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.