Operational Taxonomic Units Research Articles

Sample collecting methods for bacterial community structure analysis of scalp hair: non-invasive swabbing versus intrusive hair shaft cutting

Human skin samples for microbiome analysis are traditionally collected using a non-invasive swabbing method. Here, we compared the differences in bacterial community structures on scalp hair and scalps with samples collected using non-invasive swabbing and cutting/removal of scalp hair in 12 individuals. Hair-related samples, such as hair shafts and hair swabs, had significantly higher alpha diversity than scalp swab samples, whereas there were no significant differences between hair shafts and hair swabs. The relative abundances of the three major phyla and five major operational taxonomic units were not significantly different between the hair shaft and hair swab samples. The principal coordinate analysis plots based on weighted UniFrac distances were grouped into two clusters: samples from hair-related areas and scalp swabs, and there were significant differences only between samples from hair-related areas and scalp swabs. In addition, a weighted UniFrac analysis revealed that the sampling site-based category was a statistical category but not a hair sampling method-based category. These results suggest that scalp hair bacteria collected using non-invasive swab sampling were comparable to those collected cutting/removal of scalp hair.

SpeciateIT and vSpeciateDB: novel, fast, and accurate per sequence 16S rRNA gene taxonomic classification of vaginal microbiota

BackgroundClustering of sequences into operational taxonomic units (OTUs) and denoising methods are a mainstream stopgap to taxonomically classifying large numbers of 16S rRNA gene sequences. Environment-specific reference databases generally yield optimal taxonomic assignment.ResultsWe developed SpeciateIT, a novel taxonomic classification tool which rapidly and accurately classifies individual amplicon sequences (https://github.com/Ravel-Laboratory/speciateIT). We also present vSpeciateDB, a custom reference database for the taxonomic classification of 16S rRNA gene amplicon sequences from vaginal microbiota. We show that SpeciateIT requires minimal computational resources relative to other algorithms and, when combined with vSpeciateDB, affords accurate species level classification in an environment-specific manner.ConclusionsHerein, two resources with new and practical importance are described. The novel classification algorithm, SpeciateIT, is based on 7th order Markov chain models and allows for fast and accurate per-sequence taxonomic assignments (as little as 10 min for 107 sequences). vSpeciateDB, a meticulously tailored reference database, stands as a vital and pragmatic contribution. Its significance lies in the superiority of this environment-specific database to provide more species-resolution over its universal counterparts.

Resolving biology’s dark matter: species richness, spatiotemporal distribution, and community composition of a dark taxon

BackgroundZoology’s dark matter comprises hyperdiverse, poorly known taxa that are numerically dominant but largely unstudied, even in temperate regions where charismatic taxa are well understood. Dark taxa are everywhere, but high diversity, abundance, and small size have historically stymied their study. We demonstrate how entomological dark matter can be elucidated using high-throughput DNA barcoding (“megabarcoding”). We reveal the high abundance and diversity of scuttle flies (Diptera: Phoridae) in Sweden using 31,800 specimens from 37 sites across four seasonal periods. We investigate the number of scuttle fly species in Sweden and the environmental factors driving community changes across time and space.ResultsSwedish scuttle fly diversity is much higher than previously known, with 549 putative species detected, compared to 374 previously recorded species. Hierarchical Modelling of Species Communities reveals that scuttle fly communities are highly structured by latitude and strongly driven by climatic factors. Large dissimilarities between sites and seasons are driven by turnover rather than nestedness. Climate change is predicted to significantly affect the 47% of species that show significant responses to mean annual temperature. Results were robust regardless of whether haplotype diversity or species-proxies were used as response variables. Additionally, species-level models of common taxa adequately predict overall species richness.ConclusionsUnderstanding the bulk of the diversity around us is imperative during an era of biodiversity change. We show that dark insect taxa can be efficiently characterised and surveyed with megabarcoding. Undersampling of rare taxa and choice of operational taxonomic units do not alter the main ecological inferences, making it an opportune time to tackle zoology’s dark matter.

Diversity and Activity of Bacteria Cultured from a Cup-The Sponge Calyx nicaeensis.

Marine sponges are well-known for hosting rich microbial communities. Sponges are the most prolific source of marine bioactive compounds, which are frequently synthesized by their associated microbiota. Calyx nicaeensis is an endemic Mediterranean sponge with scarce information regarding its (bioactive) secondary metabolites. East Mediterranean specimens of mesophotic C. nicaeensis have never been studied. Moreover, no research has inspected its associated bacteria. Thus, we studied the sponge's bacterial diversity and examined bacterial interspecific interactions in search of a promising antibacterial candidate. Such novel antimicrobial agents are needed since extensive antibiotic use leads to bacterial drug resistance. Bacteria cultivation yielded 90 operational taxonomic units (OTUs). A competition assay enabled the testing of interspecific interactions between the cultured OTUs. The highest-ranked antagonistic bacterium, identified as Paenisporosarcina indica (previously never found in marine or cold habitats), was mass cultured, extracted, and separated using size exclusion and reversed-phase chromatographic methods, guided by antibacterial activity. A pure compound was isolated and identified as 3-oxy-anteiso-C15-fatty acid-lichenysin. Five additional active compounds await final cleaning; however, they are lichenysins and surfactins. These are the first antibacterial compounds identified from either the C. nicaeensis sponge or P. indica bacterium. It also revealed that the genus Bacillus is not an exclusive producer of lichenysin and surfactin.

Assessing the changes in marine microalgae diversity in the Nanji Islands Nature Reserve over the past decades using sediment eDNA

The Nanji Islands, designated as one of China’s national nature reserves, are renowned for their abundant microalgal resources. Changes in microalgae can serve as indicators of marine environmental shifts. Despite this significance, the absence of comprehensive historical records on microalgal diversity has limited the evaluation of marine environmental protection efforts. This study addresses this gap by analyzing surface sediments and sediment cores from the Nanji Islands, utilizing 210Pb dating and employing environmental DNA (eDNA) technology to trace historical shifts in the molecular diversity of microalgae. Chloroplast gene fragments were amplified using rbcL primers, and 210Pb dating determined the deepest sediment layer (78-80 cm) to date back to 1994. From the 70-72 cm layer to the 60-62 cm layer, the number of microalgal reads increased rapidly from 27,716 to 65,143, signifying enhanced abundance over the 10 years following the establishment of the nature reserve. This was followed by a deceleration in microalgal abundance growth over the subsequent 20 years, potentially reflecting variations in primary production of microalgae. Concurrently, the rise in operational taxonomic units (OTUs) and the Margalef index suggest a boost in microalgal diversity, which may be attributed to improvements in the marine environment. The microalgal community composition has shifted from a dominance of Dinoflagellata and Streptophyta to a predominance of Bacillariophyta, the change in phosphate would be one of the impact factors worth noting. This study provides foundational data on the historical changes in microalgae in the Nanji Islands and serves as a reference for exploring the relationship between environmental conditions and microalgal dynamics.

Baicalin enhances antioxidant, inflammatory defense, and microbial diversity of yellow catfish (Pelteobagrus fulvidraco) infected with Aeromonas hydrophila.

The aim of this research was to clarify the mechanism through which baicalin exerts its inhibitory effects on Aeromonas hydrophila infection. The antibacterial efficacy of baicalin was assessed by determining its minimum inhibitory concentration (MIC) against A. hydrophila. Various parameters, including the growth curve, cell wall integrity, biofilm formation, AKP content, and morphological alterations of A. hydrophila, were analyzed. In vivo experiments involved the administration of A. hydrophila 4 h postintraperitoneal injection of varying doses of baicalin to induce infection, with subsequent monitoring of mortality rates. After a 3 d period, liver, spleen, and intestinal tissues were harvested to evaluate organ indices, antioxidant and immune parameters, as well as intestinal microbial composition. The findings indicated that baicalin treatment resulted in the disruption of the cell wall of A. hydrophila, leading to the loss of its normal structural integrity. Furthermore, baicalin significantly inhibited biofilm formation and facilitated the release of intracellular proteins (P < 0.05). In vivo, baicalin enhanced the survival rates of yellow catfish infected with A. hydrophila. Compared to the control group, the liver index of yellow catfish was elevated, while the spleen and intestinal indices were reduced in the baicalin-treated group (P < 0.05). Additionally, baicalin at an appropriate dosage was found to increase levels of SOD, GSH, CAT, ACP, and AKP in yellow catfish (P < 0.05), while simultaneously decreasing MDA accumulation and the mRNA expression of inflammatory markers such as Keap1, IL1, IFN-γ, and TNF-α, (P < 0.05). Moreover, baicalin significantly enhanced the operational taxonomic unit (OTU) count in A. hydrophila-infected yellow catfish (P < 0.05), restoring the abundance of Barnesiellaceae, Enterobacteriaceae, Plesiomonas, and UBA1819 (P < 0.05). In summary, baicalin demonstrates the potential to improve the survival rate of yellow catfish subjected to A. hydrophila infection, augment antioxidant and immune responses, mitigate inflammation, and enhance intestinal microbial diversity.

Host preference explains the high endemism of ectomycorrhizal fungi in a dipterocarp rainforest.

Ectomycorrhizal (ECM) fungi are important tree symbionts within forests. The biogeography of ECM fungi remains to be investigated because it is challenging to observe and identify species. Because most ECM plant taxa have a Holarctic distribution, it is difficult to evaluate the extent to which host preference restricts the global distribution of ECM fungi. To address this issue, we aimed to assess whether host preference enhances the endemism of ECM fungi that inhabit dipterocarp rainforests. Highly similar sequences of 175 operational taxonomic units (OTUs) for ECM fungi that were obtained from Lambir Hill's National Park, Sarawak, Malaysia, were searched for in a nucleotide sequence database. Using a two-step binomial model, the probability of presence for the query OTUs and the registration rate of barcode sequences in each country were simultaneously estimated. The results revealed that the probability of presence in the respective countries increased with increasing species richness of Dipterocarpaceae and decreasing geographical distance from the study site (i.e. Lambir). Furthermore, most of the ECM fungi were shown to be endemic to Malaysia and neighbouring countries. These findings suggest that not only dispersal limitation but also host preference are responsible for the high endemism of ECM fungi in dipterocarp rainforests. Moreover, host preference likely determines the areas where ECM fungi potentially expand and dispersal limitation creates distance-decay patterns within suitable habitats. Although host preference has received less attention than dispersal limitation, our findings support that host preference has a profound influence on the global distribution of ECM fungi.

Impact of a Potent Strain of Plant Growth-Promoting Bacteria (PGPB), Bacillus subtilis S1 on Bacterial Community Composition, Enzymatic Activity, and Nitrogen Content in Cucumber Rhizosphere Soils.

Antagonistic bacterial strains from Bacillus spp. have been widely studied and utilized in the biocontrol of phytopathogens and the promotion of plant growth, but their impacts on the rhizosphere microecology when applied to crop plants are unclear. Herein, the effects of applying the antagonistic bacterium Bacillus subtilis S1 as a biofertilizer on the rhizosphere microecology of cucumbers were investigated. In a pot experiment on cucumber seedlings inoculated with S1, 3124 bacterial operational taxonomic units (OTUs) were obtained from the rhizosphere soils using high-throughput sequencing of 16S rRNA gene amplicons, and the most abundant phylum was Proteobacteria that accounted for 49.48% in the bacterial community. S1 treatment significantly reduced the abundances of soil bacterial taxa during a period of approximately 30days but did not affect bacterial diversity in the rhizosphere soils of cucumbers. The enzymatic activities of soil nitrite reductase (S-Nir) and dehydrogenase (S-DHA) were significantly increased after S1 fertilization. However, the activities of soil urease (S-UE), cellulase (S-CL), and sucrase (S-SC) were significantly reduced compared to the control group. Additionally, the ammonium- and nitrate-nitrogen contents of S1-treated soil samples were significantly lower than those of the control group. S1 fertilization reshaped the rhizosphere soil bacterial community of cucumber plants. The S-CL activity and nitrate-nitrogen content in rhizosphere soil affected by S1 inoculation play important roles in altering the abundance of rhizosphere soil microbiota.

Effects of Microplastic on the Gastrointestinal Tract and Gut microbiome of Sprague Dawley Rats

ABSTRACT Nowadays there is evidence that animals are exposed to microplastics (MPs) via food, however there is not much known about their effect on tissue of gastrointestinal tract and on gut microbiome. In this study, adult male rats were fed with polyethylene MPs, mixed with standard pellet food for 24 days at different concentrations ranging from 0 to 1000 μg/day. At the end of experiment, rats were euthanized and histopathological investigation using light and electron microscopy and the gut microbiome was analyzed using MiSeq 16S rRNA amplicon sequencing. Microscopic analysis revealed significant impact of MPs on the intestine, including visible changes to the crypt area as well as reduction in mucus secretion. Autophagic vacuoles were observed in the livers of the rats fed with MPs at 100 µg/day. Oxidative stress was evident in rats fed with 1000 µg/day of MPs, as indicated by the presence of myeloid bodies in both intestinal and hepatic cells. The gut microbiome was also affected by MPs. Although, no distinct clusters were formed when the bacterial communities in the different treatments were ordinated by multidimensional scaling (NMDS), the diversity indices including operational taxonomic unit (OTU) richness and Chao1 exhibited an increasing trend with increasing MPs concentration. Pearson correlations revealed a linear increase in the relative sequence abundance of Clostridia (R=0.036, P=0.003) with increasing MPs concentration, but not in the case of other bacterial groups,. Lactobacillus faecis was the most abundant OTU in the entire dataset, and its relative abundance decreased significantly with increasing MPs concentration. We conclude that exposure to MPs can disrupt cellular function and disturb microbiome balance, and in the event of prolonged exposure, organisms might experience unpredictable effects.

PSI-30 Effects of high canola meal diet with acidifiers supplementation on cecal microbiota of nursery pigs

Abstract Inclusion of canola meal (CM) in swine diets is typically limited stemming from the presence of glucosinolates (Gls), which can reduce growth performance and cause thyroid dysfunction in nursery pigs. While certain members of hindgut bacterial communities are capable of degrading dietary Gls, which would mitigate their detrimental effects, they have yet to be identified. Moreover, lowering the pH in the hindgut of the pig can break down Gls into less toxic compounds, potentially reducing harm. Thus, this study aimed to characterize major bacterial species populating the hindgut of nursery pigs fed a high CM diet supplemented with acidifier and encapsulated butyrate. A total of 315 nursery pigs weaned at 21 d old, average body weight (BW) 6.0 ± 1.2 kg, were assigned to one of five dietary treatments with 9 replicate pens (7 pigs/pen) in a randomized complete block design. The dietary treatments included PC: a corn-soybean meal-based diet with 20% CM, NC: a corn-soybean meal-based diet with 40% CM, NC+A: NC diet supplemented with an acidifier at 1,500 ppm, NC+B: NC diet supplemented with encapsulated butyrate at 500 ppm, and NC+A+B: NC diet supplemented with both an acidifier and encapsulated butyrate. All diets were formulated to meet or exceed the nutrient requirements of NRC (2012). At the end of the feeding trial (6 wk), one pig from each pen was euthanized to collect fresh cecal digesta. Samples were analyzed using the 16S rRNA gene through PCR-amplification of the V1-V3 regions from microbial genomic DNA, followed by Illumina MiSeq 2X300 sequencing. A comparative analysis of the highly represented taxonomic groups and operational taxonomic units (OTU) using non-parametric Kruskal-Wallis sum-rank test and Wilcoxon pairwise test identified candidate bacterial groups or species enriched across different diets. Bacillota was the predominant phylum across all diets, ranging from 83.2% to 86.3%. At the genus level, Lachnospiracea incertae sedis was less abundant in the NC diets with acidifier (Acidifier effect, P &amp;lt; 0.05), whereas Dialister was less in the NC diets with encapsulated butyrate (Butyrate effect, P &amp;lt; 0.05). The OTU Ssd-00001 (Lactobacillus amylovorus, 99.6%) tended to be greater in the cecal microbiota of pigs fed the NC diets with acidifier (Acidifier effect, P = 0.09). Interestingly, OTU Ssd-00188 (Agathobacter rectalis, 99.2%) tended to be higher in PC diet than in NC diet (P = 0.06), while its abundance was less in the cecal microbiota of pigs fed the NC diets with acidifier (Acidifier effect, P = 0.09). In conclusion, these results provide valuable insights into the microbiome of pigs fed high CM diets. Understanding the interplay between microbial communities and high CM diet with acidifier supplementation is crucial for optimizing swine diets and mitigating challenges associated with the antinutritional effects of dietary Gls from CM.

Molecular phylogeny and species diversity of the genus Dichaetophora Duda and related taxa (Diptera: Drosophilidae)

Our intensive surveys of wild drosophilids in East and Southeast Asia discovered a great species diversity (more than 100 putatively new species) of the genus Dichaetophora, which is currently comprised of 67 formally described species assigned into five species groups, i.e., agbo, tenuicauda, acutissima, sinensis and trilobita. In the present study, we delimited species from a huge amount of samples of Dichaetophora and allied taxa (the genus Mulgravea and the subgenus Dudaica of Drosophila) collected from a wide range of the Oriental and east Palearctic regions. We first sorted all specimens into morpho-species, and representative specimen(s) selected from each morpho-species were subjected to barcoding of COI (the cytochrome c oxidase subunit I gene) sequences. The applied ASAP (Assemble Species by Automatic Partitioning) analysis estimated a total of 166 to 168 MOTUs (molecular operational taxonomic units). Integrating this result with morphological evidence from re-examined, detailed structures of male terminalia, we recognized a total of 144 (109 new and 35 known) species in our sample. Out of them, 83 species representing the supraspecific taxa of Dichaetophora, Mulgravea and Dudaica were selected, along with 33 species from major genera and subgenera of Drosophila in the tribe Drosophilini, as in-group and four species from the tribe Colocasiomyini as out-group for phylogenetic reconstruction based on 12 nuclear gene markers. In the trees constructed by the maximum likelihood and Bayesian inference methods, the three focal taxa (i.e., Dichaetophora, Mulgravea and Dudaica) formed a clade provisionally called the “pan-Dichaetophora”. Within this large clade, the agbo, tenuicauda, sinensis and trilobita groups of Dichaetophora, Mulgravea and Dudaica were recovered as monophyletic groups, but Dichaetophora and its acutissima group were regarded as paraphyletic. In addition, two clusters were recognized among ungrouped species of Dichaetophora. Thus, the present study has uncovered some issues concerning the taxonomy of the pan-Dichaetophora. Such issues will be addressed elsewhere in the phylogenetic reclassification of the pan-Dichaetophora, along with descriptions/redescriptions of a large number of new/known species delimited in the present study.

Cave Myotis (Myotis velifer) consume diverse prey items and provide important ecosystem services

Abstract Insectivorous bats play an essential role as predators in natural ecosystems and contribute to pest control in agricultural landscapes. However, characterizing diets of specific bat species is difficult using conventional methods that cannot capture detailed dietary information. In this study, we used metabarcoding of the cytochrome oxidase I mitochondrial gene to analyze fecal samples of Myotis velifer and provide insight into the seasonal variation of diet from a colony located in the Chihuahuan Desert region of Texas. After filtering sequence reads, we recovered and analyzed 706 molecular operational taxonomic units (MOTUs) in the diet of M. velifer. We found 484 taxa (species and genus level) belonging to 11 insect orders in 66 fecal samples collected from March to October 2021. The orders containing the most MOTUs were Diptera (n = 353), Lepidoptera (n = 160), and Blattodea (n = 59). In their diet, we identified important insect crop pests, non-native insects, and substantial consumption of mosquitoes. Dietary composition shifted throughout seasons. Bats captured in summer months consumed the highest diversity of arthropod orders, and those captured in the spring consumed more diet items but were less diverse in arthropod orders. Our results uncovered 2 additional orders consumed by M. velifer, Blattodea and Odonata, that have not been previously described in their diet.

Dietary supplementation of Roseovarius litoreus improves survival and immune defense of the swimming crab Portunus trituberculatus

The aquaculture of the swimming crab Portunus trituberculatus is challenged by vibriosis, and Rhodobacteraceae strains play a crucial role in maintaining gut health and enhancing the immunity of crustaceans. In this study, we investigated the effects of dietary supplementation of Roseovarius litoreus, a member of Rhodobacteraceae, on the survival and immune defense of the swimming crab. The results showed that 60-day dietary supplementation of R. litoreus resulted in a marked increase in the mRNA expression of gut ZO-1. Meanwhile, R. litoreus increased zero-radius operational taxonomic unit (ZOTU) richness and changed the gut bacterial community, which was manifested by the enriched Carboxylicivirga and Roseovarius and depleted Photobacterium and Vibrio, thereby upregulating functional potentials of the gut bacterial community in the immune system and biosynthesis of other secondary metabolites. After a 72-h V. parahaemolyticus challenge, R. litoreus-supplemented crabs had an increased survival rate, reduced histological damage of hepatopancreas and gut, and improved expressions of immune-related genes and lysozyme activity. Moreover, R. litoreus-supplemented crab gut still harbored more Carboxylicivirga and Roseovarius but had fewer Vibrio gene copies than control crabs, which led to similar changes in functional potentials. Notably, all ZOTUs that were positively correlated with immune-related genes and lysozyme activity belonged to Bacteroidetes, Bacteroidales, or Carboxylicivirga. Taken together, we highlight the probiotic role of R. litoreus in the survival and immune defense of the swimming crab. These findings may contribute to the development of indigenous bacteria of the swimming crab as potential probiotics for the control of vibriosis.

Gut Microbiome variation in patients with early-stage mild-to-moderate intracerebral hemorrhage: A pilot study exploring therapeutic targets

BackgroundThe significant morbidity and mortality rates of acute intracerebral hemorrhage (ICH) are well-known around the world. The link between gut microbiota and different types of strokes is becoming more studied. The goal of this study was to look at the relationships between intestinal flora and early-stage mild-to-moderate ICH (emICH), and to provide a new perspective for adjunctive treatment of emICH. MethodsFecal samples from 100 participants with emICH (n=50) and healthy individuals (n=50) in this study were collected as well as analyzed utilizing 16S rRNA gene amplicon sequencing in order to characterize the gut microbial community. ResultsDistinct microbial communities are present within each group, with emICH patients exhibiting a diminished diversity and uniformity in their microbial profiles. A notable shift in the gut microbiota composition of emICH patients has been observed, characterized by an upsurge in pro-inflammatory microbes belonging to the Euryarchaeota phylum and a concurrent decline in beneficial Bacteroidetes species. Concurrently, significant associations and patterns among operational taxonomic units (OTUs) were identified in emICH patients. A panel of biomarkers (WAL_1855D, Methanobrevibacter, Streptococcus, Bacteroides, Coprococcus, Lachnospira) has been effectively utilized to distinguish emICH patients from healthy individuals, with an area under the curve (AUC) of 0.845. Additionally, an analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation uncovered several perturbed pathways in emICH patients, predominantly those related to metabolic processes and the inflammatory response. Moreover, predictive profiling of the microbiome's phenotypic traits suggests that emICH patients are likely to harbor a higher prevalence of Gram-negative bacteria and potential opportunistic pathogens compared to healthy controls. ConclusionsThe gut microbiota ecosystem of emICH patients is disrupted, characterized primarily by an increase in pro-inflammatory microbiota, elevated inflammatory signaling pathways, and metabolic dysregulation. Furthermore, microbiota modulation may be seen as a novel approach for the adjunctive treatment of emICH.

Genetic relationships of three species of black flies (Diptera: Simuliidae) in Taiwan

As an island about 150 km from the mainland, Taiwan would be expected to have endemic species. About 64 % of its 36 species of black flies are considered endemic, more than twice the level of endemicity that has been recorded for all insects on the island. To begin assessing the validity of the high level of endemism for the Simuliidae, we used giant chromosome banding patterns and cytochrome c oxidase I (COI) sequences, against a well-defined morphological backdrop, to evaluate three of Taiwan's black flies, Simulium chungi Takaoka & Huang, S. pingtungense Huang & Takaoka, and S. sakishimaense Takaoka. Molecular data revealed high similarity of populations of S. sakishimaense in Taiwan and at the type locality on Ishigaki Island, Japan, about 180 km to the east. Thus, populations referred to as S. sakishimaense in Taiwan are conspecific with typical S. sakishimaense in Japan, confirming their non-endemicity in Taiwan. Simulium sakishimaense might have reached Ishigaki by island hopping via Taiwan from the Chinese mainland. Chromosomes and the COI gene agree with morphology that S. sakishimaense is a member of the S. multistriatum species group although the chromosomal banding patterns do not indicate that it is distinct from S. fenestratum Edwards on the mainland. Although molecular sequences indicate S. sakishimaense is monophyletic, this taxon falls within the same Operational Taxonomic Unit as nine other members of the S. multistriatum group, including S. fenestratum. Simulium pingtungense, in agreement with morphology, is molecularly distinct from the 10 other analyzed members of the S. striatum species group, tentatively suggesting that it is endemic to Taiwan, pending analysis of samples from mainland China. Simulium chungi in Taiwan is chromosomally and molecularly unique, with larvae resembling those of S. saskishimaense. It is not, however, a member of either the S. multistriatum or S. striatum species groups. For now, the S. chungi species group remains a legitimate taxon consisting of two species. Strengthening the case for endemic taxa in Taiwan awaits analysis of key samples from the Chinese mainland.

16S rRNA phylogeny and clustering is not a reliable proxy for genome-based taxonomy in Streptomyces.

Streptomyces is among the most extensively studied genera of bacteria but its complex taxonomy remains contested and is suspected to contain significant species-level misclassification. Resolving the classification of Streptomyces would benefit many areas of applied microbiology that rely on an accurate ground truth for grouping of related organisms, including comparative genomics-based searches for novel antimicrobials. We survey taxonomic conflicts between 16S rRNA and whole genome-based Streptomyces classifications using 2276 publicly available Streptomyces genome assemblies and 48 981 publicly available full-length 16S rRNA Streptomyces sequences from silva, Greengenes, Ribosomal Database Project (RDP), and NCBI (National Centre for Biotechnology Information) databases. We construct a full-length 16S gene tree for 14 239 distinct Streptomyces sequences that resolves three major lineages of Streptomyces, but whose topology is not consistent with existing taxonomic assignments. We use these sequence data to delineate 16S and whole genome landscapes for Streptomyces, demonstrating that 16S and whole-genome classifications are frequently in disagreement, and that 16S zero-radius Operational Taxonomic Units (zOTUs) are often inconsistent with Average Nucleotide Identity (ANI)-based taxonomy. Our results strongly imply that 16S rRNA sequence data does not map to taxonomy sufficiently well to delineate Streptomyces species routinely. We propose that alternative marker sequences should be adopted by the community for classification and metabarcoding. Insofar as Streptomyces taxonomy has been determined or supported by 16S sequence data and may in parts be in error, we also propose that reclassification of the genus by alternative approaches may benefit the Streptomyces community.

Microbial Heterogeneity Identification of Cerebral Thrombi Via Metagenomic Next-Generation Sequencing-Based Strategy.

Diagnosis of the cause of cerebral thrombi is vital for recurrence prevention but also challenging. The presence of the microbiome has recently been confirmed in thrombus, suggesting a novel approach to distinguish cerebral thrombi of different origins. However, little is known about whether there is heterogeneity in microbiological colonization of cerebral thrombi of different sources. Forty patients experiencing acute ischemic stroke were included and clinical data were collected. Metagenomic next-generation sequencing was adopted to detect bacterial and genomic signatures of human cerebral thrombi samples. We found similar species diversity between the large-artery atherosclerosis thrombi and cardioembolic thrombi but different species composition and distribution of cerebral thrombus microbiota. Compared with the group with cardioembolism, the group with large-artery atherosclerosis showed a significantly higher relative abundance of Ralstonia insidiosa among the top 10 bacterial species in cerebral thrombi. Twenty operational taxonomy units were correlated with 11 clinical indicators of ischemic stroke. The Gene Ontology enrichment analysis revealed 9 different enriched biological processes (translation and carbohydrate metabolic process, etc). The enriched Kyoto Encyclopedia of Genes and Genomes pathways included ribosome, butanoate metabolism, and sulfur metabolism. This study, based on the approach of metagenomic next-generation sequencing, provides a diagnostic microbiological method to discriminate individuals with cardioembolic thrombi from those with large-artery atherosclerosis thrombi with human cerebral thrombi samples. Our findings provide a fresh perspective on microbial heterogeneity of cerebral thrombi and demonstrate biological processes and pathway features of cerebral thrombi.

Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free

BackgroundDNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads.ResultsWe show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12–15 h of sequencing.ConclusionOur results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.

Response of soil fungal-community structure and function to land conversion to agriculture in desert grassland.

Land conversion to agriculture is an important factor affecting soil ecological processes in the desert grasslands of northern China. However, soil fungal-community structure and function in response to Land conversion remain unclear. In this study, desert grassland, artificial shrubland, and land conversion were investigated in the western part of the Mu Us Sandland (Yanchi, Ningxia; Dingbian, Shaanxi). We found that land conversion significantly increased soil total carbon, nitrogen, and phosphorus, and available phosphorous and potassium contents. In the early stage of conversion to agricultural (April), soil fungal operational taxonomic units and abundance-based coverage estimator were lower than those of dessert grasslands and shrubland plots and had significant correlations with pH, electric conductivity, and available phosphorus and potassium. The dominant phyla strongly correlated with soil physicochemical properties. Concomitantly, the relative abundance of Glomeromycota was significantly lower, and the complexity of the network in the land conversion plots was lower than that in the shrubland plots. In the late stage of land conversion (September), soil fungal operational taxonomic units and abundance-based coverage estimator were lower in the conversion plots than in the desert grassland plots, with more complex network relationships compared to the desert grassland or shrubland plots. Symbiotrophic groups, a functional group of desert grassland soil fungi, can be used as a predictor of environmental change; in addition, land conversion decreases the relative abundance of arbuscular mycorrhizal functional groups. Our study highlights the response of soil fungal communities and functions to human disturbances in desert grasslands. Considering the potential of land conversion to agriculture to influence soil secondary salinization, there is a need for continued observation of soil ecological health over the time continuum of land conversion to agriculture.

Eyeing DNA barcoding for species identification of fish larvae.

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.