Summary Microsporidia are obligate intracellular eukaryotic parasites known to parasitize many species of the animal kingdom as well as some protists. However, their diversity is underestimated, in part as a consequence of the failure of ‘universal’ primers to detect them in metabarcoding studies. Besides, due to the inconsistency between taxonomy and phylogenetic data, available databases may assign incorrectly sequences obtained with high‐throughput sequencing. In this work, we developed a comprehensive reference database which positions microsporidian SSU rRNA gene sequences within a coherent ranked phylogenetic framework. We used this phylogenetic framework to study the microsporidian diversity in lacustrine ecosystems, focusing on < 150 μm planktonic size fractions. Our analysis shows a high diversity of Microsporidia, with the identification of 1531 OTUs distributed within seven clades, of which 76% were affiliated to clade IV2 and 20% to clade I (nomenclature presented hereby). About a quarter of the obtained sequences shared less than 85% identity to the closest known species, which might represent undescribed genera or families infecting small hosts. Variations in the abundance of Microsporidia were recorded between the two lakes sampled and across the sampling period, which might be explained by spatio‐temporal variations of their potential hosts such as microeukaryotes and metazooplankton.

Microsporidia are a large group of obligate intracellular eukaryotic parasites related to Fungi. Recent studies suggest that their diversity has been greatly underestimated and little is known about their hosts other than metazoans, and thus about their impact on the communities at the base of the food web. In this work, we therefore studied the diversity of Microsporidia over one year and identified potential new hosts in small-sized fractions (<150 μm) in a lake ecosystem using a metabarcoding approach coupled with co-occurrence networks and tyramide signal amplification-fluorescent in situ hybridization. Our analysis shows a great Microsporidia diversity (1 472 OTUs), with an important part of this diversity being unknown. Temporal variations of this diversity have been observed, which might follow temporal variations of their potential hosts such as protists and microzooplankton. New hosts among them were identified as well as associations with phytoplankton. Indeed, repeated infections were observed in Kellicottia (rotifers) with a prevalence of 38% (infected individuals). Microsporidia inside a Stentor (ciliate) were also observed. Finally, potential infections of the diatom Asterionella were identified (prevalence <0.1%). The microsporidian host spectrum could be therefore even more important than previously described, and their role in the functioning of lake ecosystems is undoubtedly largely unknown.

Microsporidia are a large group of obligate intracellular eukaryotic parasites. Recent studies suggest that their diversity can be huge in freshwater lake ecosystems especially in the <150 µm size fraction. However, little is known about their hosts and therefore their impact on the trophic food web functioning. In this study, single cell analysis and fluorescence microscopy were used to detect new host-parasite association within rotifer communities in lake Aydat (France). Our analysis showed the existence of a potential new species belonging to the Crispospora genus able of infecting the rotifer Kellicottia with a high prevalence (42.5%) suggesting that Microsporidia could have a great impact on the rotifer populations regulation in lakes.

Freshwater is a critical resource for human survival but severely threatened by anthropogenic activities and climate change. These changes strongly impact the abundance and diversity of the microbial communities which are key players in the functioning of these aquatic ecosystems. Although widely documented since the emergence of high‐throughput sequencing approaches, the information on these natural microbial communities is scattered among thousands of publications and it is therefore difficult to investigate the temporal dynamics and the spatial distribution of microbial taxa within or across ecosystems. To fill this gap and in the FAIR principles context we built a manually curated and standardized microbial freshwater –omics database (FreshOmics). Based on recognized ontologies (ENVO, MIMICS, GO, ISO), FreshOmics describes 29 different types of freshwater ecosystems and uses standardized attributes to depict biological samples, sequencing protocols and article attributes for more than 2487 geographical locations across 71 countries around the world. The database contains 24,808 sequence identifiers (i.e., Run_Id / Exp_ID, mainly from SRA/DDBJ SRA/ENA, GSA and MG‐RAST repositories) covering all sequence‐based ‐omics approaches used to investigate bacteria, archaea, microbial eukaryotes, and viruses. Therefore, FreshOmics allows accurate and comprehensive analyses of microbial communities to answer questions related to their roles in freshwater ecosystems functioning and resilience, especially through meta‐analysis studies. This collection also highlights different sort of errors in published works (e.g., wrong coordinates, sample type, material, spelling).

Microsporidia are a large group of obligate intracellular eukaryotic parasites related to Fungi primarily known as parasites of vertebrates and invertebrates. They are well described as parasites of organisms of interest (e.g., edible fish and crustaceans, honeybees, bioindicators such as daphnia, humans) on which they can have an important impact (e.g., reduced survival or fecundity and sex ratio distortion). However, their diversity in aquatic environments, especially in marine ecosystems, has been greatly understudied since they are not targeted by classical eukaryotic primers used in metabarcoding studies. Moreover, little is known about their hosts among protists or microzooplankton and therefore about their impact on the trophic food web functioning. In this work, we sampled 15 different sites across marine and freshwater environments, size-fractioned the samples and used microsporidian specific primers associated with metabarcoding to study the microsporidian diversity (and the associated spatial variation). Co-occurrence networks as well as Tyramide Signal Amplification-Fluorescent In Situ Hybridization were used to link potential hosts (planktonic eukaryotes < 150 μm) and Microsporidia diversity. Our analysis unraveled a large microsporidian diversity which was widely divergent between the two environments studied. In both of them, an important part of this diversity was not affiliated to a genus, suggesting an important reservoir of new microsporidian species and thus new hosts among planktonic eukaryotes. Co-occurrence networks and fluorescence microscopy showed for the first time associations between Microsporidia and dinoflagellates in freshwater and marine environments.

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