Cécile Lepère

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.

The order Nanopelagicales is the most abundant bacterioplankton lineage in freshwater lakes and exhibits typical streamlined genomic characteristi

Amplicon sequencing tools such as metabarcoding are commonly used for thorough characterisation of microbial diversity in natural samples. They mostly rely on the amplification of conserved universal markers, mainly ribosomal genes, allowing the taxonomic assignment of barcodes. However, linking taxonomic classification with functional traits is not straightforward and requires knowledge of each taxonomic group to confidently assign taxa to a given functional trait. Zoosporic parasites are highly diverse and yet understudied, with many undescribed species and host associations. However, they can have important impacts on host populations in natural ecosystems (e.g., controlling harmful algal blooms), as well as on industrial‐scale algae production, e.g. aquaculture, causing their collapse or economic losses. Here, we present ParAquaSeq, a curated database of available molecular ribosomal sequences belonging to zoosporic parasites infecting aquatic vascular plants, macroalgae and photosynthetic microorganisms, i.e. microalgae and cyanobacteria. These sequences are aligned with ancillary data and other information currently available, including details on their hosts, occurrence, culture availability and associated bibliography. The database includes 1131 curated sequences from marine, freshwater and industrial or artificial environments, and belonging to 13 different taxonomic groups, including Chytridiomycota, Oomycota, Phytomyxea, and Syndiniophyceae. The curated database will allow a comprehensive analysis of zoosporic parasites in molecular datasets to answer questions related to their occurrence and distribution in natural communities. Especially through meta‐analysis, the database serves as a valuable tool for developing effective mitigation and sustainable management strategies in the algae biomass industry, but it will also help to identify knowledge gaps for future research.

Microbial eukaryotes play a crucial role in biochemical cycles and aquatic trophic food webs. Their taxonomic and functional diversity are increasingly well described due to recent advances in sequencing technologies. However, the vast amount of data produced by -omics approaches require data-driven methodologies to make predictions about these microorganisms’ role within ecosystems. Using metatranscriptomics data, we employed a sequence similarity network-based approach to explore the metabolic specificities of microbial eukaryotes with different trophic modes in a freshwater ecosystem (Lake Pavin, France). A total of 2,165,106 proteins were clustered in connected components enabling analysis of a great number of sequences without any references in public databases. This approach coupled with the use of an in-house trophic modes database improved the number of proteins considered by 42%. Our study confirmed the versatility of mixotrophic metabolisms with a large number of shared protein families among mixotrophic and phototrophic microorganisms as well as mixotrophic and heterotrophic microorganisms. Genetic similarities in proteins of saprotrophs and parasites also suggest that fungi-like organisms from Lake Pavin, such as Chytridiomycota and Oomycetes, exhibit a wide range of lifestyles, influenced by their degree of dependence on a host. This plasticity may occur at a fine taxonomic level (e.g., species level) and likely within a single organism in response to environmental parameters. While we observed a relative functional redundancy of primary metabolisms (e.g., amino acid and carbohydrate metabolism) nearly 130,000 protein families appeared to be trophic mode-specific. We found a particular specificity in obligate parasite-related Specific Protein Clusters, underscoring a high degree of specialization in these organisms. Although no universal marker for parasitism was identified, candidate genes can be proposed at a fine taxonomic scale. We notably provide several protein families that could serve as keys to understanding host-parasite interactions representing pathogenicity factors (e.g., involved in hijacking host resources, or associated with immune evasion mechanisms). All these protein families could offer valuable insights for developing antiparasitic treatments in health and economic contexts.

The permanently anoxic waters in meromictic lakes create suitable niches for the growth of bacteria using sulphur metabolisms like sulphur oxidation. In Lake Pavin, the anoxic water mass hosts an active cryptic sulphur cycle that interacts narrowly with iron cycling, however the metabolisms of the microorganisms involved are poorly known. Here we combined metagenomics, single‐cell genomics, and pan‐genomics to further expand our understanding of the bacteria and the corresponding metabolisms involved in sulphur oxidation in this ferruginous sulphide‐ and sulphate‐poor meromictic lake. We highlighted two new species within the genus Sulfurimonas that belong to a novel clade of chemotrophic sulphur oxidisers exclusive to freshwaters. We moreover conclude that this genus holds a key‐role not only in limiting sulphide accumulation in the upper part of the anoxic layer but also constraining carbon, phosphate and iron cycling.

The advent of high‐throughput sequencing has led to the discovery of a considerable diversity of microbial eukaryotes in aquatic ecosystems, nevertheless, their function and contribution to the trophic food web functioning remain poorly characterized especially in freshwater ecosystems. Based on metabarcoding data obtained from a meromictic lake ecosystem (Pavin, France), we performed a morpho‐physio‐phenological traits‐based approach to infer functional groups of microbial eukaryotes. Metatranscriptomic data were also analysed to assess the metabolic potential of these groups across the diel cycle, size fraction, sampling depth, and periods. Our analysis highlights a huge microbial eukaryotic diversity in the monimolimnion characterized by numerous saprotrophs expressing transcripts related to sulfur and nitrate metabolism as well as dissolved and particulate organic matter degradation. We also describe strong seasonal variations of microbial eukaryotes in the mixolimnion, especially for parasites and mixoplankton. It appears that the water mixing (occurring during spring and autumn) which benefits photosynthetic host communities also promotes parasitic fungi dissemination and over‐expression of genes involved in the zoospore phototaxis and stage transition in the parasitic cycle. Mixoplanktonic haptophytes over‐expressing photosynthesis‐, endocytosis‐ and phagosome‐linked genes under nutrient limitation also suggest that phagotrophy may provide them an advantage over non‐phagotrophic phytoplankton.

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).