Low-abundance OTUs Research Articles

Exposure to nanoplastics and nanomaterials either single and combined affects the gill-associated microbiome of the Antarctic soft-shelled clam Laternula elliptica

Nanoplastics and engineering nanomaterials (ENMs) are contaminants of emerging concern (CECs), increasingly being detected in the marine environment and recognized as a potential threat for marine biota at the global level including in polar areas. Few studies have assessed the impact of these anthropogenic nanoparticles in the microbiome of marine invertebrates, however combined exposure resembling natural scenarios has been overlooked. The present study aimed to evaluate the single and combined effects of polystyrene nanoparticles (PS NP) as proxy for nanoplastics and nanoscale titanium dioxide (nano-TiO2) on the prokaryotic communities associated with the gill tissue of the Antarctic soft-shell clam Laternula elliptica, a keystone species of marine benthos Wild-caught specimens were exposed to two environmentally relevant concentrations of carboxylated PS NP (PS–COOH NP, ∼62 nm size) and nano-TiO2 (Aeroxide P25, ∼25 nm) as 5 and 50 μg/L either single and combined for 96h in a semi-static condition.Our findings show a shift in microbiome composition in gills of soft-shell clams exposed to PS NP and nano-TiO2 either alone and in combination with a decrease in the relative abundance of OTU1 (Spirochaetaceae). In addition, an increase of gammaproteobacterial OTUs affiliated to MBAE14 and Methylophagaceae (involved in ammonia denitrification and associated with low-quality water), and the OTU Colwellia rossensis (previously recorded in polluted waters) was observed. Our results suggest that nanoplastics and nano-TiO2 alone and in combination induce alterations in microbiome composition by promoting the increase of negative taxa over beneficial ones in the gills of the Antarctic soft-shell clam. An increase of two low abundance OTUs in PS-COOH NPs exposed clams was also observed. A predicted gene function analysis revealed that sugar, lipid, protein and DNA metabolism were the main functions affected by either PS-COOH NP and nano-TiO2 exposure. The molecular functions involved in the altered affiliated OTUs are novel for nano-CEC exposures.

Does filter pore size introduce bias in DNA sequence-based plankton community studies?

The cell size of microbial eukaryotic plankton normally ranges from 0.2 to 200 μm. During the past decade, high-throughput sequencing of DNA has been revolutionizing their study on an unprecedented scale. Nonetheless, it is currently unclear whether we can accurately, effectively, and quantitatively depict the microbial eukaryotic plankton community using size-fractionated filtration combined with environmental DNA (eDNA) molecular methods. Here we assessed the microbial eukaryotic plankton communities with two filtering strategies from two subtropical reservoirs, that is one-step filtration (0.2–200 μm) and size-fractionated filtration (0.2–3 and 3–200 μm). The difference of 18S rRNA gene copy abundance between the two filtering treatments was less than 50% of the 0.2–200 μm microbial eukaryotic community for 95% of the total samples. Although the microbial eukaryotic plankton communities within the 0.2–200 μm and the 0.2–3 and 3–200 μm size fractions had approximately identical 18S rRNA gene copies, there were significant differences in their community composition. Furthermore, our results demonstrate that the systemic bias introduced by size-fractionation filtration has more influence on unique OTUs than shared OTUs, and the significant differences in abundance between the two eukaryotic plankton communities largely occurred in low-abundance OTUs in specific seasons. This work provides new insights into the use of size-fractionation in molecular studies of microbial eukaryotes populating the plankton.

Performance determinants of unsupervised clustering methods for microbiome data

BackgroundIn microbiome data analysis, unsupervised clustering is often used to identify naturally occurring clusters, which can then be assessed for associations with characteristics of interest. In this work, we systematically compared beta diversity and clustering methods commonly used in microbiome analyses. We applied these to four published datasets where highly distinct microbiome profiles could be seen between sample groups, as well a clinical dataset with less clear separation between groups.ResultsAlthough no single method outperformed the others consistently, we did identify the key scenarios where certain methods can underperform. Specifically, the Bray Curtis (BC) metric resulted in poor clustering in a dataset where high-abundance OTUs were relatively rare. In contrast, the unweighted UniFrac (UU) metric clustered poorly on dataset with a high prevalence of low-abundance OTUs. To explore these hypotheses about BC and UU, we systematically modified the properties of the poorly performing datasets and found that this approach resulted in improved BC and UU performance. Based on these observations, we rationally combined BC and UU to generate a novel metric. We tested its performance while varying the relative contributions of each metric and also compared it with another combined metric, the generalized UniFrac distance. The proposed metric showed high performance across all datasets.ConclusionsOur systematic evaluation of clustering performance in these five datasets demonstrates that there is no existing clustering method that universally performs best across all datasets. We propose a combined metric of BC and UU that capitalizes on the complementary strengths of the two metrics.syUurMUnqpLuEn-KfNXmwVideo abstract

The yellow perch (Perca flavescens) microbiome revealed resistance to colonisation mostly associated with neutralism driven by rare taxa under cadmium disturbance

BackgroundDisentangling the dynamics of microbial interactions within communities improves our comprehension of metacommunity assembly of microbiota during host development and under perturbations. To assess the impact of stochastic variation of neutral processes on microbiota structure and composition under disturbance, two types of microbial habitats, free-living (water), and host-associated (skin and gut) were experimentally exposed to either a constant or gradual selection regime exerted by two sublethal cadmium chloride dosages (CdCl2). Yellow Perch (Perca flavescens) was used as a piscivorous ecotoxicological model. Using 16S rDNA gene based metataxonomics, quantitative diversity metrics of water, skin and gut microbial communities were characterized along with development and across experimental conditions.ResultsAfter 30 days, constant and gradual selection regimes drove a significant alpha diversity increase for both skin and gut microbiota. In the skin, pervasive negative correlations between taxa in both selection regimes in addition to the taxonomic convergence with the environmental bacterial community, suggest a loss of colonisation resistance resulting in the dysbiosis of yellow perch microbiota. Furthermore, the network connectivity in gut microbiome was exclusively maintained by rare (low abundance) OTUs, while most abundant OTUs were mainly composed of opportunistic invaders such as Mycoplasma and other genera related to fish pathogens such as Flavobacterium. Finally, the mathematical modelling of community assembly using both non-linear least squares models (NLS) based estimates of migration rates and normalized stochasticity ratios (NST) based beta-diversity distances suggested neutral processes drove by taxonomic drift in host and water communities for almost all treatments. The NLS models predicted higher demographic stochasticity in the cadmium-free host and water microbiomes, however, NST models suggested higher ecological stochasticity under perturbations.ConclusionsNeutral models agree that water and host-microbiota assembly promoted by rare taxa have evolved predominantly under neutral processes with potential involvement of deterministic forces sourced from host filtering and cadmium selection. The early signals of perturbations in the skin microbiome revealed antagonistic interactions by a preponderance of negative correlations in the co-abundance networks. Our findings enhance our understanding of community assembly host-associated and free-living under anthropogenic selective pressure.

Rain-Fed Granite Rock Basins Accumulate a High Diversity of Dormant Microbial Eukaryotes.

Rain fed granite rock basins are ancient geological landforms of worldwide distribution and structural simplicity. They support habitats that can switch quickly from terrestrial to aquatic along the year. Diversity of animals and plants, and the connexion between communities in different basins have been widely explored in these habitats, but hardly any research has been carried out on microorganisms. The aim of this study is to provide the first insights on the diversity of eukaryotic microbial communities from these environments. Due to the ephemeral nature of these aquatic environments, we predict that the granitic basins should host a high proportion of dormant microeukaryotes. Based on an environmental DNA diversity survey, we reveal diverse communities with representatives of all major eukaryotic taxonomic supergroups, mainly composed of a diverse pool of low abundance OTUs. Basin communities were very distinctive, with alpha and beta diversity patterns non-related to basin size or spatial distance respectively. Dissimilarity between basins was mainly characterised by turnover of OTUs. The strong microbial eukaryotic heterogeneity observed among the basins may be explained by a complex combination of deterministic factors (diverging environment in the basins), spatial constraints, and randomness including founder effects. Most interestingly, communities contain organisms that cannot coexist at the same time because of incompatible metabolic requirements, thus suggesting the existence of a pool of dormant organisms whose activity varies along with the changing environment. These organisms accumulate in the pools, which turns granitic rock into high biodiversity microbial islands whose conservation and study deserve further attention.

Rare Plankton Subcommunities Are Far More Affected by DNA Extraction Kits Than Abundant Plankton.

Advances in high-throughput sequencing technologies allow a more complete study of microbial plankton community composition and diversity, especially in the rare microbial biosphere. The DNA extraction of plankton is a key step for such studies; however, little is known about its influences on the abundant or rare microbial biosphere. Our aim was to quantify the influences of different DNA extraction kits on abundant and rare plankton in the surface waters of a reservoir and provide a reference for the comparisons between microbial community studies with different extraction methods. We evaluated the influence of five common commercial kits on DNA quality, microbial community diversity and composition, and the reproducibility of methods using both 16S and 18S rRNA genes amplicon sequencing. Our data showed that results of Fast DNA Spin Kit for Soil (MPF) had higher α diversity for bacteria and high DNA quality, indicating that it is the most suitable approach for bacterioplankton diversity study. However, DNeasy Blood & Tissue Kit (QD) and QIAamp DNA Mini Kit (QQ) methods could produce results that are easier to replicate for bacteria and eukaryotes, respectively, and were more comparable between studies. The use of different DNA extraction kits had larger influence on the rare taxa compared with abundant taxa. Therefore, the comparability between studies that employed different extraction methods can be improved by removing low-abundance or less-representative OTUs. Collectively, this study provides a comprehensive assessment of the biases associated with DNA extraction for plankton communities from a freshwater reservoir. Our results may guide researchers in experimental design choices for DNA-based ecological studies in aquatic ecosystem.

Metabarcoding reveals that rhizospheric microbiota of Quercus pyrenaica is composed by a relatively small number of bacterial taxa highly abundant

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests; however, these forests show an advanced stage of deterioration in the Iberian Peninsula. Plant-associated microorganisms play an essential role improving their host’s fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for oak-forest improvement. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of different melojo-oak forest formations through pyrosequencing of 16S rRNA gene amplicons. We found that potentially active bacterial communities were as rich and diverse as total bacterial communities, but different in terms of relative abundance patterns in some of the studied areas. Both core microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak’s rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in identifying major players for the development of bacterial bioinoculants.

Identification of Fungal Communities Within the Tar Spot Complex of Corn in Michigan via Next-Generation Sequencing.

Tar spot is a fungal disease complex of corn that has been destructive and yield limiting in Central and South America for nearly 50 years. Phyllachora maydis, the causal agent of tar spot, is an emerging corn pathogen in the United States, first reported in 2015 from major corn producing regions of the country. The tar spot disease complex putatively includes Monographella maydis (syn. Microdochium maydis), which increases disease damage through the development of necrotic halos surrounding tar spot lesions. These necrotic halos, termed "fish-eye" symptoms, have been identified in the United States, though Monographella maydis has not yet been confirmed. A recent surge in disease severity and loss of yield attributed to tar spot in the United States has led to increased attention and expanded efforts to understand the disease complex and how to manage it. In this study, next-generation sequencing of the internal transcribed spacer-1 (ITS1) ribosomal DNA was used to identify fungal taxa that distinguish tar spot infections with or without fish-eye symptoms. Fungal communities within tar spot only lesions were significantly different from communities having fish-eye symptoms. Two low abundance OTUs were identified as Microdochium sp., however, neither were associated with fish-eye symptom development. Interestingly, a single OTU was found to be significantly more abundant in fish-eye lesions compared to tar spot lesions and had a 91% ITS1 identity to Neottiosporina paspali. In addition, the occurrence of this OTU was positively associated with Phyllachora maydis fish-eye symptom networks, but not in tar spot symptom networks. Neottiosporina paspali has been reported to cause necrotic lesions on various monocot grasses. Whether the related fungus we detected is part of the tar-spot complex of corn and responsible for fish-eye lesions remains to be tested. Alternatively, many OTUs identified as Phyllachora maydis, suggesting that different isolate genotypes may be capable of causing both tar spot and fish-eye symptoms, independent of other fungi. We conclude that Monographella maydis is not required for fish-eye symptoms in tar spot of corn.

Total and Active Core Microbiomes of Melojo Oak Are Composed by a Minority of Bacterial Taxa Highly Abundant

Melojo oak (Quercus pyrenaica Willd.) is a key tree species of Mediterranean forests with high landscape value in the Iberian Peninsula. However, these forests exhibit an advanced stage of deterioration and their distribution area has been seriously reduced over the last decades. Plant-associated microorganisms play an essential role improving their host’s fitness, hence, a better understanding of oak rhizospheric microbiome, especially of those active members, could be the first step towards microbiome-based approaches for the improvement of the survival and growth of oak trees. Here we reported, for the first time, the diversity of total (DNA-based) and potentially active (RNA-based) bacterial communities of three types of Quercus pyrenaica Willd. forest formations through 454 pyrosequencing of 16S rDNA amplicons, and defined the rhizospheric core microbiome of Q. pyrenaica at Sierra Nevada National and Natural Park during spring season. In addition rRNA:rDNA ratio was calculated as a proxy of the contribution of each core taxon to the rhizosphere environment. We found that potentially active bacterial communities were as rich and diverse as DNA-based populations, but different in terms of relative abundance patterns in some of the studied areas. Both microbiomes were dominated by a relatively small percentage of OTUs, most of which showed positive correlation between both libraries. However, the uncoupling between abundance (rDNA) and potential activity (rRNA) for some taxa suggests that the most abundant taxa are not always the most active, and that low-abundance OTUs may have a strong influence on oak’s rhizospheric ecology. Thus, measurement of rRNA:rDNA ratio could be helpful in characterizing rhizospheric bacterial communities, their potential involvement in the functioning of the soil ecosystem and, therefore, in identifying major bacterial players for the development of microbiome-based strategies for future afforestation tasks.

Bamboo Specialists from Two Mammalian Orders (Primates, Carnivora) Share a High Number of Low-Abundance Gut Microbes.

Bamboo specialization is one of the most extreme examples of convergent herbivory, yet it is unclear how this specific high-fiber diet might selectively shape the composition of the gut microbiome compared to host phylogeny. To address these questions, we used deep sequencing to investigate the nature and comparative impact of phylogenetic and dietary selection for specific gut microbial membership in three bamboo specialists-the bamboo lemur (Hapalemur griseus, Primates: Lemuridae), giant panda (Ailuropoda melanoleuca, Carnivora: Ursidae), and red panda (Ailurus fulgens, Carnivora: Musteloideadae), as well as two phylogenetic controls-the ringtail lemur (Lemur catta) and the Asian black bear (Ursus thibetanus). We detected significantly higher Shannon diversity in the bamboo lemur (10.029) compared to both the giant panda (8.256; p = 0.0001936) and the red panda (6.484; p = 0.0000029). We also detected significantly enriched bacterial taxa that distinguished each species. Our results complement previous work in finding that phylogeny predominantly governs high-level microbiome community structure. However, we also find that 48 low-abundance OTUs are shared among bamboo specialists, compared to only 8 OTUs shared by the bamboo lemur and its sister species, the ringtail lemur (Lemur catta, a generalist). Our results suggest that deep sequencing is necessary to detect low-abundance bacterial OTUs, which may be specifically adapted to a high-fiber diet. These findings provide a more comprehensive framework for understanding the evolution and ecology of the microbiome as well as the host.

A Small Number of Low-abundance Bacteria Dominate Plant Species-specific Responses during Rhizosphere Colonization.

Plant growth can be affected by soil bacteria. In turn, plants are known to influence soil bacteria through rhizodeposits and changes in abiotic conditions. We aimed to quantify the phylotype richness and relative abundance of rhizosphere bacteria that are actually influenced in a plant species-specific manner and to determine the role of the disproportionately large diversity of low-abundance bacteria belonging to the rare biosphere (<0.1 relative abundance) in this process. In addition, we aimed to determine whether plant phylogeny has an influence on the plant species-specific rhizosphere bacterial community. For this purpose, 19 herbaceous plant species from five different plant orders were grown in a common soil substrate. Bacterial communities in the initial soil substrate and the established rhizosphere soils were compared by 16S rRNA gene amplicon sequencing. Only a small number of bacterial operational taxonomic units (OTUs, 97% sequence identity) responded either positively (ca. 1%) or negatively (ca. 1%) to a specific plant species. On average, 91% of plant-specific positive response OTUs comprised bacteria belonging to the rare biosphere, highlighting that low-abundance populations are metabolically active in the rhizosphere. In addition, low-abundance OTUs were in terms of their summed relative abundance major drivers of the bacterial phyla composition across the rhizosphere of all tested plant species. However, no effect of plant phylogeny could be observed on the established rhizosphere bacterial communities, neither when considering differences in the overall established rhizosphere communities nor when considering plant species-specific responders only. Our study provides a quantitative assessment of the effect of plants on their rhizosphere bacteria across multiple plant orders. Plant species-specific effects on soil bacterial communities involved only 18–111 bacterial OTUs out of several 1000s; this minority may potentially impact plant growth in plant–bacteria interactions.

Identification of a Core Bacterial Community within the Large Intestine of the Horse

The horse has a rich and complex microbial community within its gastrointestinal tract that plays a central role in both health and disease. The horse receives much of its dietary energy through microbial hydrolysis and fermentation of fiber predominantly in the large intestine/hindgut. The presence of a possible core bacterial community in the equine large intestine was investigated in this study. Samples were taken from the terminal ileum and 7 regions of the large intestine from ten animals, DNA extracted and the V1-V2 regions of 16SrDNA 454-pyrosequenced. A specific group of OTUs clustered in all ileal samples and a distinct and different signature existed for the proximal regions of the large intestine and the distal regions. A core group of bacterial families were identified in all gut regions with clear differences shown between the ileum and the various large intestine regions. The core in the ileum accounted for 32% of all sequences and comprised of only seven OTUs of varying abundance; the core in the large intestine was much smaller (5-15% of all sequences) with a much larger number of OTUs present but in low abundance. The most abundant member of the core community in the ileum was Lactobacillaceae, in the proximal large intestine the Lachnospiraceae and in the distal large intestine the Prevotellaceae. In conclusion, the presence of a core bacterial community in the large intestine of the horse that is made up of many low abundance OTUs may explain in part the susceptibility of horses to digestive upset.

Water mass-specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing

Bacterial assemblages from subsurface (100 m depth), meso- (200-1000 m depth) and bathy-pelagic (below 1000 m depth) zones at 10 stations along a North Atlantic Ocean transect from 60°N to 5°S were characterized using massively parallel pyrotag sequencing of the V6 region of the 16S rRNA gene (V6 pyrotags). In a dataset of more than 830,000 pyrotags, we identified 10,780 OTUs of which 52% were singletons. The singletons accounted for less than 2% of the OTU abundance, whereas the 100 and 1000 most abundant OTUs represented 80% and 96% respectively of all recovered OTUs. Non-metric Multi-Dimensional Scaling and Canonical Correspondence Analysis of all the OTUs excluding the singletons revealed a clear clustering of the bacterial communities according to the water masses. More than 80% of the 1000 most abundant OTUs corresponded to Proteobacteria of which 55% were Alphaproteobacteria, mostly composed of the SAR11 cluster. Gammaproteobacteria increased with depth and included a relatively large number of OTUs belonging to Alteromonadales and Oceanospirillales. The bathypelagic zone showed higher taxonomic evenness than the overlying waters, albeit bacterial diversity was remarkably variable. Both abundant and low-abundance OTUs were responsible for the distinct bacterial communities characterizing the major deep-water masses. Taken together, our results reveal that deep-water masses act as bio-oceanographic islands for bacterioplankton leading to water mass-specific bacterial communities in the deep waters of the Atlantic.