A Goldilocks Principle for the Gut Microbiome: Taxonomic Resolution Matters for Microbiome-Based Classification of Colorectal Cancer

Despite being highly preventable, colorectal cancer remains a leading cause of cancer-related death in the United States. Low-cost, noninvasive detection methods could greatly improve our ability to identify and treat early stages of disease.

Genomic Evaluation of the Genus Coltivirus Indicates Genetic Diversity among Colorado Tick Fever Virus Strains and Demarcation of a New Species

The type species of the genus Coltivirus, Colorado tick fever virus (CTFV), was discovered in 1943 and is the most common tick-borne viral infection in the Western US. Despite its long history, very little is known about the molecular diversity of viruses classified within the species Colorado tick fever coltivirus. Previous studies have suggested genetic variants and potential serotypes of CTFV, but limited genetic sequence information is available for CTFV strains. To address this knowledge gap, we report herein the full-length genomes of five strains of CTFV, including Salmon River virus and California hare coltivirus (CTFV-Ca). The sequence from the full-length genome of Salmon River virus identified a high genetic identity to the CTFV prototype strain with >90% amino acid identity in all the segments except segment four, suggesting Salmon River virus is a strain of the species Colorado tick fever coltivirus. Additionally, analysis suggests that segment four has been associated with reassortment in at least one strain. The CTFV-Ca full-length genomic sequence was highly variable from the prototype CTFV in all the segments. The genome of CTFV-Ca was most similar to the Eyach virus, including similar segments six and seven. These data suggest that CTFV-Ca is not a strain of CTFV but a unique species. Additional sequence information of CTFV strains will improve the molecular surveillance tools and provide additional taxonomic resolution to this understudied virus.

Revealing microbial species diversity using sequence capture by hybridization

Targeting small parts of the 16S rDNA phylogenetic marker by metabarcoding reveals microorganisms of interest but cannot achieve a taxonomic resolution at the species level, precluding further precise characterizations. To identify species behind operational taxonomic units (OTUs) of interest, even in the rare biosphere, we developed an innovative strategy using gene capture by hybridization. From three OTU sequences detected upon polyphenol supplementation and belonging to the rare biosphere of the human gut microbiota, we revealed 59 nearly full-length 16S rRNA genes, highlighting high bacterial diversity hidden behind OTUs while evidencing novel taxa. Inside each OTU, revealed 16S rDNA sequences could be highly distant from each other with similarities down to 85 %. We identified one new family belonging to the order Clostridiales , 39 new genera and 52 novel species. Related bacteria potentially involved in polyphenol degradation have also been identified through genome mining and our results suggest that the human gut microbiota could be much more diverse than previously thought.

The Edible Plant Microbiome represents a diverse genetic reservoir with functional potential in the human host

Plant microbiomes have been extensively studied for their agricultural relevance on growth promotion and pathogenesis, but little is known about their role as part of the diet when fresh fruits and vegetables are consumed raw. Most studies describing these communities are based on 16S rRNA gene amplicon surveys, limiting our understanding of the taxonomic resolution at the species level and functional capabilities. In this study, we characterized microbes colonizing tomatoes, spinach, brined olives, and dried figs using shotgun metagenomics. We recovered metagenome-assembled genomes of novel lactic acid bacteria from green olives and identified high intra- and inter-specific diversity of Pseudomonas in tomatoes. All samples were colonized by Pseudomonas, consistent with other reports with distinct community structure. Functional characterization showed the presence of enzymes involved in vitamin and short chain fatty acid metabolism and degradation of diverse carbohydrate substrates including plant fibers. The dominant bacterial members were isolated, sequenced, and mapped to its metagenome confirming their identity and indicating the microbiota is culturable. Our results reveal high genetic diversity, previously uncultured genera, and specific functions reflecting a likely plant host association. This study highlights the potential that plant microbes can play when consumed as part of our diet and proposes these as transient contributors to the gut microbiome.

New insight into the taxonomic resolution of the genus Bythotrephes Leydig (Crustacea: Cladocera) based on molecular data from Central Europe

The taxonomic status of the genus Bythotrephes Leydig (Crustacea: Cladocera) has been debated since the second half of the XIX century. The most widespread view of recent decades has been that Bythotrephes is a monotypic genus, which was support by preliminary molecular data. However, the recent detailed morphological revision of this genus clearly distinguishes at least seven species. Therefore, we performed a multi-lake survey in Central Europe to give new insight into the taxonomic status of Bythotrephes by combining genetic analysis with traditional morphology-based taxonomy. Based on the morphology we identified two species in Central Europe, B. brevimanus and B. lilljeborgi, as well as hybrid forms. For the genetic analysis, we used newly obtained 113 sequences of mtDNA COI gene of the 535-bp length Bythotrephes from Central Europe and sequences downloaded from GenBank. There were no significant differences between all analyzed sequences, which supports the hypothesis that Bythotrephes is a monotypic genus, with only one highly polymorphic species. On the other hand, the results of our work could point out that the COI gene is insufficient to evaluate the taxonomic status of Bythotrephes. Nonetheless, we have identified 29 new haplotypes of mtDNA COI, and one which was the same as the haplotype found in North America and Finland. Furthermore, this haplotype was the source variant from which most other haplotypes were derived.

The bacterial hitchhiker's guide to COI: Universal primer-based COI capture probes fail to exclude bacterial DNA, but 16S capture leaves metazoa behind

Environmental DNA (eDNA) metabarcoding from water samples has, in recent years, shown great promise for biodiversity monitoring. However, universal primers targeting the cytochrome oxidase I (COI) marker gene popular in metazoan studies have displayed high levels of nontarget amplification. To date, enrichment methods bypassing amplification have not been able to match the detection levels of conventional metabarcoding. This study evaluated the use of universal metabarcoding primers as capture probes to either isolate target DNA, or to remove nontarget DNA, prior to amplification by using biotinylated versions of universal metazoan and bacterial barcoding primers, namely metazoan COI and bacterial 16S. Additionally, each step of the protocol was assessed by amplifying both COI and bacterial 16S to investigate the effect on the metazoan and bacterial communities. Bacterial abundance increased in response to the captures (COI library), while the quality of the captured DNA was improved. The metazoan-based probe captured bacterial DNA in a range that was also amplifiable with the 16S primers, demonstrating the ability of universal capture probes to isolate larger fragments of DNA from eDNA. This concept could be applied to metazoan metabarcoding, by using a truly conserved site without a high-level taxonomic resolution as a target of capture, to isolate DNA spanning over a nearby barcoding region, which can then be processed through conventional metabarcoding by amplification protocol.

First Report of Three Tylenchidae Taxa from Southern Alberta, Canada

Members of the family Tylenchidae are highly abundant in soil habitats, including agricultural settings, where they play key ecological roles. In the present study, we identified three Tylenchidae species, namely Basiria bhabi, Coslenchus acceptus, and Filenchus vulgaris, using integrative taxonomy. The detailed morphological and morphometric characteristics, distribution, and host associations of each species were also discussed. Phylogenetic analyses of these populations with other Tylenchidae nematodes indicated the presence of divergent lineages in Filenchus and Basiria, whereas Coslenchus appeared to be a monophyletic genus. Herein, we aim to grow awareness about this common but least studied group of nematodes. The species reported in this study are new records for Canada, revealing that the identified nematode diversity in our cultivated areas is relatively underrepresented. Our analyses also provided greater taxonomic resolution and captured rare taxa that might have been missed or misidentified in prior nematode inventory surveys. These findings will add to our understanding of the nematofauna of southern Alberta, thereby providing a more complete picture of existing nematode diversity present in the fields of this highly cultivated region.

Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians ecological traits (e.g., nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding-analysis of extra-organismal DNA released into the environment-allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160-311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata, and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set Amph16S had the highest resolution among the tested sets. Finally, we applied Amph16S to actual metabarcoding and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.