity and abundance were predicted to manifest mainly at the scale of infracommunities, whereas the effects of host‐related and environmental factors were predicted to manifest mainly at the scale of component and compound communities. This study found that, in general, diversity and abundance of flea and mite assemblages at two lower hierarchical levels (infracommunities and component communities) were affected by host‐related, parasite‐related and environmental factors, and compound communities were affected mainly by host‐related and environmental factors. The effects of factors differed between fleas and mites: in fleas, community structure and abundance depended on host diversity to a greater extent than in mites. In addition, the effects of factors differed among parasite assemblages harboured by different host species.     

Enteric helminths of perch (Perca fluviatilis L.) and yellow perch (Perca flavescens Mitchill): stochastic or predictable assemblages?

Component communities of perch (Perca fluviatilis L) in Eurasia and the North American yellow perch (Perca flavescens Mitchill) were examined to determine the nature of their parasite communities. The scale of this investigation is continental and includes data collected across the distribution of each host species. Data were compiled from the literature and from 5 sample sites in North America. Four parasite species were found to occur frequently in the helminth component communities of P. flavescens. The cestodes Bothriocephalus cuspidatus and Proteocephalus pearsei, the digenean Crepidostomum cooperi, and the nematode Dichelyne cotylophora comprised a suite of species of which some or all occurred in most samples. Similarly, a group of 4 predictable parasite species was identified for P. fluviatilis in Eurasia, the digenean Bunodera luciopercae, the nematode Camallanus lacustris, the cestode Proteocephalus percae, and the acanthocephalan Acanthocephalus lucii. Specificity was not a requirement for predictability. Despite geographical isolation for millions of years, and different fish species interactions within and between continents, the predictability of these parasite assemblages indicates they are shaped by a biology, especially feeding patterns, common to both perch species. This is evidence that parasite assemblages comprised of nonhost-specific parasites in freshwater fishes are not merely stochastic assemblages but have key components that are predictable at this broad continental scale.     

Impact of virioplankton on archaeal and bacterial community richness as assessed in seawater batch cultures

During cruises in the tropical Atlantic Ocean (January to February 2000) and the southern North Sea (December 2000), experiments were conducted to monitor the impact of virioplankton on archaeal and bacterial community richness. Prokaryotic cells equivalent to 10 to 100% of the in situ abundance were inoculated into virus-free seawater, and viruses equivalent to 35 to 360% of the in situ abundance were added. Batch cultures with microwave-inactivated viruses and without viruses served as controls. The apparent richness of archaeal and bacterial communities was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified 16S rRNA gene fragments. Although the estimated richness of the prokaryotic communities generally was greatly reduced within the first 24 h of incubation due to confinement, the effects of virus amendment were detected at the level of individual operational taxonomic units (OTUs) in the T-RFLP patterns of both groups, Archaea and BACTERIA: One group of OTUs was detected in the control samples but was absent from the virus-treated samples. This negative response of OTUs to virus amendment probably was caused by viral lysis. Additionally, we found OTUs not responding to the amendments, and several OTUs exhibited variable responses to the addition of inactive or active viruses. Therefore, we conclude that individual members of pelagic archaeal and bacterial communities can be differently affected by the presence of virioplankton.     

Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces

New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.     

Impact of flooding on soil bacterial communities associated with poplar (Populus sp.) trees

Soil bacterial communities were analyzed in different habitats (bulk soil, rhizosphere, rhizoplane) of poplar tree microcosms (Populus tremulaxP. alba) using cultivation-independent methods. The roots of poplar trees regularly experience flooded and anoxic conditions. Therefore, we also determined the effect of flooding on microbial communities in microcosm experiments. Total community DNA was extracted and bacterial 16S rRNA genes were amplified by PCR and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and sequencing. Clone libraries were created from all three habitats under both unflooded and flooded conditions resulting in a total of 281 sequences. Numbers of different sequences (<97% similarity) in the different habitats represented 16-55% of total bacterial species richness determined from the nonparametric richness estimator Chao1. According to the number of different terminal restriction fragments (T-RFs), all of the different habitats contained approximately 20 different operational taxonomic units (OTUs), except the flooded rhizoplane habitat whose community contained less OTUs. Results of cloning and T-RFLP analysis generally supported each other. Correspondence analysis of T-RFLP patterns showed that the bacterial communities were different in bulk soil, rhizosphere and rhizoplane and changed upon flooding. For example OTUs representing Bacillus sp. were highest in the unflooded bulk soil and rhizosphere. Sequences related to Aquaspirillum, in contrast, were predominant on the poplar roots and in the rhizosphere of flooded microcosms but were rarely found in the other habitats.