Marina Chauvet

Microsporidia are highly diverse, widespread parasites related to fungi and several groups of zoosporic protistan parasites. They infect a broad range of hosts, from protists to vertebrates, in many environments including most, if not all, aquatic habitats. Their genomes range from relatively conventional to highly reduced, yet they share key cellular structures and traits underlying infection and host association. Overall microsporidian diversity, including both ‘long‐branched’ and ‘short‐branched’ lineages, is poorly sampled; their environmental diversity and host range remain therefore largely unexplored. Although short sequences are often insufficient to distinguish between species, recent targeted metabarcoding studies have revealed unexpectedly high microsporidian diversity in both oceanic and lacustrine ecosystems, along with significant spatial and temporal variation. These studies have also identified several previously unrecognized hosts, including heterotrophic protists, phytoplankton and microzooplankton, all key components of the base of aquatic food webs. These findings suggest that Microsporidia, like other parasitic groups, may strongly influence the structure and functioning of aquatic ecosystems. We therefore advocate increased research on Microsporidia across aquatic habitats. Integrative research combining phylogenomics, long‐read sequencing and ecology studies will be essential to fully characterize their diversity, host associations and ecological impacts.

Despite their ubiquity, the diversity and dynamics of Microsporidia in aquatic ecosystems have long been overlooked. Here, we applied 18S rRNA gene metabarcoding targeting the V1‐V3 region for Microsporidia and V4 region for other eukaryotes to sedimentary ancient DNA to reconstruct the temporal dynamics of Microsporidia and their eukaryotic hosts over the past 138 years in Lake Aydat (France). We detected remarkably high microsporidian diversity, with 1976 Operational Taxonomic Units (OTUs). However, 54% of these OTUs could not be assigned at the genus level, indicating substantial, as yet undescribed diversity. The remaining OTUs were affiliated predominantly with three genera: Crispospora , Pseudoberwaldia , and Vittaforma. Despite only one formally described species in each of these genera, we show a high species‐level diversity forming putative freshwater lake‐specific clades, which are likely associated with new hosts. Using co‐occurrence network analysis combined with published host records, we identify rotifers as candidate reservoirs for lacustrine Microsporidia. The microsporidian community underwent substantial temporal changes, marked by a major turnover in the early 2000s. This shift coincides with decades of strong anthropogenic pressure on Lake Aydat, including untreated sewage inputs, nutrient enrichment from agro‐pastoral activities, and wetland destruction. As obligate intracellular parasites, Microsporidia are tightly linked to the ecology and population dynamics of their hosts, suggesting that the observed community shifts primarily reflect changes in host communities.

Microbial eukaryotes play a vital role in biogeochemical cycles and aquatic food webs. Over past decades, their taxonomic diversity has been investigated using short-read DNA metabarcoding, which, while effective, is hampered by the limited resolution of the targeted regions. In this study, we utilised both short-read (Illumina) and long-read (PacBio) metabarcoding approaches to analyse eukaryotic diversity in a lake ecosystem over one year, allowing for a direct comparison of the results obtained from each method. Our metabarcoding analysis revealed a high degree of congruence between long-read and short-read data at broad taxonomic levels. However, at finer taxonomic scales, such as the genus level, long-read sequencing achieved higher resolution, enabling more precise identification of microbial eukaryotes. This enhanced taxonomic resolution proved especially valuable for tracking seasonal dynamics within key groups, including ciliates and chytrid fungi. The resolution offered by long-read sequencing provides a more detailed picture of the microbial eukaryotic community, thereby facilitating the exploration of ecological interactions at a finer scale. For example, this approach allowed us to monitor the dynamics of the chytrid genus Zygorhizidium in relation to various diatom genera, which are primary targets for chytrid parasitism.

Les microsporidies constituent un vaste groupe de parasites eucaryotes unicellulaires intracellulaires obligatoires apparentés aux champignons, connus principalement comme parasites de vertébrés et d’invertébrés. En effet, celles-ci sont couramment décrites comme parasites d’organismes d’intérêt tels que les insectes auxiliaires, les poissons et crustacés comestibles, ou encore l’Homme. Ces parasites sont présents dans tous types d’environnements, mais plus de 69% des microsporidies sont retrouvées en milieux aquatiques. Dans ces milieux, les microsporidies sont connues pour impacter les communautés de crustacés et de poissons mais il n’existe en revanche que très peu de descriptions de microsporidies parasites d’organismes à la base du fonctionnement des réseaux trophiques (e.g. protistes, microplancton). Leur impact sur la structure et le fonctionnement des écosystèmes aquatiques est donc largement méconnu et non pris en compte. L’objectif de ces travaux de thèse était donc de révéler l’étendue de la diversité des microsporidies et de leur spectre d’hôtes dans les écosystèmes aquatiques. Pour cela, nous avons étudié la diversité des microsporidies au cours du temps et dans différents environnements via une approche de métabarcoding que nous avons préalablement adaptée à ce groupe de parasites. Nous avons également évalué quels pouvaient être leurs hôtes parmi les protistes et les organismes planctoniques à l’aide de différentes approches complémentaires (réseaux de cooccurrence, hybridation in situ, isolement de couples hôte-parasite). Nous avons ainsi révélé une diversité insoupçonnée de microsporidies dans les environnements aquatiques, dont une proportion non négligeable n’était affiliée à aucun genre, montrant ainsi qu’une part importante de cette diversité reste encore à décrire. Nous avons également identifié différents nouveaux hôtes dans ces environnements, dont le rôle est majeur dans le fonctionnement des réseaux trophiques. En effet, nous avons pu identifier des hôtes parmi les rotifères, les ciliés ou encore les dinoflagellés. Nous émettons donc l’hypothèse que les microsporidies, à l’instar d’autres parasites, ont le potentiel d’impacter la structure et le fonctionnement des réseaux trophiques aquatiques, en raison de leur diversité dans ces écosystèmes et leur spectre d’hôte particulièrement large. La contribution relative de ces parasites dans la régulation des communautés à la base du fonctionnement des réseaux trophiques reste maintenant à mettre en lumière.

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.

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. 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.

Summary: 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 (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.