Monitoring the ecological status of rivers with diatom eDNA metabarcoding: A comparison of taxonomic markers and analytical approaches for the inference of a molecular diatom index.

Recently, several studies demonstrated the usefulness of diatom eDNA metabarcoding as an alternative to assess the ecological quality of rivers and streams. However, the choice of the taxonomic marker as well as the methodology for data analysis differ between these studies, hampering the comparison of their results and effectiveness. The aim of this study was to compare two taxonomic markers commonly used in diatom metabarcoding and three distinct analytical approaches to infer a molecular diatom index. We used the values of classical morphological diatom index as a benchmark for this comparison. We amplified and sequenced both a fragment of the rbcL gene and the V4 region of the 18S rRNA gene for 112 epilithic samples from Swiss and French rivers. We inferred index values using three analytical approaches: by computing it directly from taxonomically assigned sequences, by calibrating de novo the ecovalues of all metabarcodes, and by using a supervised machine learning algorithm to train predictive models. In general, the values of index obtained using the two "taxonomy-free" approaches, encompassing molecular assignment and machine learning, were closer correlated to the values of the morphological index than the values based on taxonomically assigned sequences. The correlations of the three analytical approaches were higher in the case of rbcL compared to the 18S marker, highlighting the importance of the reference database which is more complete for the rbcL marker. Our study confirms the effectiveness of diatom metabarcoding as an operational tool for rivers ecological quality assessment and shows that the analytical approaches by-passing the taxonomic assignments are particularly efficient when reference databases are incomplete.

Diatom DNA metabarcoding to assess the effect of natural radioactivity in mineral springs on ASV of benthic diatom communities.

Little is still known about the low dose effects of radiation on the microbial communities in the environment. Mineral springs are ecosystems than can be affected by natural radioactivity. These extreme environments are, therefore, observatories for studying the influence of chronic radioactivity on the natural biota. In these ecosystems we find diatoms, unicellular microalgae, playing an essential role in the food chain. The present study aimed to investigate, using DNA metabarcoding, the effect of natural radioactivity in two environmental compartments (i.e. spring sediments and water) on the genetic richness, diversity and structure of diatom communities in 16 mineral springs in the Massif Central, France. Diatom biofilms were collected during October 2019, and a 312 bp region of the chloroplast gene rbcL (coding for the Ribulose Bisphosphate Carboxylase) used as a barcode for taxonomic assignation. A total of 565 amplicon sequence variants (ASV) were found. The dominant ASV were associated with Navicula sanctamargaritae, Gedaniella sp., Planothidium frequentissimum, Navicula veneta, Diploneis vacillans, Amphora copulata, Pinnularia brebissonii, Halamphora coffeaeformis, Gomphonema saprophilum, and Nitzschia vitrea, but some of the ASVs could not be assigned at the species level. Pearson correlation failed to show a correlation between ASV' richness and radioactivity parameters. Non-parametric MANOVA analysis based on ASVs occurrence or abundances revealed that geographical location was the main factor influencing ASVs distribution. Interestingly, 238U was the second factor that explained diatom ASV structure. Among the ASVs in the mineral springs monitored, ASV associated with one of the genetic variants of Planothidium frequentissimum was well represented in the springs and with higher levels of 238U, suggesting its high tolerance to this particular radionuclide. This diatom species may therefore represent a bio-indicator of high natural levels of uranium.

Fish eDNA metabarcoding from aquatic biofilm samples: Methodological aspects.

Fish eDNA metabarcoding is usually performed from filtered water samples. The volume of filtered water depends on the study scope and can rapidly become time consuming according to the number of samples that have to be processed. To avoid time allocated to filtration, passive DNA samplers have been used to recover fish eDNA from marine environments faster. In freshwater ecosystems, aquatic biofilms were used to catch eDNA from macroinvertebrates. Here, we test the capacity of aquatic biofilms to entrap fish eDNA in a large lake and, therefore, the possibility to perform fish eDNA metabarcoding from this matrix compared to the traditional fish eDNA approach from filtered water samples. Methodological aspects of the use of aquatic biofilms for fish eDNA metabarcoding (e.g. PCR replicates, biological replicates, bioinformatics pipeline, reference database and taxonomic assignment) were validated against a mock community. When using biofilms from habitats sheltered from wind and waves, biofilm and water approach provided similar inventories. Richness and diversity were comparable between both approaches. Approaches differed only for rare taxa. Our results illustrate the capacity of aquatic biofilms to act as passive eDNA samplers of fish eDNA and, therefore, the possibility to use biofilms to monitor fish communities efficiently from biofilms. Furthermore, our results open up avenues of research to study a diversity of biological groups (among which bioindicators as diatoms, macroinvertebrates and fish) from eDNA isolated from a single environmental matrix reducing sampling efforts, analysis time and costs.

Paleoreconstructions of ciliate communities reveal long-term ecological changes in temperate lakes.

Ciliates are unicellular heterotrophic organisms that play a key role in aquatic planktonic and benthic food webs. Advances in sedimentary DNA (sed-DNA) analysis offer the possibility to integrate these bioindicators in paleoenvironmental reconstructions. In this study, we used the top-bottom paleolimnological approach and metabarcoding techniques applied to sed-DNA to compare the recent and past (i.e. prior to major anthropogenic impacts) ciliate communities of 48 lakes located along an elevation gradient. Our results show an overall decline in the ß-diversity in recent time, especially in lowland lakes, which are more strongly exposed to local human pressures. Analyses of the functional groups indicate important restructuration of the food web, including the recent increase in mixotrophs. Moreover, changes in the benthic ciliates were consistent with the widespread increase in deep water anoxia. Our results provided evidence that sed-DNA can uncover information about past ciliate communities on a wide variety of lakes. Overall, our study demonstrates the potential of using ciliates as new paleoindicators, integrating information from the pelagic to the benthic zones, and providing valuable insights into ecosystem functioning through a trait-based functional community approach. As paleoindicator, they thus offer a more holistic view on the long-term changes of aquatic ecosystems.

DNA sequence and taxonomic gap analyses to quantify the coverage of aquatic cyanobacteria and eukaryotic microalgae in reference databases: Results of a survey in the Alpine region.

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.

Exploring the capacity of aquatic biofilms to act as environmental DNA samplers: Test on macroinvertebrate communities in rivers.

Aquatic biofilms are heterogeneous assemblages of microorganisms surrounded by a matrix of extracellular polymeric substances (EPS). Recent studies suggest that aquatic biofilms can physically act as sorptive sponges of DNA. We took the opportunity from already available samples of stone biofilms and macroinvertebrates specimens collected in parallel at the same sites to test the capacity of biofilms to act as DNA samplers of macroinvertebrate communities in streams. Macroinvertebrate communities are usually studied with metabarcoding using the DNA extracted from their bodies bulk samples, which remains a time-consuming approach and involves the destruction of all individual specimens from the samples. The ability of biofilms to capture DNA was explored on 19 rivers sites of a tropical island (Mayotte Island, France). First, macroinvertebrate specimens were identified based on their morphological characteristics. Second, DNA was extracted from biofilms, and macroinvertebrate communities were targeted using a standard COI barcode. The resulting morphological and molecular inventories were compared. They provided comparable structures and diversities for macroinvertebrate communities when one is working with the unassigned OTU data. After taxonomic assignment of the OTU data, diversity and richness were no longer correlated. The ecological assessment derived from morphological bulk samples was conserved by the biofilms samples. We also showed that the biofilm method allows to detect a higher diversity for some organisms (Cnidaria), that is hardly accessible with the morphological method. The results of this study exploring the DNA signal captured by natural biofilms are encouraging. However, a more detailed study integrating more replicates and comparing the biodiversity signal based on both morphological and molecular bulk macroinvertebrate samples to the one captured by biofilms will be necessary. Better understanding how the DNA signal captured by natural biofilms represents the biodiversity of a given sampling site is necessary before considering its use for bioassessment applications.

Cyprus' diatom diversity and the association of environmental and anthropogenic influences for ecological assessment of rivers using DNA metabarcoding.

Human activities are the leading cause of environmental impairments. Appropriate biomonitoring of ecosystems is needed to assess these activities effectively. In freshwater ecosystems, periphytic and epilithic biofilms have diatom assemblages. These assemblages respond rapidly to environmental changes, making diatoms valuable bioindicators. For this reason, freshwater biomonitoring programs are currently using diatoms (e.g., Water Framework Directive). In the past ten years, DNA metabarcoding coupled with next-generation sequencing and bioinformatics represents a complementary approach for diatom biomonitoring. In this study, this approach is used for the first time in Cyprus by considering the association of environmental and anthropogenic pressures to diatom assemblages. Statistical analysis was then applied to identify the environmental (i.e., river types, geo-morphological) and anthropogenic (i.e., physicochemical, human land-use pressures) variables' role in the observed diatom diversity. Results indicate differences in diatom assemblages between intermittent and perennial rivers. Achnanthidium minutissimum was more abundant in intermittent rivers; whereas Amphora pediculus and Planothidium caputium in perennial ones. Additionally, we could demonstrate the correlation between nutrients (e.g., nitrogen, phosphorus), stations' local characteristics (e.g., elevation), and land use activities on the observed differences in diatom diversity. Finally, we conclude that multi-stressors and anthropogenic pressures together as multiple stressors have a significant statistical relationship to the observed diatom diversity and play a pivotal role in determining Cyprus' rivers' ecological status.

Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers.

Our study of 164 diatom samples from Catalonia (NE Spain) is the first to evaluate the applicability of DNA metabarcoding, based on high throughput sequencing (HTS) using a 312-bp rbcL marker, for biomonitoring Mediterranean rivers. For this, we compared the values of a biotic index (IPS) and the ecological status classes derived from them, between light microscope-based (LM) and HTS methods. Very good correspondence between methods gives encouraging results concerning the applicability of DNA metabarcoding for Catalan rivers for the EU Water Framework Directive (WFD). However, in 10 sites, the ecological status class was downgraded from "Good"/"High" obtained by LM to "Moderate"/"Poor"/"Bad" by HTS; these "critical" sites are especially important, because the WFD requires remedial action by water managers for any river with Moderate or lower status. We investigated the contribution of each species to the IPS using a "leave-one-out" sensitivity analysis, paying special attention to critical sites. Discrepancies in IPS between LM and HTS were mainly due to the misidentification and overlooking in LM of a few species, which were better recovered by HTS. This bias was particularly important in the case of Fistulifera saprophila, whose clear underrepresentation in LM was important for explaining 8 out of the 10 critical sites and probably reflected destruction of weakly-silicified frustules during sample preparation. Differences between species in the rbcL copy number per cell affected the relative abundance obtained by HTS for Achnanthidium minutissimum, Nitzschia inconspicua and Ulnaria ulna, which were also identified by the sensitivity analysis as important for the WFD. Only minor IPS discrepancies were attributed to the incompleteness of the reference library, as most of the abundant and influential species (to the IPS) were well represented there. Finally, we propose that leave-one-out analysis is a good method for identifying priority species for isolation and barcoding.

A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms.

Diatoms are a compulsory biological quality element in the ecological assessment of rivers according to the Water Framework Directive. The application of current official indices requires the identification of individuals to species or lower rank under a microscope based on the valve morphology. This is a highly time-consuming task, often susceptible of disagreements among analysts. In alternative, the use of DNA metabarcoding combined with High-Throughput Sequencing (HTS) has been proposed. The sequences obtained from environmental DNA are clustered into Operational Taxonomic Units (OTUs), which can be assigned to a taxon using reference databases, and from there calculate biotic indices. However, there is still a high percentage of unassigned OTUs to species due to the incompleteness of reference libraries. Alternatively, we tested a new taxonomy-free approach based on diatom community samples to assess rivers. A combination of three machine learning techniques is used to build models that predict diatom OTUs expected in test sites, under reference conditions, from environmental data. The Observed/Expected OTUs ratio indicates the deviation from reference condition and is converted into a quality class. This approach was never used with diatoms neither with OTUs data. To evaluate its efficiency, we built a model based on OTUs lists (HYDGEN) and another based on taxa lists from morphological identification (HYDMORPH), and also calculated a biotic index (IPS). The models were trained and tested with data from 81 sites (44 reference sites) from central Portugal. Both models were considered accurate (linear regression for Observed and Expected richness: R2 ≈ 0.7, interception ≈ 0.8) and sensitive to global anthropogenic disturbance (Rs2 > 0.30 p < 0.006 for global disturbance). Yet, the HYDGEN model based on molecular data was sensitive to more types of pressures (such as, changes in land use and habitat quality), which gives promising insights to its use for bioassessment of rivers.

Diatom DNA metabarcoding for ecological assessment: Comparison among bioinformatics pipelines used in six European countries reveals the need for standardization.

Ecological assessment of lakes and rivers using benthic diatom assemblages currently requires considerable taxonomic expertise to identify species using light microscopy. This traditional approach is also time-consuming. Diatom metabarcoding is a promising alternative and there is increasing interest in using this approach for routine assessment. However, until now, analysis protocols for diatom metabarcoding have been developed and optimised by research groups working in isolation. The diversity of existing bioinformatics methods highlights the need for an assessment of the performance and comparability of results of different methods. The aim of this study was to test the correspondence of outputs from six bioinformatics pipelines currently in use for diatom metabarcoding in different European countries. Raw sequence data from 29 biofilm samples were treated by each of the bioinformatics pipelines, five of them using the same curated reference database. The outputs of the pipelines were compared in terms of sequence unit assemblages, taxonomic assignment, biotic index score and ecological assessment outcomes. The three last components were also compared to outputs from traditional light microscopy, which is currently accepted for ecological assessment of phytobenthos, as required by the Water Framework Directive. We also tested the performance of the pipelines on the two DNA markers (rbcL and 18S-V4) that are currently used by the working groups participating in this study. The sequence unit assemblages produced by different pipelines showed significant differences in terms of assigned and unassigned read numbers and sequence unit numbers. When comparing the taxonomic assignments at genus and species level, correspondence of the taxonomic assemblages between pipelines was weak. Most discrepancies were linked to differential detection or quantification of taxa, despite the use of the same reference database. Subsequent calculation of biotic index scores also showed significant differences between approaches, which were reflected in the final ecological assessment. Use of the rbcL marker always resulted in better correlation among molecular datasets and also in results closer to these generated using traditional microscopy. This study shows that decisions made in pipeline design have implications for the dataset's structure and the taxonomic assemblage, which in turn may affect biotic index calculation and ecological assessment. There is a need to define best-practice bioinformatics parameters in order to ensure the best representation of diatom assemblages. Only the use of similar parameters will ensure the compatibility of data from different working groups. The future of diatom metabarcoding for ecological assessment may also lie in the development of new metrics using, for example, presence/absence instead of relative abundance data.

The impact of OTU sequence similarity threshold on diatom-based bioassessment: A case study of the rivers of Mayotte (France, Indian Ocean).

Extensive studies on the taxonomic resolution required for bioassessment purposes have determined that resolution above species level (genus, family) is sufficient for their use as indicators of relevant environmental pressures. The high-throughput sequencing (HTS) and meta-barcoding methods now used for bioassessment traditionally employ an arbitrary sequence similarity threshold (SST) around 95% or 97% to cluster sequences into operational taxonomic units, which is considered descriptive of species-level resolution. In this study, we analyzed the effect of the SST on the resulting diatom-based ecological quality index, which is based on OTU abundance distribution along a defined environmental gradient, ideally avoiding taxonomic assignments that could result in high rates of unclassified OTUs and biased final values. A total of 90 biofilm samples were collected in 2014 and 2015 from 51 stream sites on Mayotte Island in parallel with measures of relevant physical and chemical parameters. HTS sequencing was performed on the biofilms using the rbcL region as the genetic marker and diatom-specific primers. Hierarchical clustering was used to group sequences into OTUs using 20 experimental SST levels (80%-99%). An OTU-based quality index (IdxOTU) was developed based on a weighted average equation using the abundance profiles of the OTUs. The developed IdxOTU revealed significant correlations between the IdxOTU values and the reference pressure gradient, which reached maximal performance using an SST of 90% (well above species level delimitation). We observed an interesting and important trade-off with the power to discriminate between sampling sites and index stability that will greatly inform future applications of the index. Taken together, the results from this study detail a thoroughly optimized and validated approach to generating robust, reproducible, and complete indexes that will greatly facilitate effective and efficient environmental monitoring.

Benthic Diatom Communities in an Alpine River Impacted by Waste Water Treatment Effluents as Revealed Using DNA Metabarcoding.

Freshwater ecosystems are continuously affected by anthropogenic pressure. One of the main sources of contamination comes from wastewater treatment plant (WWTP) effluents that contain wide range of micro- and macropollutants. Chemical composition, toxicity levels and impact of treated effluents (TEs) on the recipient aquatic ecosystems may strongly differ depending on the wastewater origin. Compared to urban TEs, hospital ones may contain more active pharmaceutical substances. Benthic diatoms are relevant ecological indicators because of their high species and ecological diversity and rapid response to human pressure. They are routinely used for water quality monitoring. However, there is a knowledge gap on diatom communities' development and behavior in treated wastewater in relation to prevailing micro- and macropollutants. In this study, we aim to (1) investigate the response of diatom communities to urban and hospital TEs, and (2) evaluate TEs effect on communities in the recipient river. Environmental biofilms were colonized in TEs and the recipient river up- and downstream from the WWTP output to study benthic diatoms using DNA metabarcoding combined with high-throughput sequencing (HTS). In parallel, concentrations of nutrients, pharmaceuticals and seasonal conditions were recorded. Diatom metabarcoding showed that benthic communities differed strongly in their diversity and structure depending on the habitat. TE sites were generally dominated by few genera with polysaprobic preferences belonging to the motile guild, while river sites favored diverse communities from oligotrophic and oligosaprobic groups. Seasonal changes were visible to lower extent. To categorize parameters important for diatom changes we performed redundancy analysis which suggested that communities within TE sites were associated to higher concentrations of beta-blockers and non-steroidal anti-inflammatory drugs in urban effluents vs. antibiotics and orthophosphate in hospital effluents. Furthermore, indicator species analysis showed that 27% of OTUs detected in river downstream communities were indicator for urban or hospital TE sites and were absent in the river upstream. Finally, biological diatom index (BDI) calculated to evaluate the ecological status of the recipient river suggested water quality decrease linked to the release of TEs. Thus, in-depth assessment of diatom community composition using DNA metabarcoding is proposed as a promising technique to highlight the disturbing effect of pollutants in Alpine rivers.

Diat.barcode, an open-access curated barcode library for diatoms.

Diatoms (Bacillariophyta) are ubiquitous microalgae which produce a siliceous exoskeleton and which make a major contribution to the productivity of oceans and freshwaters. They display a huge diversity, which makes them excellent ecological indicators of aquatic ecosystems. Usually, diatoms are identified using characteristics of their exoskeleton morphology. DNA-barcoding is an alternative to this and the use of High-Throughput-Sequencing enables the rapid analysis of many environmental samples at a lower cost than analyses under microscope. However, to identify environmental sequences correctly, an expertly curated reference library is needed. Several curated libraries for protists exists; none, however are dedicated to diatoms. Diat.barcode is an open-access library dedicated to diatoms which has been maintained since 2012. Data come from two sources (1) the NCBI nucleotide database and (2) unpublished sequencing data of culture collections. Since 2017, several experts have collaborated to curate this library for rbcL, a chloroplast marker suitable for species-level identification of diatoms. For the latest version of the database (version 7), 605 of the 3482 taxonomical names originally assigned by the authors of the rbcL sequences were modified after curation. The database is accessible at https://www6.inra.fr/carrtel-collection_eng/Barcoding-database .

Boosting DNA metabarcoding for biomonitoring with phylogenetic estimation of operational taxonomic units' ecological profiles.

DNA metabarcoding has been introduced as a revolutionary way to identify organisms and monitor ecosystems. However, the potential of this approach for biomonitoring remains partially unfulfilled because a significant part of the sampled DNA cannot be affiliated to species due to incomplete reference libraries. Thus, biotic indices, which are based on the estimated abundances of species in a community and their ecological profiles, can be inaccurate. We propose to compute biotic indices using phylogenetic imputation of operational taxonomic units (OTUs') ecological profiles (OTU-PITI approach). First, OTUs sequences are inserted within a reference phylogeny. Second, OTUs' ecological profiles are estimated on the basis of their phylogenetic relationships with reference species whose ecology is known. Based on these ecological profiles, biotic indices can be computed using all available OTUs. Using freshwater diatoms as a case study, we show that short DNA barcodes can be placed accurately within a phylogeny and their ecological preferences estimated with a satisfactory level of precision. In the light of these results, we tested the approach with a data set of 139 environmental samples of benthic river diatoms for which the same biotic index (specific sensitivity index) was calculated using (a) traditional microscopy, (b) OTUs with taxonomic assignment approach, (c) OTUs with phylogenetic estimation of ecological profiles (OTU-PITI) and (d) OTU with taxonomic assignment completed by the phylogenetic approach (OTU-PITI) for unclassified OTUs. Using traditional microscopy as a reference, we found that the combination of the OTUs' taxonomic assignment completed by the phylogenetic method performed satisfactorily and substantially better than the other methods tested.

Survival and growth of Stenotrophomonas maltophilia in free-living amoebae (FLA) and bacterial virulence properties.

Stenotrophomonas maltophilia is found ubiquitously in the environment and is an important emerging nosocomial pathogen. S. maltophilia has been recently described as an Amoebae-Resistant Bacteria (ARB) that exists as part of the microbiome of various free-living amoebae (FLA) from waters. Co-culture approaches with Vermamoeba vermiformis demonstrated the ability of this bacterium to resist amoebal digestion. In the present study, we assessed the survival and growth of six environmental and one clinical S. maltophilia strains within two amoebal species: Acanthamoeba castellanii and Willaertia magna. We also evaluated bacterial virulence properties using the social amoeba Dictyostelium discoideum. A co-culture approach was carried out over 96 hours and the abundance of S. maltophilia cells was measured using quantitative PCR and culture approach. The presence of bacteria inside the amoeba was confirmed using confocal microscopy. Our results showed that some S. maltophilia strains were able to multiply within both amoebae and exhibited multiplication rates up to 17.5 and 1166 for A. castellanii and W. magna, respectively. In contrast, some strains were unable to multiply in either amoeba. Out of the six environmental S. maltophilia strains tested, one was found to be virulent. Surprisingly, this strain previously isolated from a soil amoeba, Micriamoeba, was unable to infect both amoebal species tested. We further performed an assay with a mutant strain of S. maltophilia BurA1 lacking the efflux pump ebyCAB gene and found the mutant to be more virulent and more efficient for intra-amoebal multiplication. Overall, the results obtained strongly indicated that free-living amoebae could be an important ecological niche for S. maltophilia.

DNA metabarcoding and microscopic analyses of sea turtles biofilms: Complementary to understand turtle behavior.

Sea turtles are distributed in tropical and subtropical seas worldwide. They play several ecological roles and are considered important indicators of the health of marine ecosystems. Studying epibiotic diatoms living on turtle shells suggestively has great potential in the study of turtle behavior because diatoms are always there. However, diatom identification at the species level is time consuming, requires well-trained specialists, and there is a high probability of finding new taxa growing on turtle shells, which makes identification tricky. An alternative approach based on DNA barcoding and high throughput sequencing (HTS), metabarcoding, has been developed in recent years to identify species at the community level by using a DNA reference library. The suitabilities of morphological and molecular approaches were compared. Diatom assemblages were sampled from seven juvenile green turtles (Chelonia mydas) from Mayotte Island, France. The structures of the epibiotic diatom assemblages differed between both approaches. This resulted in different clustering of the turtles based on their diatom communities. Metabarcoding allowed better discrimination between turtles based on their epibiotic diatom assemblages and put into evidence the presence of a cryptic diatom diversity. Microscopy, for its part, provided more ecological information of sea turtles based on historical bibliographical data and the abundances of ecological guilds of the diatom species present in the samples. This study shows the complementary nature of these two methods for studying turtle behavior.

River biofilm community changes related to pharmaceutical loads emitted by a wastewater treatment plant.

Wastewater treatment plants (WWTP) are the main sources of a broad spectrum of pharmaceuticals found in freshwater ecosystems. These pollutants raise environmental health concerns because of their highly bioactive nature and their chronic releases. Despite this, pharmaceuticals' effects on aquatic environments are poorly defined. Biofilms represent a major part of the microbial life in rivers and streams. They can drive key metabolic cycles and their organizations reflect exposures to changing chemical, physical, and biological constraints. This study estimated the concentrations, over a 3-year period, of ten pharmaceuticals and five nutrients in a river contaminated by a conventional WWTP fed by urban and hospital wastewaters. Variations in these concentrations were related to biofilm bacterial community dynamics. Rock biofilms had developed over defined periods and were harvested at four locations in the river from the up- and downstream WWTP discharge point. Pharmaceuticals were found in all locations in concentrations ranging from not being detected to 192 ng L-1. Despite the high dilution factor of the WWTP effluents by the receiving river, pharmaceuticals were found more concentrated downstream than upstream the WWTP. Shifts in bacterial community structures linked to the environmental emission of pharmaceuticals were superior to seasonal community changes. A community structure from a site located downstream but close to the WWTP was more strongly associated with high pharmaceutical loads and different from those of biofilm samples from the WWTP upstream or far downstream sites. These latter sites were more strongly associated with high nutrient contents. Low environmental concentrations of pharmaceuticals can thus be transferred from WWTP effluents to a connected stream and induce bacterial aquatic community changes over time.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

Virulence test using nematodes to prescreen Nocardia species capable of inducing neurodegeneration and behavioral disorders.

BACKGROUND: Parkinson's disease (PD) is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain-some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. Nocardia are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system (CNS) invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are N. farcinica, N. nova, N. brasiliensis and N. cyriacigeorgica. Some studies have highlighted the ability of N. cyriacigeorgica to induce Parkinson's disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. METHODS: Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. RESULTS: The nematode Caenorhabditis elegans allowed us to demonstrate that Nocardia strains belonging to N. cyriacigeorgica and N. farcinica species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of Nocardia strains to induce neurodegeneration. These behavioral disorders were induced by N. cyriacigeorgica species (N. cyriacigeorgica GUH-2 and N. cyriacigeorgica 44484) and N. farcinica 10152. DISCUSSION: We conclude that C. elegans is a good model for detecting Nocardia strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to characterize the induced behavioral disorders and bacterial dissemination.

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/.