We propose a routine protocol based on size fractionation of pelagic samples and the use of the fluorochrome calcofluor white (which binds to ␤-1,3 and ␤-1,4 polysaccharides) for diagnosing, identifying, and counting chitinaceous fungal parasites (i.e., the sporangia of chytrids) of phytoplankton. The protocol was applied to freshwater samples collected during different seasons (spring and summer/autumn) in two lakes whose trophic statuses varied. Because few samples were collected (i.e., two dates per site), the findings are considered preliminary and mainly a "proof of concept" rather than a valid comparison of sites versus seasons. The results from the proposed protocol indicate higher diversity of infected host and parasite communities than in previous studies. Chytrid epidemics were omnipresent, infecting diverse phytoplankton host communities, primarily diatoms, chlorophytes, and colonial and filamentous cyanobacteria. The diversity and numerical abundance of sporangia and of hosts, and the prevalence of infection (range, <1 to 24% of total host cells) as well, increased from the oligotrophic Lake Pavin to the eutrophic Lake Aydat, while the temporal changes in parasites were apparently more influenced by the host community composition. We conclude that the proposed protocol offers a valid method for the quantitative ecology of chytrid epidemics in aquatic ecosystems and food web dynamics.Fungal infections are recurrent in aquatic ecosystems (15,42,46). Organisms belonging to the order of Chytridiales (i.e., chytrids) are known mainly as phytoplankton parasites (10,22,44). Recently, we have unveiled a large reservoir of unexpected fungal diversity in freshwater lakes, primarily of chytrids (31-33). Parasitic chytrids and other zoosporic true fungi sensu Barr (3) produce zoospores and are often host specific, highly infective, and extremely virulent (13, 16). They are considered relevant not only for the evolution of their hosts but also for the population dynamics and successions of phytoplankton communities (14, 48). Studies of chytrid fungal parasitism carried out in the English Lake District indicate that infection of diatoms, desmids, and other green algae is fairly common (10). Significant chytrid parasitism has also been recorded in other lakes, affecting primarily the diatom Asterionella formosa (12,29,37,48). However, fungal parasitism on plankton has rarely been studied and has mostly been restricted to descriptive taxonomy. Full descriptions of parasitic chytrids have been given since the middle of the last century (8-9) but even today, their impact on the dynamics of host populations and the related biogeochemical cycling and energetics remain largely unknown (16), mainly because of methodological difficulties (17).Various approaches have been used to study fungal parasites, but routine techniques for reliably identifying and counting these organisms are lacking in the context of aquatic microbial ecology (31-33). Some of them have been misidentified as protistan bacterivorous nanoflagellates, e...

Water samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of taxa.

This study is the first assessment of the quantitative impact of parasitic chytrids on a planktonic food web. We used a carbon-based food web model of Lake Pavin (Massif Central, France) to investigate the effects of chytrids during the spring diatom bloom by developing models with and without chytrids. Linear inverse modelling procedures were employed to estimate undetermined flows in the lake. The Monte Carlo Markov chain linear inverse modelling procedure provided estimates of the ranges of model-derived fluxes. Model results support recent theories on the probable impact of parasites on food web function. In the lake, during spring, when ‘inedible’ algae (unexploited by planktonic herbivores) were the dominant primary producers, the epidemic growth of chytrids significantly reduced the sedimentation loss of algal carbon to the detritus pool through the production of grazer-exploitable zoospores. We also review some theories about the potential influence of parasites on ecological network properties and argue that parasitism contributes to longer carbon path lengths, higher levels of activity and specialization, and lower recycling. Considering the “structural asymmetry” hypothesis as a stabilizing pattern, chytrids should contribute to the stability of aquatic food webs.

Fungi are a highly complex group of organisms of the kingdom Eumycota (i.e. the true-fungi) and other fungus-like organisms traditionally studied by mycologists, such as slime molds (Myxomycota) and oomycota (

This study presents an original rDNA PCR and microscopic survey of pelagic freshwater fungal communities, and was designed to unveil the diversity of true Fungi (i.e. the kingdom Eumycota) in three contrasting lake ecosystems (Lakes Pavin, Aydat and Vassivière) located in the French Massif Central. Three clone libraries were constructed from samples collected in the euphotic layers of the lakes during spring 2007. Phylogenetic analysis of the combined data from the three lakes clustered our sequences into thee divisions: Chytridiomycota (50% of total sequences), Ascomycota (40%) and Basidiomycota (10% in Pavin and Aydat only). Several sequences were assigned to a novel Chytridiomycota clade first recovered in Lake Pavin in 2005. Most of the sequences retrieved in the investigated lakes were affiliated with known fungal species, most of which were apparently well adapted to thrive in the pelagic realm. Their main functions (i.e. parasitism and saprophytism), putatively inferred from the closest relatives of the retrieved molecular sequences, were confirmed by microscopic approaches and by enrichment experiments with pollen grains. The occurrence of three fungal forms (zoosporic, yeast and mycelial) was associated with different trophic modes, establishing fungi as strong potential competitors for various niches in pelagic ecosystems, primarily in relation to the processing of particulate organic matter and the production of propagule food sources for grazers. For the first time, this study provides insight into the diversity and the associated functions of all members of the Kingdom Eumycota investigated in the whole plankton fraction of aquatic ecosystems.

Microbial parasites typically are characterized by their small size, short generation time, and high rates of reproduction, with a simple life cycle occurring generally within a single host. They are diverse and ubiquitous in aquatic ecosystems, comprising viruses, prokaryotes, and eukaryotes. Recently, environmental 18S rDNA surveys of microbial eukaryotes have unveiled major infecting agents in pelagic systems, consisting primarily of chytrids (Chytridiomycota). Chytrids are external eucarpic parasites that infect diverse prokaryotic and eukaryotic algae, primarily diatoms and filamentous species. They produce specialized rhizoidal systems within host cells, i.e. the nutrient conveying system for the formation of fruit bodies (sporangia) from which propagules (motile uniflagellated zoospores) are released into the environment. In this review, we summarize the ecological potential of parasites of phytoplankton and infer the implications for food web dynamics. We focus on chytrids, together with other parasitic eukaryotes, with special emphasis on (1) the role of microparasites in driving the structure of phytoplankton communities, (2) the role of chytrid zoospores in matter and energy transfer, and (3) the potential consequences of infections for food web dynamics. We raise the question of genetic potential from host -parasite interactions and also of how environmental factors might affect the host -parasite relationships in the pelagic realm.

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Studies on freshwater Perkinsea are scarce compared to their marine counterparts; they are therefore not well ecologically characterized. In this study, we investigated the diversity, distribution and ecological role of Perkinsea in freshwater ecosystems. Our approach included (1) the phylogenetic analyses of near full-length SSU and LSU sequences of freshwater Perkinsea, (2) a meta-analysis of public Perkinsea 18S ribosomal RNA gene sequences available from the freshwater environments (25 lakes, 4 rivers), (3) microscopic observations of Perkinsea associated with planktonic communities and (4) single amplified genome analysis. Whereas Perkinsea appear to be rare in river ecosystems (85 reads), they are found in almost all of the lakes studied. However, their diversity does vary considerably between lakes (from 0 to 2 463 Operational Taxonomic Units (OTUs)). Phylogenetic analysis showed that the Parvilucifera/Dinovorax/Snorkelia and Perkinsus/Xcellia/Gadixcellia clades resulted from an initial speciation event. This second clade is further split into well-supported, monophyletic groups, including a clade dominated by freshwater representatives, which is further structured into three distinct subclades: freshwater clade 1, freshwater clade 2 and a freshwater and brackish clade. The Perkinsea Single Amplified Genome (SAG) as well as most of the abundant Operational Taxonomic Units (OTUs) fall into freshwater clade 2. The tyramide signal amplification-fluorescent in situ hybridization method showed an internal association between Perkinsea and the colonial phytoplankton Sphaerocystis. The Single Amplified Genome (SAG) annotation contained 698 genes and gene ontology terms could be assigned to 486 protein-coding genes. Although the number of genes appears to be low (10.6% of the entire gene set assessed by BUSCO), the analysis of the proteome revealed some putative secreted virulence factors. This study showed a large distribution of Perkinsea across lake ecosystems and potential parasitic association with phytoplankton. However, further investigations are needed for a better knowledge on the role of these microorganisms in freshwater ecosystems.