Conventional morphology-based identification is commonly used for routine assessment of freshwater ecosystems. However, cost and time efficient techniques such as high-throughput sequencing (HTS) based approaches may resolve the constraints encountered in conducting morphology-based surveys. Here, we characterized stream macroinvertebrate species diversity and community composition via metabarcoding and morphological analysis from environmental samples collected from the Shigenobu River Basin in Ehime Prefecture, Japan. We compared diversity metrics and assessed both approaches' ability to evaluate the relationship between macroinvertebrate community and environmental variables. In total, we morphologically identified 45 taxa (3 families, six subfamilies, 31 genera, and five species) from 8,276 collected individuals from ten study sites. We detected 44 species by metabarcoding, with 35 species collapsed into 11 groups matching the morphologically identified taxa. A significant positive correlation between logged depth (number of HTS reads) and abundance of morphological taxa was observed, which implied that quantitative data can be used for subsequent analyses. Relatively higher estimates of alpha diversity were calculated from the metabarcoding data in comparison to morphology-based data. However, beta diversity estimates between metabarcoding and morphology data based on both incidence and abundance-based matrices were correlated proving that community differences between sampling sites were preserved in the molecular data.Also, both models were significant, but metabarcoding data (93%) explained a relatively higher percentage of variation in the relationship between community composition and the environmental variables than morphological data (91%). Overall, we present both the feasibility and limitations of HTS-driven estimations of taxonomic richness, community composition, and diversity metrics, and that metabarcoding was proven comparable and more sensitive against morphology-based analysis for stream macroinvertebrate biodiversity assessment and environmental monitoring.

10Conventional morphology-based identification is commonly used for routine assessment of freshwater 11 ecosystems. However, cost and time efficient techniques such as high-throughput sequencing (HTS) based 12 approaches may resolve the constraints encountered in conducting morphology-based surveys. Here, we 13 characterized stream macroinvertebrate species diversity and community composition via metabarcoding and 14 morphological analysis from environmental samples collected from the Shigenobu River Basin in Ehime 15 Prefecture, Japan. We compared diversity metrics and assessed both approaches' ability to evaluate the 16 relationship between macroinvertebrate community and environmental variables. In total, we morphologically 17 identified 45 taxa (3 families, six subfamilies, 31 genera, and five species) from 8,276 collected individuals 18 from ten study sites. We detected 44 species by metabarcoding, with 35 species collapsed into 11 groups 19 matching the morphologically identified taxa. A significant positive correlation between logged depth (number 20 of HTS reads) and abundance of morphological taxa was observed, which implied that quantitative data can 21 be used for subsequent analyses. Relatively higher estimates of alpha diversity were calculated from the 22 metabarcoding data in comparison to morphology-based data. However, beta diversity estimates between 23 metabarcoding and morphology data based on both incidence and abundance-based matrices were 24 correlated proving that community differences between sampling sites were preserved in the molecular data. 25 Also, both models were significant, but metabarcoding data (93%) explained a relatively higher percentage of 26 variation in the relationship between community composition and the environmental variables than 27 morphological data (91%). Overall, we present both the feasibility and limitations of HTS-driven estimations 28 of taxonomic richness, community composition, and diversity metrics, and that metabarcoding was proven 29 comparable and more sensitive against morphology-based analysis for stream macroinvertebrate biodiversity 30 assessment and environmental monitoring. 31 32 community composition 33 42 al., 2016). 43 Conventional morphological analysis is most commonly used in routine monitoring programs evaluating 44 environmental quality changes. However, this is not only time consuming but has serious issues with accuracy, 45 and consistency in the level of taxonomic identification that highly depends on taxonomic expertise (Hajibabaei 46 et al., 2011). Specifically, small organisms such as the larval stages of stream macroinvertebrates frequently 47 used for river biomonitoring are often difficult or impossible to identify at finer taxonomic resolution (e.g., 48 species level) (Sweeny et al., 2011). A promising alternative approach is DNA metabarcoding -a combination 49 of amplicon-based high-throughput sequencing (HTS) analysis and DNA taxonomy (Hebert et al., 2003). High-50 throughput amplicon sequencing can process large number ...

Sediment bypass tunnels (SBTs) are guiding structures used to reduce sediment accumulation in reservoirs during high flows by transporting sediments to downstream reaches during operation. Previous studies monitoring the ecological effects of SBT operations on downstream reaches suggest a positive influence of SBTs on riverbed sediment conditions and macroinvertebrate communities based on traditional morphology-based surveys. Morphology-based macroinvertebrate assessments are costly and time-consuming, and the large number of morphologically cryptic, small-sized and undescribed species usually results in coarse taxonomic identification. Here, we used DNA metabarcoding analysis to assess the influence of SBT operations on macroinvertebrates downstream of SBT outlets by estimating species diversity and pairwise community dissimilarity between upstream and downstream locations in dam-fragmented rivers with operational SBTs in comparison to dam-fragmented (i.e., no SBTs) and free-flowing rivers (i.e., no dam). We found that macroinvertebrate community dissimilarity decreases with increasing operation time and frequency of SBTs. These factors of SBT operation influence changes in riverbed features, e.g. sediment relations, that subsequently effect the recovery of downstream macroinvertebrate communities to their respective upstream communities. Macroinvertebrate abundance using morphologically-identified specimens was positively correlated to read abundance using metabarcoding. This supports and reinforces the use of quantitative estimates for diversity analysis with metabarcoding data.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.

Functional redundancy is a key ecosystem property representing the fact that different taxa contribute to an ecosystem in similar ways through the expression of redundant functions. The redundancy of potential functions (or genome-level functional redundancy $${{{{{{\rm{FR}}}}}}}_{g}$$ FR g ) of human microbiomes has been recently quantified using metagenomics data. Yet, the redundancy of expressed functions in the human microbiome has never been quantitatively explored. Here, we present an approach to quantify the proteome-level functional redundancy $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p in the human gut microbiome using metaproteomics. Ultra-deep metaproteomics reveals high proteome-level functional redundancy and high nestedness in the human gut proteomic content networks (i.e., the bipartite graphs connecting taxa to functions). We find that the nested topology of proteomic content networks and relatively small functional distances between proteomes of certain pairs of taxa together contribute to high $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p in the human gut microbiome. As a metric comprehensively incorporating the factors of presence/absence of each function, protein abundances of each function and biomass of each taxon, $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p outcompetes diversity indices in detecting significant microbiome responses to environmental factors, including individuality, biogeography, xenobiotics, and disease. We show that gut inflammation and exposure to specific xenobiotics can significantly diminish the $${{{{{{\rm{FR}}}}}}}_{p}$$ FR p with no significant change in taxonomic diversity.