Highest Number Of Operational Taxonomic Units Research Articles

The Effect of Spatio‐Temporal Sampling and Biological Replication on the Detection of Kelp Forest Fish Communities Using eDNA Metabarcoding

ABSTRACTEnvironmental DNA (eDNA) metabarcoding surveys can support the acquisition of extensive biodiversity data to support ecosystem monitoring and conservation actions. However, the optimization of eDNA metabarcoding project design is essential to capture spatio‐temporal heterogeneity of eDNA signals and maximize diversity detection. In this study, we developed a system‐specific approach to detect fish communities in kelp forests, by analyzing fine‐scale spatio‐temporal patterns in eDNA signals at two sites along the South African coastline, as well as testing the effect of biological replication and pooling of replicates on species detection. At each site, samples were collected at two stations along the shoreline at two depth zones, and this was repeated at two time points (24 h apart). We detected 113 operational taxonomic units (OTUs) across 32 families, but fewer than 20% of OTUs could be assigned to species, indicating that barcode reference libraries need to be drastically improved. We detected significant differences in communities across small spatial scales (< 600 m) and time points, suggesting that to best capture a site's diversity patterns, samples should be collected at multiple points and times within at least 24 h. To detect ~80% of the fish community, including some low abundance species, a minimum of four samples appear sufficient. In addition, a higher number of OTUs (76 vs. 65) were found in individual replicates than in any of the pools. However, pooling samples prior to sequencing can still detect valuable broad‐scale biodiversity patterns for monitoring and can offset the decrease in data resolution with the benefit of accumulating comprehensive data from increased sampling efforts over time. As a pilot investigation into how best to maximize kelp forest‐associated fish communities, this study provides a basis for optimizing sampling design for coastal eDNA‐based surveys in southern Africa and strengthens the development of long‐term eDNA monitoring programs to better support conservation and management actions.

A preliminary study into the detection of fish environmental DNA in selected South African estuaries

Environmental DNA (eDNA) is a powerful technique for the non-invasive and comprehensive monitoring of entire ecosystems. Despite its many advantages, the use of eDNA in South Africa as an assessment tool for fish biodiversity is largely underutilised. This study was performed as an initial screening to assess the efficiency of the eDNA method for characterising fish communities in South African waters using the metabarcoding approach. Water samples were collected from eight estuaries and vacuum filtered using 0.45 μm nylon membrane filters. DNA was extracted from the filters, and sample extracts underwent PCR assaying using MiFish primers for the 12S rRNA gene, prior to being sequenced on the Illumina MiSeq platform. The resulting sequence data produced 1 574 092 raw reads, which resulted in 119 operational taxonomic units (OTUs) after filtering and clustering. Taxonomic assignment of the OTUs produced four different taxonomic groups and one group representing unidentified sequences. The group with the highest number of OTUs (54%) was assigned to fish taxa consisting of 12 families and 22 genera identified across all estuaries, with only 16 taxa identified to species level. As this was a preliminary study, few samples were collected, and therefore several recommendations for a more comprehensive study design are provided. Overall, these results support the hypothesis that the eDNA metabarcoding approach can efficiently detect fish species in South African estuarine environments.

Marine Bacterioplankton Community Dynamics and Potentially Pathogenic Bacteria in Seawater around Jeju Island, South Korea, via Metabarcoding.

Understanding marine bacterioplankton composition and distribution is necessary for improving predictions of ecosystem responses to environmental change. Here, we used 16S rRNA metabarcoding to investigate marine bacterioplankton diversity and identify potential pathogenic bacteria in seawater samples collected in March, May, September, and December 2013 from two sites near Jeju Island, South Korea. We identified 1343 operational taxonomic units (OTUs) and observed that community diversity varied between months. Alpha- and Gamma-proteobacteria were the most abundant classes, and in all months, the predominant genera were Candidatus Pelagibacter, Leisingera, and Citromicrobium. The highest number of OTUs was observed in September, and Vibrio (7.80%), Pseudoalteromonas (6.53%), and Citromicrobium (6.16%) showed higher relative abundances or were detected only in this month. Water temperature and salinity significantly affected bacterial distribution, and these conditions, characteristic of September, were adverse for Aestuariibacter but favored Citromicrobium. Potentially pathogenic bacteria, among which Vibrio (28 OTUs) and Pseudoalteromonas (six OTUs) were the most abundant in September, were detected in 49 OTUs, and their abundances were significantly correlated with water temperature, increasing rapidly in September, the warmest month. These findings suggest that monthly temperature and salinity variations affect marine bacterioplankton diversity and potential pathogen abundance.

Bacterial community structure and predicted function in the rhizosphere of wild and cultivated date palms: Effects of Fertilizers on Composition and Functionality

This study investigated bacterial diversity in date palm rhizosphere and assessed the influence of different fertilizers on diversity using metagenomics. A total of 6356 Operational Taxonomic Units (OTUs) and 1164425 sequences were analyzed across 27 samples. The findings revealed variations in microbial community phylogeny among similar cultivars from different farms, except for the Khalas cultivar, suggesting minor influence of genotype on microbial community structure. Wild date palms exhibited more unique OTUs, in contrast, cultivated date palms had a higher number of OTUs and diversity. Moreover, different fertilizer treatments had varying effects on bacterial diversity. For example, organic and bio-organic fertilizers positively influenced specific bacterial groups, including delta-proteobacteria, acidobacteria-Gp3, Anaerolinaea, and Clostridia. Conversely, combining high concentrations of chemical fertilizers with other types did not show significant effects. Classes such as Bacilli, Nitrospira, Deltaproteobacteria, Spartobacteria, and Thermomacrobia exhibited high relative abundances in treatments with high chemical fertilizer concentrations. Our analysis revealed potential pathways related to carbon, nitrogen, phosphorus, phenol, sulfur, and antimicrobial compounds. Interestingly, these pathways and functions varied across different date palm cultivars. Our findings suggest that date palm cultivars shape the rhizosphere by selectively influencing bacterial communities consistently across other locations. These modulated bacterial communities can potentially provide enhanced benefits to the host. Importantly, this study was the first to investigate the soil bacterial diversity of wild date palms, giving valuable insights into the microbial community associated with this specific context.

Observation of the Gut Microbiota Profile in C57BL/6 Mice Induced by Plasmodium berghei ANKA Infection.

The genus of Plasmodium parasites can cause malaria, which is a prevalent infectious disease worldwide, especially in tropical and subtropical regions. C57BL/6 mice infected with P. berghei ANKA (PbA) will suffer from experimental cerebral malaria (ECM). However, the gut microbiota in C57BL/6 mice has rarely been investigated, especially regarding changes in the intestinal environment caused by infectious parasites. P. berghei ANKA-infected (PbA group) and uninfected C57BL/6 (Ctrl group) mice were used in this study. C57BL/6 mice were infected with PbA via intraperitoneal injection of 1 × 106 infected red blood cells. Fecal samples of two groups were collected. The microbiota of feces obtained from both uninfected and infected mice was characterized by targeting the V4 region of the 16S rRNA through the Illumina MiSeq platform. The variations in the total gut microbiota composition were determined based on alpha and beta diversity analyses of 16S rRNA sequencing. The raw sequences from all samples were generated and clustered using ≥ 97% sequence identity into many microbial operational taxonomic units (OTUs). The typical microbiota composition in the gut was dominated by Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia at the phylum level. Bacteroidetes and Verrucomicrobia were considerably decreased after PbA infection compared with the control group (Ctrl), while Firmicutes and Proteobacteria were increased substantially after PbA infection compared with Ctrl. The alpha diversity index showed that the observed OTU number was increased in the PbA group compared with the Ctrl group. Moreover, the discreteness of the beta diversity revealed that the PbA group samples had a higher number of OTUs than the Ctrl group. LEfSe analysis revealed that several potential bacterial biomarkers were clearly related to the PbA-infected mice at the phylogenetic level. Several bacterial genera, such as Acinetobacter, Lactobacillus, and Lachnospiraceae_NK4A136_group, were overrepresented in the PbA-infected fecal microbiota. Meanwhile, a method similar to gene coexpression network construction was used to generate the OTU co-abundance units. These results indicated that P. berghei ANKA infection could alter the gut microbiota composition of C57BL/6 mice. In addition, potential biomarkers should offer insight into malaria pathogenesis and antimalarial drug and malaria vaccine studies.

Diversity and Spatiotemporal Dynamics of Fungal Communities in the Rhizosphere Soil of Cotton in the Arid Region of Northwest China.

This study aimed to investigate the fungal diversity and its temporal and spatial dynamics in the rhizosphere soil of healthy cotton by high-throughput sequencing. We studied species richness, composition, and distribution of cotton rhizosphere fungal community with respect to location (Alaer, Kuerle, Tumushuke, Hami, Shihezi, Wusu, and Jinghe) and plant growth period (seedling stage, bud stage, flowering stage, and boll-opening stage) using the methods of PCR-based high-throughput sequencing and real-time quantitative PCR. A total of 1,838,454 fungal nuclear ribosomal internal transcribed spacer region sequences (rRNA ITS) were obtained from all cotton plants sampled at different growth stages in the seven locations in Xinjiang. The most abundant fungal group in the cotton rhizosphere was the Ascomycota (78.72%), followed by the Zygomycota (9.56%) and Basidiomycota (2.77%). These sequences revealed an enormous number of operational taxonomic units (OTUs) in cotton (1802 unique OTUs), with 67-464 OTUs in a single cotton sample, at a 3% threshold and a sequencing depth of 30,000 sequences. We identified 33 classes and 389 genera from the resulting 1,800,714 sequences. Sordariomycetes was the most frequent class in all samples, followed by Leotiomycetes and Eurotiomycetes. There were some differences in OTUs among different growth stages, but the differences were not significant, with 382 OTUs (14.66%) being common to each of the stages. A marked difference in the diversity of fungi in the rhizosphere soil of cotton was evident among the different locations, with the highest number of OTUs being detected in Jinghe (1084 OTUs) and clusters of OTUs representative of northern and eastern Xinjiang being detected. There were significantly more tags of Mortierella in Jinghe and Wusu than in the other sampling sites. The dynamics of the rhizosphere fungal communities were influenced by sampling sites. To the best of our knowledge, the current study is the first application of PCR-based Illumina to characterize and compare the fungal biodiversity in multiple rhizosphere soil samples from cotton.

Actinobacteria associated with Chinaberry tree are diverse and show antimicrobial activity

Many actinobacteria produce secondary metabolites that include antimicrobial compounds. Since most of the actinobacteria cannot be cultivated, their antimicrobial potential awaits to be revealed. We hypothesized that the actinobacterial endophyte communities inside Melia toosendan (Chinaberry) tree are diverse, include strains with antimicrobial activity, and that antimicrobial activity can be detected using a cultivation independent approach and co-occurrence analysis. We isolated and identified actinobacteria from Chinaberry, tested their antimicrobial activities, and characterized the communities using amplicon sequencing and denaturing gradient gel electrophoresis as cultivation independent methods. Most of the isolates were identified as Streptomyces spp., whereas based on amplicon sequencing the most abundant OTU was assigned to Rhodococcus, and Tomitella was the most diverse genus. Out of the 135 isolates, 113 inhibited the growth of at least one indicator organism. Six out of the 7577 operational taxonomic units (OTUs) matched 46 cultivated isolates. Only three OTUs, Streptomyces OTU4, OTU11, and OTU26, and their corresponding isolate groups were available for comparing co-occurrences and antimicrobial activity. Streptomyces OTU4 correlated negatively with a high number of OTUs, and the isolates corresponding to Streptomyces OTU4 had high antimicrobial activity. However, for the other two OTUs and their corresponding isolate groups there was no clear relation between the numbers of negative correlations and antimicrobial activity. Thus, the applicability of co-occurrence analysis in detecting antimicrobially active actinobacteria could not be proven.

Spatial Variation of -communities in the Low Oxygen Waters of Prado Hydroelectric (South-West of Colombia)

Denitrification is a process of reduction of nitrate to dinitrogen by facultative anaerobic microorganisms, which have functional genes encoding denitrification enzymes (reductases). The nosZ gene encoding the enzyme that reduces nitrous oxide to dinitrogen was utilized in this research, as molecular marker for denitrifying communities into low oxygen waters of Prado reservoir. Our objective was to analyze and compare the composition (richness and abundance) of nosZ-type denitrifiers in relationship with physicochemical variables (oxygen, pH, temperature, nitrate, nitrite and ammonium) in three areas of this dam: Isla del Sol, Lozanía and Tomogó which are distant and have different anthropogenic influences. For this, we performed DNA extraction, amplification, 454 pyrosequencing and phylogenetic analysis of nosZ gene. The Chao1 estimator and Shannon index were used for compare richness and diversity of nosZ gene; and the relationship between compositions of operational taxonomic units (OTU) with physicochemical variables was established by canonical correspondence analysis (CCA). In the reservoir 45 nosZ-OTUs to species level (80% similarity) were detected. Lozanía had the highest number of OTUs (25) and greatest diversity (SCHAO1: 35; H: 2.0), compared to the other areas sampling. The phylogenetic analysis showed the presence of many OTUs (28 of 45) with low similarity to Proteobacteria group and high similarity with sequences of environmental clones reported previously. The CCA showed that the nosZ-community composition of Prado dam was related with low pH (6.2), oxygen (0.01mg / L) and nitrate (<0.25 mg / L) recorded in the water column.

DGGE and multivariate analysis of a yeast community in spontaneous cocoa fermentation process.

Cocoa bean is the main raw material used in the production of chocolate. In southern Bahia, Brazil, cocoa farming and processing is an important economic activity. The fermentation of cocoa is the processing stage that yields important chocolate flavor precursors and complex microbial involvement is essential for this process. In this study, PCR-denaturing gradient gel electrophoreses (DGGE) was used to investigate the diversity of yeasts present during the spontaneous fermentation of cocoa in southern Bahia. The DGGE analysis revealed a richness of 8 to 13 distinct bands of varied intensities among the samples; and samples taken at 24, 36, and 48 h into the fermentation process were found to group with 70% similarity and showed the greatest diversity of bands. Hierarchical clustering showed that all samples had common operational taxonomic units (OTUs) and the highest number of OTUs was found in the 48 h sample. Variations in pH and temperature observed within the fermenting mass over time possibly had direct effects on the composition of the existing microbial community. The findings reported here indicate that a heterogeneous yeast community is involved in the complex cocoa fermentation process, which is known to involve a succession of specialized microorganisms.

Assessing the dynamic of microbial communities during leaf decomposition in a low-order stream by microscopic and molecular techniques

Although a number of studies have indicated that microbes are key players in nutrient cycling, limitations on how to accurately assess their diversity have constrained further knowledge on the role of microbial diversity in organic matter decomposition in streams. Microbial diversity on leaf litter of Alnus glutinosa was assessed by microscopic analysis of bacterial cells and released fungal conidia, and by the number of operational taxonomic units (OTUs) from denaturing gradient gel electrophoresis using two different primer pairs targeting the rDNA of fungi (ITS2 and 5' end of the 18S region) and bacteria (V3 region and V6-V8 regions). Fingerprints of fungal and bacterial DNA showed a higher diverse microbial community on decomposing leaves than that assessed by microscopy-based techniques. Higher number of OTUs was obtained with primers targeting the ITS2 region of fungi, but the selected primers for bacteria showed similar number of OTUs. A succession of fungal or bacterial taxa throughout leaf decomposition was found, regardless of the chosen primer. These microbial communities ensured a rapid decomposition of submerged leaf litter (k=-0.045 day(-1)). Fungal biomass (up to 58 mg g(-1) AFDM) contributed with more than 98% to the total microbial biomass, supporting a greater role of fungi than bacteria in leaf-litter decomposition in streams.

Detection and Phylogenetic Relationships of Highly Diverse Uncultured Acidobacterial Communities in Altamira Cave Using 23S rRNA Sequence Analyses

Acidobacteria have been established as a novel and widespread bacterial phylum consistently detected during 16S rDNA-based molecular surveys. Recently, representatives of the phylum Acidobacteria were found to make up a large percentage of cloned sequences recovered from paleolithic paintings. The present study addresses the intraphylum diversity of Acidobacteria in Altamira Cave. In contrast to previous studies, we have focused on 23S rRNA-based phylogenetic analyses, exploiting the high information content of the 23S RNA gene. Comparison with existing studies was facilitated by the preparation of corresponding 16S rRNA clone libraries. Fourty-one distinct operational taxonomic units (OTU) could be identified. Of the eight described acidobacterial subgroups, five were represented in the cave; only subgroups 1, 2, and 8 were not found. Subgroup 4 was the group harbouring the highest number of OTUs. Various clusters that could not be assigned to any of the established subgroups, but were clearly grouped within the phylum and appeared to represent new taxa at elevated phylogenetic levels, were detected. Results from this study give a first insight into the enormous acidobacterial diversity existing in a single hypogean environment containing unique paleolithic paintings, the Altamira Cave. Novel taxa have been found within the phylum, suggesting a higher diversity of Acidobacteria than previously suspected even at a local scale.