Microviridae is a subset of single-stranded DNA (ssDNA) viruses infecting bacteria. This group of phages has been previously observed to be very abundant (representing >90% of the total known viral metagenomic sequences) in Lake Bourget. However, this observation was made only during one period (in summer) and from a single sample collected at a single depth (near surface). This result suggests the importance of these viruses, poorly examined thus far, especially in fresh waters. In this study, performed on the two largest natural lakes in France (e.g. Lakes Annecy and Bourget), Microviridae structure was determined each month throughout the year (2011) using PCR-DGGE, with primers that target the major-capsid-protein-encoding gene VP1; cloning/sequencing was used to investigate their diversity. Our results confirm that Microviridae are diverse in peri-alpine lakes and are mainly represented by gokushoviruses. We also found for the first time ssDNA viruses belonging to Alpavirinae, another subfamily within Microviridae recently proposed by Krupovic and Forterre (2011), generally prophages infecting members of the Phylum Bacteroidetes. Our data also support highly variable community composition and dynamics of individual components whose patterns were different between lakes, suggesting distinct host communities and/or abiotic influences between the two ecosystems. We point out that most of the major observed ssDNA Microviridae viruses display boom-bust patterns (with a sharp increase/decline) in their dynamics, with high relative abundances, suggesting brutal control of hosts and rapid regulation of the host community structure.

When considering microbial biotic interactions, viruses as well as eukaryotic grazers are known to be important components of aquatic microbial food webs. It might be the same for bacterivorous bacteria but these groups have been comparatively less studied. This is typically the case of the Bdellovibrio and like organisms (BALOs), which are obligate bacterial predators of other bacteria. Recently, the abundance and distribution of three families of this functional group were investigated in perialpine lakes, revealing their presence and quantitative importance. Here, a more in-depth analysis is provided for Lake Geneva regarding the diversity of these bacterial predators at different seasons, sites and depths. We reveal a seasonal and spatial (vertical) pattern for BALOs. They were also found to be relatively diverse (especially Bdellovibrionaceae) and assigned to both known and unknown phylogenetic clusters. At last we found that most BALOs were positively correlated to other bacterial groups, mainly Gram-negative, in particular Myxococcales (among which many are predators of other microbes). This study is the first shedding light on this potentially important bacterial killing group in a large and deep lake.

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.

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.