scholarly journals First Evidence That Nematode Communities in Deadwood Are Related to Tree Species Identity and to Co-Occurring Fungi and Prokaryotes

2021 ◽  
Vol 9 (7) ◽  
pp. 1454
Author(s):  
Julia Moll ◽  
Friederike Roy ◽  
Claus Bässler ◽  
Jacob Heilmann-Clausen ◽  
Martin Hofrichter ◽  
...  
Keyword(s):  
Tree Species ◽  
Forest Ecosystems ◽  
Amplicon Sequencing ◽  
Wood Properties ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Species Identity ◽  
Nematode Communities ◽  
Terrestrial Environments ◽  
Nematode Diversity

Nematodes represent a diverse and ubiquitous group of metazoans in terrestrial environments. They feed on bacteria, fungi, plants, other nematodes or parasitize a variety of animals and hence may be considered as active members of many food webs. Deadwood is a structural component of forest ecosystems which harbors many niches for diverse biota. As fungi and bacteria are among the most prominent decomposing colonizers of deadwood, we anticipated frequent and diverse nematode populations to co-occur in such ecosystems. However, knowledge about their ability to colonize this habitat is still limited. We applied DNA-based amplicon sequencing (metabarcoding) of the 18S rRNA gene to analyze nematode communities in sapwood and heartwood of decaying logs from 13 different tree species. We identified 247 nematode ASVs (amplicon sequence variants) from 27 families. Most of these identified families represent bacterial and fungal feeders. Their composition strongly depended on tree species identity in both wood compartments. While pH and water content were the only wood properties that contributed to nematodes’ distribution, co-occurring fungal and prokaryotic (bacteria and archaea) α- and β-diversities were significantly related to nematode communities. By exploring thirteen different tree species, which exhibit a broad range of wood characteristics, this study provides first and comprehensive insights into nematode diversity in deadwood of temperate forests and indicates connectivity to other wood-inhabiting organisms.

scholarly journals Forfeiting the founder effect: turnover defines biofilm community succession

2018 ◽  
Author(s):  
Colin J. Brislawn ◽  
Emily B. Graham ◽  
Karl Dana ◽  
Peter Ihardt ◽  
Sarah J. Fansler ◽  
...  
Keyword(s):  
Bacterial Colonization ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Community Succession ◽  
Ecological Processes ◽  
Functional Capabilities ◽  
Resolution Imaging ◽  
Successional Stages ◽  
Almost All

ABSTRACTMicrobial community succession is a fundamental process that effects underlying functions of almost all ecosystems; yet the roles and fates of the most abundant colonizers are poorly understood. Does early abundance spur long term persistence? How do deterministic and stochastic processes influence the roles of founder species? We performed a succession experiment within a hypersaline microbial mat ecosystem to investigate how ecological processes contributed to the turnover of founder species. Bacterial and micro-eukaryotic founder species were identified from primary succession and tracked through a defined maturation period using 16S and 18S rRNA gene amplicon sequencing in combination with high resolution imaging that utilized stable isotope tracers to evaluate basic functional capabilities. The majority of the founder species did not maintain high relative abundances in later stages of succession. Turnover (versus nestedness) was the dominant process shaping the final community structure. We also asked if different ecological processes acted on bacteria versus eukaryotes during successional stages and found that deterministic and stochastic forces corresponded more with eukaryote and bacterial colonization, respectively. Our results show that taxa from different kingdoms, that share habitat in the tight spatial confines of a biofilm, were influenced by different ecological forces and time scales of succession.

scholarly journals Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

2021 ◽  
Author(s):  
Kevin Xu Zhong ◽  
Anna Cho ◽  
Christophe M. Deeg ◽  
Amy M. Chan ◽  
Curtis A. Suttle
Keyword(s):  
18S Rrna ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Model Organisms ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Oyster Spat ◽  
Guide Rna ◽  
16S Rna ◽  
Eukaryotic Microbes

Abstract BackgroundThe microbiome affects the health of plants and animals, including humans, and has many biological, ecological and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. ResultsTo overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that >96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. ConclusionCCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Keywords: Eukaryotic microbiome, 18S rRNA gene, Microeukaryote, CRISPR-Cas, Taxon-specific single-guide RNA, gRNA-target-site, CasOligo, CCSAS

scholarly journals New Insights Into Nematode DNA-metabarcoding as Revealed by the Characterization of Artificial and Spiked Nematode Communities

Diversity ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 52 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nancy de Sutter ◽  
Nicole Viaene ◽  
Annelies Haegeman
Keyword(s):  
Species Richness ◽  
18S Rrna ◽  
Nematode Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Soil Nematode ◽  
Mock Community ◽  
Nematode Communities ◽  
Dna Metabarcoding ◽  
Soil Nematode Communities

Nematodes are ideal biological indicators to monitor soil biodiversity and ecosystem functioning. For this reason, they have been receiving increasing attention from a broad range of scientists. The main method to characterize soil nematode communities until at least genus level is still based on microscopic observations of nematode morphology. Such an approach is time-consuming, labor-intensive, and requires specialized personnel. The first studies on the potential use of DNA-metabarcoding to characterize nematode communities showed some shortcomings: under- or overestimation of species richness caused by failure to detect a number of nematode species or caused by intraspecific sequence variants increasing the number of OTUs (operational taxonomic units) or ‘molecular’ species, and flaws in quantification. We set up experiments to optimize this metabarcoding approach. Our results provided new insights such as the drastic effect of different DNA-extraction methods on nematode species richness due to variation in lysis efficacy. Our newly designed primer set (18S rRNA gene, V4-V5 region) showed in silico an improved taxonomic coverage compared with a published primer set (18S rRNA gene, V6-V8 region). However, results of DNA-metabarcoding with the new primer set showed less taxonomic coverage, and more non-nematode reads. Thus, the new primer set might be more suitable for whole soil faunal analysis. Species-specific correction factors calculated from a mock community with equal amounts of different nematode species were applied on another mock community with different amounts of the same nematode species and on a biological sample spiked with four selected nematode species. Results showed an improved molecular quantification. In conclusion, DNA-metabarcoding of soil nematode communities is useful for monitoring shifts in nematode composition but the technique still needs further optimization to enhance its precision.

scholarly journals Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

scholarly journals Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Feces

2008 ◽  
Vol 75 (1) ◽  
pp. 108-112 ◽  
Author(s):  
L. S. Waldron ◽  
B. C. Ferrari ◽  
M. R. Gillings ◽  
M. L. Power
Keyword(s):  
Species Identification ◽  
Restriction Fragment ◽  
Rflp Analysis ◽  
Cost Effective ◽  
18S Rrna Gene ◽  
Individual Species ◽  
Rrna Gene ◽  
Species Identity ◽  
Cryptosporidium Hominis ◽  
Detection And Identification

ABSTRACT Effective management of human cryptosporidiosis requires efficient methods for detection and identification of the species of Cryptosporidium isolates. Identification of isolates to the species level is not routine for diagnostic assessment of cryptosporidiosis, which leads to uncertainty about the epidemiology of the Cryptosporidium species that cause human disease. We developed a rapid and reliable method for species identification of Cryptosporidium oocysts from human fecal samples using terminal restriction fragment polymorphism (T-RFLP) analysis of the 18S rRNA gene. This method generated diagnostic fragments unique to the species of interest. A panel of previously identified isolates of species was blind tested to validate the method, which determined the correct species identity in every case. The T-RFLP profiles obtained for samples spiked with known amounts of Cryptosporidium hominis and Cryptosporidium parvum oocysts generated the two expected diagnostic peaks. The detection limit for an individual species was 1% of the total DNA. This is the first application of T-RFLP to protozoa, and the method which we developed is a rapid, repeatable, and cost-effective method for species identification.

scholarly journals Morphological and Molecular Identification of Fusarium oxysporum Sch. Isolated From Guava Wilt in Bangladesh

2012 ◽  
Vol 41 (1) ◽  
pp. 49-54 ◽  
Author(s):  
M Zakir Hussain ◽  
MA Rahman ◽  
Mohammad Nurul Islam ◽  
MA Latif ◽  
MA Bashar
Keyword(s):  
Fusarium Oxysporum ◽  
Psidium Guajava ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Species Identity ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Serious Disease ◽  
Species Specific

Wilt of guava plants (Psidium guajava L.) is a serious disease in Bangladesh. Sixteen isolates of Fusarium oxysporum Sch. were collected from the root and stem fragments of guava plants growing in six districts of Bangladesh. Species identity was based on the colony character, nature of conidiogenous cell, morphology of microconidia, macroconidia and chlamydospores. Eleven isolates were confirmed as F. oxysporum through polymerase chain reaction (PCR) using species specific primers designed from the conserved regions of 18S rRNA gene. DOI: http://dx.doi.org/10.3329/bjb.v41i1.11082 Bangladesh J. Bot. 41(1): 49-54, 2012 (June)

scholarly journals Sasso Pisano Geothermal Field Environment Harbours Diverse Ktedonobacteria Representatives and Illustrates Habitat-Specific Adaptations

2021 ◽  
Vol 9 (7) ◽  
pp. 1402
Author(s):  
Sania Arif ◽  
Corinna Willenberg ◽  
Annika Dreyer ◽  
Heiko Nacke ◽  
Michael Hoppert
Keyword(s):  
Hot Springs ◽  
Hot Spring ◽  
Amplicon Sequencing ◽  
Heavy Metal Resistance ◽  
18S Rrna Gene ◽  
Metal Resistance ◽  
Geothermal Field ◽  
Rrna Gene ◽  
Metabolic Potential ◽  
Eukaryotic Diversity

The hydrothermal steam environment of Sasso Pisano (Italy) was selected to investigate the associated microbial community and its metabolic potential. In this context, 16S and 18S rRNA gene partial sequences of thermophilic prokaryotes and eukaryotes inhabiting hot springs and fumaroles as well as mesophilic microbes colonising soil and water were analysed by high-throughput amplicon sequencing. The eukaryotic and prokaryotic communities from hot environments clearly differ from reference microbial communities of colder soil sites, though Ktedonobacteria showed high abundances in various hot spring samples and a few soil samples. This indicates that the hydrothermal steam environments of Sasso Pisano represent not only a vast reservoir of thermophilic but also mesophilic members of this Chloroflexi class. Metabolic functional profiling revealed that the hot spring microbiome exhibits a higher capability to utilise methane and aromatic compounds and is more diverse in its sulphur and nitrogen metabolism than the mesophilic soil microbial consortium. In addition, heavy metal resistance-conferring genes were significantly more abundant in the hot spring microbiome. The eukaryotic diversity at a fumarole indicated high abundances of primary producers (unicellular red algae: Cyanidiales), consumers (Arthropoda: Collembola sp.), and endoparasite Apicomplexa (Gregarina sp.), which helps to hypothesise a simplified food web at this hot and extremely nutrient-deprived acidic environment.

Inconsistent effects of nitrogen canopy enrichment and soil warming on black spruce epiphytic phyllosphere bacterial communities, taxa, and functions

2020 ◽  
Author(s):  
Rim Khlifa ◽  
Daniel Houle ◽  
Hubert Morin ◽  
Steven Kembel
Keyword(s):  
Climate Change ◽  
Boreal Forest ◽  
Bacterial Communities ◽  
Forest Ecosystems ◽  
Black Spruce ◽  
Amplicon Sequencing ◽  
Nitrogen Addition ◽  
Rrna Gene ◽  
Climate Change Scenarios ◽  
Soil Warming

Phyllosphere microbial communities have received considerable attention given their important influence on their plant hosts and on ecosystem functioning. In a context where climate change threatens the sustainability of ecosystems, it is important to understand how phyllosphere microbes will respond to changes in their environment. We used 16S rRNA gene amplicon sequencing to quantify phyllosphere bacterial communities of black spruce exposed to nitrogen canopy enrichment and soil warming in the boreal forest of Quebec, Canada. The treatments were applied from April to September 2015 and the sampling was done in September. Neither treatment influenced the overall community structure and diversity of black spruce phyllosphere bacterial communities. However, some bacterial taxa and inferred microbial functions did differ among treatments, revealing in particular a stronger response of some bacteria to soil warming rather than nitrogen enrichment. Our results suggest that soil warming could potentially induce more changes in phyllosphere bacterial taxa abundances and functions than nitrogen addition, with potential consequences for microbial diversity and boreal forest ecosystem function under likely climate change scenarios. Our study suggests avenues for further research to integrate a more mechanistic understanding of the importance of phyllosphere microbes for black spruce and boreal forest ecosystems.

scholarly journals Ontogeny, species identity, and environment dominate microbiome dynamics in wild populations of kissing bugs (Triatominae)

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Joel J. Brown ◽  
Sonia M. Rodríguez-Ruano ◽  
Anbu Poosakkannu ◽  
Giampiero Batani ◽  
Justin O. Schmidt ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Pathogenic Bacteria ◽  
Life Stage ◽  
Blood Feeding ◽  
18S Rrna Gene ◽  
Significant Loss ◽  
Individual Variability ◽  
Rrna Gene ◽  
Species Identity ◽  
Microbiome Composition

Abstract Background Kissing bugs (Triatominae) are blood-feeding insects best known as the vectors of Trypanosoma cruzi, the causative agent of Chagas’ disease. Considering the high epidemiological relevance of these vectors, their biology and bacterial symbiosis remains surprisingly understudied. While previous investigations revealed generally low individual complexity but high among-individual variability of the triatomine microbiomes, any consistent microbiome determinants have not yet been identified across multiple Triatominae species. Methods To obtain a more comprehensive view of triatomine microbiomes, we investigated the host-microbiome relationship of five Triatoma species sampled from white-throated woodrat (Neotoma albigula) nests in multiple locations across the USA. We applied optimised 16S rRNA gene metabarcoding with a novel 18S rRNA gene blocking primer to a set of 170 T. cruzi-negative individuals across all six instars. Results Triatomine gut microbiome composition is strongly influenced by three principal factors: ontogeny, species identity, and the environment. The microbiomes are characterised by significant loss in bacterial diversity throughout ontogenetic development. First instars possess the highest bacterial diversity while adult microbiomes are routinely dominated by a single taxon. Primarily, the bacterial genus Dietzia dominates late-stage nymphs and adults of T. rubida, T. protracta, and T. lecticularia but is not present in the phylogenetically more distant T. gerstaeckeri and T. sanguisuga. Species-specific microbiome composition, particularly pronounced in early instars, is further modulated by locality-specific effects. In addition, pathogenic bacteria of the genus Bartonella, acquired from the vertebrate hosts, are an abundant component of Triatoma microbiomes. Conclusion Our study is the first to demonstrate deterministic patterns in microbiome composition among all life stages and multiple Triatoma species. We hypothesise that triatomine microbiome assemblages are produced by species- and life stage-dependent uptake of environmental bacteria and multiple indirect transmission strategies that promote bacterial transfer between individuals. Altogether, our study highlights the complexity of Triatominae symbiosis with bacteria and warrant further investigation to understand microbiome function in these important vectors.

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