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Next Generation Sequencing Technologies (NGS), particularly metabarcoding, are valuable tools for authenticating foodstuffs and detecting eventual fraudulent practices such as species substitution. This technique, mostly used for the analysis of prokaryotes in several environments (including food), is in fact increasingly applied to identify eukaryotes (e.g., fish, mammals, avian, etc.) in multispecies food products. Besides the "wet-lab" procedures (e.g., DNA extraction, PCR, amplicon purification, etc.), the metabarcoding workflow includes a final "dry-lab" phase in which sequencing data are analyzed using a bioinformatic pipeline (BP). BPs play a crucial role in the accuracy, reliability, and interpretability of the metabarcoding results. Choosing the most suitable BP for the analysis of metabarcoding data could be challenging because it might require greater informatics skills than those needed in standard molecular analysis. To date, studies comparing BPs for metabarcoding data analysis in foodstuff authentication are scarce. In this study, we compared the data obtained from two previous studies in which fish burgers and insect-based products were authenticated using a customizable, ASV-based, and command-line interface BP (BP1) by analyzing the same data with a customizable but OTU-based and graphical user interface BP (BP2). The final sample compositions were compared statistically. No significant difference in sample compositions was highlighted by applying BP1 and BP2. However, BP1 was considered as more user-friendly than BP2 with respect to data analysis streamlining, cost of analysis, and computational time consumption. This study can provide useful information for researchers approaching the bioinformatic analysis of metabarcoding data for the first time. In the field of food authentication, an effective and efficient use of BPs could be especially useful in the context of official controls performed by the Competent Authorities and companies' self-control in order to detect species substitution and counterfeit frauds.

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As wheat (Triticum aestivum) is an important staple food across the world, preservation of stable yields and increased productivity are major objectives in breeding programs. Drought is a global concern because its adverse impact is expected to be amplified in the future due to the current climate change. Here, we analyzed the effects of edaphic, environmental, and host factors on the wheat root microbiomes collected in soils from six regions in Belgium. Amplicon sequencing analysis of unplanted soil and wheat root endosphere samples indicated that the microbial community variations can be significantly explained by soil pH, microbial biomass, wheat genotype, and soil sodium and iron levels. Under drought stress, the biodiversity in the soil decreased significantly, but increased in the root endosphere community, where specific soil parameters seemingly determine the enrichment of bacterial groups. Indeed, we identified a cluster of drought-enriched bacteria that significantly correlated with soil compositions. Interestingly, integration of a functional analysis further revealed a strong correlation between the same cluster of bacteria and ß-glucosidase and osmoprotectant proteins, two functions known to be involved in coping with drought stress. By means of this in silico analysis, we identified amplicon sequence variants (ASVs) that could potentially protect the plant from drought stress and validated them in planta. Yet, ASVs based on 16S rRNA sequencing data did not completely distinguish individual isolates because of their intrinsic short sequences. Our findings support the efforts to maintain stable crop yields under drought conditions through implementation of root microbiome analyses.

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The coastal region of the East China Sea (ECS) is a famous "hotspot" for harmful algal blooms (HABs) in China. We hypothesize that such frequent occurrences of diverse HABs in the ECS are determined by the presence of unique HAB species in this region. In this project, we identified 3966 amplicon sequence variants (ASVs) representing 35 classes in six protist phyla/divisions. Among the 237 annotated protist species, we identified 58 HAB species, of which 23 HAB species had never been previously reported in the ECS. Many HAB species also displayed unique spatial distribution patterns in the ECS. Notably, we identified three HAB species Prorocentrum donghaiense, Lebouridinium glaucum and Noctiluca scintillans in the site S05-1 with substantially elevated abundance, suggesting that this sampling site was experiencing a multiple-species HAB event. This study was the first attempt in applying ASV-based metabarcoding analysis in studying protist and HAB species in the ECS.

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Insects host a highly diverse microbiome, which plays a crucial role in insect life. However, the composition and diversity of microbiomes associated with Neotropical freshwater insects is virtually unknown. In addition, the extent to which diversification of this microbiome is associated with host phylogenetic divergence remains to be determined. Here, we present the first comprehensive analysis of bacterial communities associated with six closely related species of Neotropical water striders in Panama. We used comparative phylogenetic analyses to assess associations between dominant bacterial linages and phylogenetic divergence among species of water striders. We found a total of 806 16S rRNA amplicon sequence variants (ASVs), with dominant bacterial taxa belonging to the phyla Proteobacteria (76.87%) and Tenericutes (19.51%). Members of the α- (e.g., Wolbachia) and γ- (e.g., Acinetobacter, Serratia) Proteobacteria, and Mollicutes (e.g., Spiroplasma) were predominantly shared across species, suggesting the presence of a core microbiome in water striders. However, some bacterial lineages (e.g., Fructobacillus, Fluviicola and Chryseobacterium) were uniquely associated with different water strider species, likely representing a distinctive feature of each species' microbiome. These findings indicate that both host identity and environmental context are important drivers of microbiome diversity in water striders. In addition, they suggest that diversification of the microbiome is associated with diversification in water striders. Although more research is needed to establish the evolutionary consequences of host-microbiome interaction in water striders, our findings support recent work highlighting the role of bacterial community host-microbiome codiversification.

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The structure of microbial communities, microalgae, heterotrophic protozoa and fungi contributes to characterize food webs and productivity and, from an anthropogenic point of view, the qualitative characteristics of water bodies. Traditionally, in freshwater environments many investigations have been directed to the study of pelagic microalgae ("phytoplankton") and periphyton (i.e., photosynthetic and mixotrophic protists) through the use of light microscopy (LM). While the number of studies on bacterioplankton communities have shown a substantial increase after the advent of high-throughput sequencing (HTS) approaches, the study of the composition, structure, and spatio-temporal patterns of microbial eukaryotes in freshwater environments was much less widespread. Moreover, the understanding of the correspondence between the relative phytoplankton abundances estimated by HTS and LM is still incomplete. Taking into account these limitations, this study examined the biodiversity and seasonality of the community of eukaryotic microplankton in the epilimnetic layer of a large and deep perialpine lake (Lake Garda) using HTS. The analyses were carried out at monthly frequency during 2014 and 2015. The results highlighted the existence of a rich and well diversified community and the presence of numerous phytoplankton taxa that were never identified by LM in previous investigations. Furthermore, the relative abundances of phytoplankton estimated by HTS and LM showed a significant relationship at different taxonomic ranks. In the 2 years of investigation, the temporal development of the whole micro-eukaryotic community showed a clear non-random and comparable distribution pattern, with the main taxonomic groups coherently distributed in the individual seasons. In perspective, the results obtained in this study highlight the importance of HTS approaches in assessing biodiversity and the relative importance of the main protist groups along environmental gradients, including those caused by anthropogenic impacts (e.g., eutrophication and climate change).

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Temperature is a prominent abiotic environmental variable that drives the adaptive trajectories of animal lineages and structures the composition of animal communities. Global temperature regimes are expected to undergo rapid shifts in the next century, yet for many animal taxa we lack an understanding of the consequences of these predicted shifts for animal populations. In this review, we synthesize recent evidence that temperature variation shapes the composition and function of animal gut microbiomes, key regulators of host physiology, with potential consequences for host population responses to climate change. Several recent studies spanning a range of animal taxa, including Chordata, Arthropoda, and Mollusca, have reported repeatable associations between temperature and the community composition and function of the gut microbiome. In several cases, the same microbiome responses to temperature have been observed across distantly related animal taxa, suggesting the existence of conserved mechanisms underlying temperature-induced microbiome plasticity. Extreme temperatures can disrupt the stability of alpha-diversity within the gut microbiomes individual hosts and generate beta-diversity among microbiomes within host populations. Microbiome states resulting from extreme temperatures have been associated, and in some cases causally linked, with both beneficial and deleterious effects on host phenotypes. We propose routes by which temperature-induced changes in the gut microbiome may impact host fitness, including effects on colonization resistance in the gut, on host energy and nutrient assimilation, and on host life history traits. Cumulatively, available data indicate that disruption of the gut microbiome may be a mechanism by which changing temperatures will impact animal fitness in wild-living populations.

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Background: Oral microbiota has been linked to both health and diseases. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested. Results: Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed amplicon sequence variants (254 ± 53 in saliva and 175 ± 37 in tongue; P = 8.9e-7, Kruskal-Wallis test) and Shannon index (6.0 ± 0.4 in saliva and 5.4 ± 0.3 in tongue; P = 2.0e-7, Kruskal-Wallis test). Fusobacterium periodonticum, Saccharibacteria sp. 352, Streptococcus oralis subsp . dentisani, Prevotella melaninogenica, Granulicatella adiacens, Campylobacter concisus, and Haemophilus parainfluenzae were the core operational taxonomic units (OTUs) common to both sites. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differed significantly in terms of bacterial composition, they showed inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.

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The phylogenetic depth at which arbuscular mycorrhizal (AM) fungi harbor a coherent ecological niche is unknown, which has consequences for operational taxonomic unit (OTU) delineation from sequence data and the study of their biogeography. We tested how changes in AM fungi community composition across habitats (beta diversity) vary with OTU phylogenetic resolution. We inferred exact sequence variants (ESVs) to resolve phylotypes at resolutions finer than provided by traditional sequence clustering and analyzed beta diversity profiles up to order-level sequence clusters. At the ESV level, we detected the environmental predictors revealed with traditional OTUs or at higher genetic distances. However, the correlation between environmental predictors and community turnover steeply increased at a genetic distance of c. 0.03 substitutions per site. Furthermore, we observed a turnover of either closely or distantly related taxa (respectively at or above 0.03 substitutions per site) along different environmental gradients. This study suggests that different axes of AM fungal ecological niche are conserved at different phylogenetic depths. Delineating AM fungal phylotypes using DNA sequences should screen different phylogenetic resolutions to better elucidate the factors that shape communities and predict the fate of AM symbioses in a changing environment.

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