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International audience; 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

There is an urgent need to evaluate the effects of anthropogenic pressures and climatic change on fish populations’ dynamics. When monitored in lakes, the spawning of fish is generally assessed using traditional, mostly destructive or damaging, methods as gillnetting and collection of fertilized eggs.Over the last decade, environmental DNA (eDNA) based methods have been widely developed for the detection of aquatic species, offering a non-invasive alternative method to conventional biomonitoring tools. In particular, the emergence of new methods as the droplet digital PCR (ddPCR) offer the possibility to quantify an absolute eDNA signal in a very sensitive way and at a low cost.Here, we developed and implemented a quantitative eDNA method to monitor the spawning activity of two fish species, European perch and whitefish. ddPCR protocols were formalized based on existing and newly designed COI primers, and were applied during four spawning periods in lake Geneva.The results demonstrate the efficiency of eDNA coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for the targeted species. In addition, the use of a control species (i.e., quantification of the eDNA signal of a fish that does not reproduce during the monitoring period) was shown to be relevant to clearly discriminate fluctuations of the eDNA signal associated to the spawning activity from the baseline eDNA signal. For future implementation, we recommend using an integrative sampling strategy (e.g., pooled samples for a give station) to smooth the local variability of the eDNA signal. These results show that we reached an operational level to use these non-invasive eDNA methods to monitor the spawning periods of these two fish species in large lakes.

The objective of this protocol is to provide a reliable and replicable method for the DNA extraction of lake micro-plankton to be used for downstream DNA analysis. This protocol is one of those proposed by the Eco-AlpsWater consortium to promote the implementation of High Throughput Sequencing (HT S) of environmental DNA (eDNA) in the biomonitoring and ecological assessment of water bodies. The extraction is performed from samples filtered through Sterivex cartridges (Sterivex™ GP 0.22μm) and stored at -20°C, as described in the protocol dx.doi.org/10.17504/protocols.io.xn6fmhe, and with the use of the DNeasy® PowerWater Sterivex Kit (QIAGEN) with specific modifications adapted to plankton DNA extraction. The application proposed here, in the context of EcoAlpsWater, aims at comparing DNA inventories to traditional phytoplanktonic inventories and at characterizing more broadly the micro-planktonic diversity through eDNA analysis (including bacteria). This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (http://www.alpine-space.eu/projects/eco-alpswater/en/home) contributed to the optimization of this protocol.

Anthropogenic pressures and more recently climatic change have increased the interest to study the impact of environmental changes on the key stages of fish life cycle. In lake Geneva, a deep peri-alpine lake, climate change and phosphorous level are known to have consequences on salmonid and percid populations, including key species for recreational and commercial fisheries, whose stocks are subject to significant fluctuations. To follow these stock variations, the spawning activity of European perch (Perca fluviatilis) and whitefish (Coregonus lavaretus) is monitored in this lake since several years using traditional methods, unfortunately mostly destructive or damaging (e.g. gillnetting and collection of fertilized eggs). DNA isolated from the environment (eDNA) has been widely developed for the detection of specific species or whole biological communities, and this non-invasive method offers an alternative to conventional surveying tools. Until recently, the methods used for eDNA analysis (e.g. qPCR, metabarcoding) could be limited by their sensitivity, quantification limit or price, but the emergence of new methods, such as the droplet digital PCR (ddPCR), offers the possibility to quantify an absolute eDNA signal in a very sensitive way and at a lower cost. Here, we show for the first time the applicability of an eDNA method to monitor the spawning activity of two fish species in a lake by using ddPCR. During two spawning seasons for whitefish and one spawning season for European perch, water samples were collected every week from the subsurface, simultaneously to traditional monitoring sampling, and filtered through sterile cartridges. The eDNA was then extracted and analyzed using ddPCR, targeting the mitochondrial DNA of the two fish species. The results demonstrate the efficiency of eDNA coupled with ddPCR to identify the timing and duration of the spawning periods, as well as the peak of the spawning activity for whitefish and European perch in Lake Geneva. This study shows that we have reached an operational level to use this non-invasive eDNA monitoring of the spawning activity of these fish species in lakes.

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

The objective of this protocol is the sampling and the filtration of water samples through 0.45 µm Sterivex™ filter units, and their buffer preservation prior to DNA extraction. This protocol is used upstream to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target fish eDNA. The use of preservation buffer allows sample preservation for up to several hours at room temperature, several days at 4°C and several months at -20°C, and thus the possibility to filter samples on site. This protocol allows to filter approximately between 0.7 L and 5 L through a Sterivex™ filter unit depending on the water sample characteristics and the chosen filtration method (syringe or vacuum pump).

The objective of this protocol is DNA extraction from water samples filtered through 0.45 µm Sterivex™ filter units and preserved in 2 mL of buffer (dx.doi.org/10.17504/protocols.io.br5rm856). Extraction is performed using NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications adapted to DNA extraction from Sterivex™ filter units. This protocol is used upstream to molecular biology analysis (e.g. qPCR, metabarcoding, ddPCR) to specifically target fish eDNA.

High-throughput sequencing of sedimentary DNA (sed-DNA) was utilized to reconstruct the temporal dynamics of microbial eukaryotic communities (MECs) at a centennial scale in two re-oligotrophicated lakes that were exposed to different levels of phosphorus enrichment. The temporal changes within the MECs were expressed in terms of richness, composition and community structure to investigate their relationships with two key forcing factors (i.e., nutrient enrichment and climate warming). Various groups, including Apicomplexa, Cercozoa, Chrysophyceae, Ciliophora, Chlorophyceae and Dinophyceae, responded to phosphorus enrichment levels with either positive or negative impacts on their richness and relative abundance. For both lakes, statistical modelling demonstrated that phosphorus concentration ([P]) was a dominant contributor to MECs modifications before the 1980s; after the mid-80s, the contribution of air temperature changes increased and potentially surpassed the contribution of [P]. Co-occurrence network analysis revealed that some clusters of taxa (i.e., modules) composed mainly of Dinophyceae and unclassified Alveolata were strongly correlated to air temperature in both lakes. Overall, our data showed that sed-DNA constitutes a precious archive of information on past biodiversity changes, allowing the study of the dynamics of numerous eukaryotic groups that were not traditionally considered in paleo-reconstructions.

The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-plankton to be used for downstream DNA analysis. This protocol is one of those proposed by the EcoAlpsWater consortium to promote the implementation of High Throughput Sequencing (HT S) of environmental DNA (eDNA) in the biomonitoring and ecological assessment of water bodies. The application proposed here, in the context of EcoAlpsWater, aims at comparing DNA inventories to traditional phytoplanktonic inventories and at characterizing more broadly the micro-planktonic diversity through eDNA analysis (including bacteria). The sampling strategy is similar to that used for classical phytoplankton survey focusing on the euphotic zone, however the procedure for filtration and preservation is adapted for DNA samples. This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (http://www.alpine-space.eu/projects/eco-alpswater/en/home) contributed to the optimization of this protocol.

International audience; The effectiveness of environmental protection measures is based on the early identification and diagnosis of anthropogenic pressures. Similarly, restoration actions require precise monitoring of changes in the ecological quality of ecosystems, in order to highlight their effectiveness. Monitoring the ecological quality relies on bioindicators, which are organisms revealing the pressures exerted on the environment through the composition of their communities. Their implementation, based on the morphological identification of species, is expensive because it requires time and experts in taxonomy. Recent genomic tools should provide access to reliable and high-throughput environmental monitoring by directly inferring the composition of bioindicators' communities from their DNA (metabarcoding). The French-Swiss program SYNAQUA (INTERREG France-Switzerland 2017-2019) proposes to use and validate the tools of environmental genomic for biomonitoring and aims ultimately at their implementation in the regulatory bio-surveillance. SYNAQUA will test the metabarcoding approach focusing on two bioindicators, diatoms, and aquatic oligochaetes, which are used in freshwater biomonitoring in France and Switzerland. To go towards the renewal of current biomonitoring practices, SYNAQUA will (1) bring together different actors: scientists, environmental managers, consulting firms, and biotechnological companies, (2) apply this approach on a large scale to demonstrate its relevance, (3) propose robust and reliable tools, and (4) raise public awareness and train the various actors likely to use these new tools. Biomonitoring approaches based on such environmental genomic tools should address the European need for reliable, higher-throughput monitoring to improve the protection of aquatic environments under multiple pressures, guide their restoration , and follow their evolution.

The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-plankton to be used for downstream DNA analysis. This protocol is one of those proposed by the EcoAlpsWater consortium to promot ethe implementation of High Throughput Sequencing (HTS) of environmental DNA (eDNA) in the biomonitoring and ecological assesment of water bodies. The application proposed here, in the context of EcoAlpsWater, aims at comparing DNA inventories to traditional phytoplanktonic inventories and at characterizing more broadly the micro-planktonic diversity through eDNA analysis. The sampling strategy is similar to that used for classical phytoplankton survey focusing on the euphotic zone, however the procedure for filtration and preservation is adapted for DNA samples. This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (involving 12 partners : http://www.alpine-space.eu/projects/eco-alpswater/en/home) have contributed to the optimization of this protocol.

The work was done in collaboration with V. Lecomte (GRAIE). We thank B. Montuelle, F. Rimet, R. Sommaruga, and anonymous reviewers for their constructive comments on the manuscript.; International audience; 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.

International audience; Benthic diatom communities are used in the ecological assessment of Portuguese rivers through the calculation of an autecological index, the IPS (Indice de Polluosensibilite Specifique), officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals at species level. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing techniques offer a promising alternative to this classical approach, decreasing expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers, by comparing the classifications of the IPS obtained with morphological and molecular approaches. A total of 88 samples were collected in four river types and a quality gradient in central Portugal in the spring of 2017, following the Water Framework Directive (WFD) standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. Molecular approach included eDNA extraction followed by PCR amplification and DNA high-throughput sequencing (Illumina MiSeq) using a diatom-specific 312 bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned using the R-Syst::diatom reference library. We also tested the effect of a correction factor (CF) based on biovolume for the quantification of molecular data. When comparing the number of species identified, morphological, molecular without and with CF inventories revealed 306, 125 and 111 species, respectively. Molecular data showed 67% of unassigned OTUs. These results were mainly due to the incompleteness of the reference library, which currently represents the major difficulty in taxonomic assignment of DNA sequences in metabarcoding. Regarding IPS values, the results indicated a good correlation between morphological and molecular methods, particularly when applying the CF. Furthermore, Principal Coordinates analysis plot based on species abundances revealed a quality gradient in all 3 methodologies (morphological, molecular without and with CF). These results support the hypothesis that DNA metabarcoding is a valid approach for ecological quality assessment of Portuguese rivers. Yet, there is still work to be done on this new methodology to enable a smooth transition between the traditional and this new approach without misspend accumulated knowledge from the last decades on water quality assessment.

Two experiments were conducted to test whether viruses, small auto- and heterotrophic nanoflagellates were key factors regulating at relatively short-term (4 days) and nearby periods (April vs. May) the bacterial community in surface waters of Lake Geneva. 2.5 L containing polycarbonate bottles were incubated in situ with either μm filtered water with additions of either virus-free water or a viral concentrate. Abundances of viruses, prokaryotes and small autotrophs were obtained each day using flow cytometry, while bacterial richness was assessed using 16S rDNA PCR-DGGE and auto- and heterotrophic flagellates counted with epifluorescence microscopy at t0 and t96. Transmission electron microscopy was also used to assess virus-induced bacterial mortality at the start and the end of the experiments. Cloning-sequencing was applied on PCR products obtained after excision of selected DGGE bands to highlight more specifically the identity of bacteria of interest in the context of the experiment. The autotrophs and grazer presence and/or the virus enrichment resulted in different effects on the structure of the bacterial community and the impact was also different with the period. In May, bacterial structure changes seemed to be related to the impact or influence of the eukaryotes (including nanoflagellate grazers), while viruses might have a higher impact on the bacterial community structure the month before. This study provides new persuasive evidence that the presence of viruses and small eukaryotes are likely to drive bacterial community composition and shifts on the short-term in lacustrine ecosystems. More interestingly, such effects seem to be different between viruses and grazers, the ones sustaining, the others reducing bacterial community composition.

Summary Decennial changes in Planktothrix rubescens diversity and dynamics were reconstructed by applying molecular tools to analyse DNA and RNA extracted from lake sediments. The sediments studied were sampled from a deep peri-alpine lake that has experienced both dramatic shifts in trophic conditions and large-scale climatic changes. Palaeolimnological proxies were combined with statistical modelling to investigate the relative influence of phosphorus concentrations and temperature changes on the extent of Planktothrix blooms over the last century. Phylogenetic analysis revealed that the overall composition of the cyanobacterial community changed over the transition from oligotrophic to eutrophic conditions. When the relative abundance of Planktothrix decreased in the 1970s, concomitant with eutrophication, total cyanobacterial abundance remained high and more Anabaena and Microcystis sequences were detected. In spite of such drastic environmental changes, the lake provided a constant niche for one particular Planktothrix species, which was consistently present from the 1920s to the present day. Phosphorus concentration was found to be the dominant driver of the relative abundance of P. rubescens, with the highest abundances observed during mesotrophic conditions. The relative role of climate was nutrient-dependent, with warmer springs having a positive effect on P. rubescens abundance only during mesotrophic periods. Overall, this study confirms that analysis of genetic signatures preserved in sediment archives allows assessment of key palaeoenvironmental indicator species that have no diagnostic microscopic cellular features in the sediment record. In the case of cyanobacteria, palaeogenetics offer unique opportunities to anticipate how future climate change might affect the response of P. rubescens to phosphorus concentration.

International audience; In this study, we evaluated the driving forces exerted by a large set of environmental and biological parameters on the spatial and temporal dynamics of archaeal community structure in two neighbouring peri-alpine lakes that differ in terms of trophic status. We analysed monthly data from a 2-year sampling period at two depths corresponding to the epi- and hypolimnetic layers. The archaeal communities seemed to be mainly composed of ammonia-oxidizing archaea belonging to the thaumarchaeotal phylum. The spatio-temporal dynamics of these communities were very similar in the two lakes and were characterized by (1) disparities in archaeal community structure in both time and space and (2) no seasonal reproducibility between years. The archaeal communities were regulated by a complex combination of abiotic factors, including temperature, nutrients, chlorophyll a and dissolved oxygen, and biotic factors such as heterotrophic nanoflagellates and ciliates. However, in most cases, these factors explained

International audience; Mangroves are threatened ecosystems that provide numerous ecosystem services, especially through their wide biodiversity, and their bioremediation capacity is a challenging question in tropical areas. In a mangrove in Mayotte, we studied the potential role of microbial biofilm communities in removing nutrient loads from pre-treated wastewater. Microbial community samples were collected from tree roots, sediments, water, and from a colonization device, and their structure and dynamics were compared in two areas: one exposed to sewage and the other not. The samples from the colonization devices accurately reflected the natural communities in terms of diversity. Communities in the zone exposed to sewage were characterized by more green algae and diatoms, higher bacteria densities, as well as different compositions. In the area exposed to sewage, the higher cell densities associated with specific diversity patterns highlighted adapted communities that may play a significant role in the fate of nutrients.